Delivering Nitric Oxide with Nanoparticles John F. Quinn, Michael R. Whittaker, Thomas P. Davis PII: DOI: Reference:
S0168-3659(15)00097-8 doi: 10.1016/j.jconrel.2015.02.007 COREL 7556
To appear in:
Journal of Controlled Release
Received date: Revised date: Accepted date:
23 October 2014 30 January 2015 4 February 2015
Please cite this article as: John F. Quinn, Michael R. Whittaker, Thomas P. Davis, Delivering Nitric Oxide with Nanoparticles, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.02.007
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ACCEPTED MANUSCRIPT Delivering Nitric Oxide with Nanoparticles John F. Quinn 1, Michael R. Whittaker 1 and Thomas P. Davis*1,2
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ARC Centre of Excellence in Convergent Bio-Nano Science and Technology. Monash
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Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 399 Royal Parade, Parkville, Victoria 3152, Australia.
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK. correspondence author: [email protected]; tel +61 3 9903 9260
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Abstract
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While best known for its important signalling functions in human physiology, nitric oxide is also of considerable therapeutic interest. As such, nanoparticle-based systems which enable
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the sustained exogenous delivery of nitric oxide have been the subject of considerable investigation in recent years. Herein we review the various nanoparticle systems that have been used to date for nitric oxide delivery, and explore the array of potential therapeutic applications that have been reported. Specifically, we discuss the modification of sol-gel based silica particles, functionalised metal / metal oxide nanoparticles, polymer-coated metal nanoparticles, dendrimers, micelles and star polymers to impart nitric oxide release capability. We also consider the various areas in which therapeutic applications are envisaged: wound healing, antimicrobial applications, cardiovascular treatments, sexual medicine and cancer treatment.
Finally, we discuss possible future directions for this
versatile and potentially important technology. .
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ACCEPTED MANUSCRIPT 1. Introduction The gasotransmitters (nitric oxide, carbon monoxide and hydrogen sulphide) are a small
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family of otherwise gaseous molecules with an important role in intra- and extracellular
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signalling.1 The role of gaseous molecules in cell signalling was first identified in the 1980s,2
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though the term “gasotransmitter” only came into common usage in 2001.3 Of the three molecules so far identified, nitric oxide (NO) has been the subject of the most considerable
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investigation, and has been shown to have a role in many different physiological events.4 For instance, nitric oxide signalling is important in pain perception,5 sleep control and
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regulation6, vasodilation7, mucus production8, sphincter contraction and relaxation9 regulation of erection10 and the proper functioning of the immune system11.
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Deficiency or overproduction of NO is associated with a number of pathologies.
For
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instance, reduced NO is associated with hypertension in preeclampsia12 and Prinzmetal’s
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angina.13 Elevated levels of NO have also been associated with autoimmune diseases such as rheumatoid arthritis14, systemic lupus erythematosus15 and multiple sclerosis.16 High levels of NO are also related to the processes in transplant rejection17 and in septic shock.18 Clearly
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appropriate regulation of NO in the body is important in maintaining good overall health. In addition to its role in biological signalling processes and in various pathologies, NO has also demonstrated some potential as a therapeutic agent. In particular, exogenous delivery of NO has been demonstrated to have some activity against tumour cells,19 and to be potentially useful in antimicrobial applications.20 For instance, exogenous NO has been applied successfully in the dissipation of biofilms.21 With increased antibiotic resistance becoming a major clinical problem, new antimicrobials which release NO may be of clinical benefit either alone or when applied alongside traditional antibiotics.
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ACCEPTED MANUSCRIPT As NO is a gaseous radical species, direct delivery, while possible, has significant limitations. Instead, it is generally necessary to deliver NO using a reactive precursor of some
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description. Although there are a number of different chemistries available, organic nitrates,
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S-nitrosothiols, metal complexes and N-diazeniumdiolates have received much attention as
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potential small molecule agents for the delivery of NO in a therapeutic context (see Scheme 1).22 One of the principle ways to alter the biodistribution and pharmacokinetic properties of NO donors is to incorporate them into a nanoparticulate form. Friedman and co-workers have
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previously noted that the most important features of an NO delivery system are that the
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material should be easily applied, and be capable of delivering NO over a therapeutically meaningful time interval.23 Issues of cost, storage stability and transportability also need to be
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considered (it is these issues that render the use of gaseous NO particularly impractical).
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Herein we review the current state of the art for nitric oxide delivery using nanoparticles, looking at both the preparation of the particles, post assembly modification to incorporate the
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NO delivering moiety, and in vitro and in vivo studies of the particles. We also explore possible future strategies for the preparation of NO delivering nanoparticles, and examine
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some potential future directions for this important technology.
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ACCEPTED MANUSCRIPT Scheme 1. Common small molecule nitric oxide donors. 2. Preparation of NO-Releasing Nanoparticles
These may be broadly categorized as (i) sol-gel derived silica
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delivery of NO.24
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To date, studies have been conducted on a number of different vehicles for the exogenous
nanoparticles; (ii) surface functionalised metal / metal oxide nanoparticles; (iii) polymer coated metal nanoparticles; (iv) dendrimers; (v) micelles; and (vi) core cross-linked star
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polymers (see Scheme 2).
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Scheme 2. Main classes of nanomaterials applied in the exogenous delivery of nitric oxide. 2a. Nitric Oxide Delivery from Sol-gel Derived Silica Nanoparticles In important preliminary work Meyerhoff and coworkers investigated the functionalization of fumed slilica particles with N-diazeniumdiolate groups as a method for preparing NO releasing particles.25 Although with a size range of 0.2-0.3 µm the particles prepared were
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ACCEPTED MANUSCRIPT not strictly nanoparticles, the study provides an important proof of principle and serves as a starting point for the numerous nanoparticle investigations that have followed. The silica
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particles in question were first surface modified with amine containing silylation reagents to
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provide particles having either primary or secondary amine functionality (or both) on the
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surface. The particles were then exposed to nitric oxide gas to form N-diazeniumdiolates, with secondary amines proving to be the best precursor for stable N-diazeniumdiolate formation. The authors found the N-diazeniumdiolate conversion was maximised in the
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presence of a low concentration of base to maintain the secondary amine in a deprotonated
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state while causing minimal cleavage of the silyl ether groups. These particular particles were applied in thromboresistant tubing, and led to a decrease in the number of thrombi and a
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lower degree of platelet activation when imbedded in a polyurethane coating on the inner
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wall of tubing used in a rabbit extracorporeal blood circulation system. The approaches in
nanoparticles.
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this work have been revisited in numerous subsequent studies to form NO releasing
Substantial early work in the preparation nanoparticles capable of delivering an NO payload
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was conducted by the groups of Schoenfisch and Friedman. In their initial study, Schoenfisch and co-workers applied a similar approach to that which was used by Meyerhoff on fumed silica, this being the formation of NO releasing N-diazeniumdiolate groups on the particles.26 Sol-gel based silica particles with secondary amine functionality were first synthesized by the condensation of tetraethoxysilane (TEOS) or tetramethoxysilane (TMOS) in the presence of N-(6-aminohexyl)aminopropyltrimethoxysilane
(AHAP3), or
(aminoethylamino-
methyl)phenethyltrimethoxysilane
(AEMP3),
N-(2-aminoethyl)-3-
aminopropyltrimethoxysilane (AEAP3).
The authors observed that by variation of the
proportion of amino functional monomer they were also able to control the diameter of the resulting silica nanoparticles. For instance, using TEOS alone resulted in particles with 6
ACCEPTED MANUSCRIPT diameter of 250 nm. By the addition of 10% AHAP3 to the reaction mixture the particle diameter was substantially reduced to 20 nm. Conversely, addition of 10% AEAP3 resulted
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in particles of 500 nm diameter. The particles prepared were essentially non-porous, as
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determined using surface area and pore volume analysis. It is worth noting that there was an
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upper limit to the amount of secondary amine that could readily be incorporated in the systems based on TEOS (20%), as at higher amounts of secondary amine functional monomer significant particle aggregation occurred. The authors attributed this phenomenon
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to hydrogen bonding between the amino groups and silanols. In order to abrogate this issue,
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the authors applied the faster condensing TMOS to decrease the likelihood of aggregation during synthesis.
The strategy was successful with particles having up to 87% amino
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functional monomer being successfully prepared.
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Having prepared sol gel particles with secondary amino groups, the authors then converted these secondary amino groups to N-diazeniumdiolate groups by exposing to elevated
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pressures (5 atm) of NO gas for three days. When the resulting particles were exposed to aqueous conditions the N-diazeniumdiolate moieties were capable of releasing NO, with the
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half-life depending on the specific amine used and the particle size. The total amount of NO released was, as expected, dependent on the total amount of amino functional monomer used in the particle synthesis. The authors demonstrated that the particles could be incorporated into tecophilic polyurethane and silicone rubber, and that the hydrophilicity of the host polymer influenced the kinetics of the NO release. These results demonstrated that particle size, chemical structure and the localised chemical environment all influenced the capability of the particles to release NO, and provide tunability of NO release. For instance, a subsequent study from the same group has examined incorporating these particles into electrospun polyurethane fibers as a potential method for preparing porous coatings with NO release capability.27 7
ACCEPTED MANUSCRIPT Friedman et al. employed an approach wherein nitric oxide was effectively frozen inside a glassy sol-gel matrix, from where NO could be released through the thermal degradation of
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nitrite ions in the presence of reducing sugars such as glucose and tagatose. (see Scheme 3).23
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The particular sol gel matrix employed was again based on either TMOS or TEOS. The
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particles prepared were characterised using transmission electron microscopy, and were relatively uniform in both size and shape (diameter = 10 nm) (see Fig 1 a). Sustained release of NO from the particles was characterised by the use of an NO specific probe and the NO
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specific fluorescence dye 4,5-diaminofluorescein (see Fig 1b). By judicious selection of the
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matrix components and reaction protocol, the authors were able to demonstrate sustained delivery of NO over a variety of different time frames. Varying molecular weight
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polyethylene glycol (PEG) was added to the matrix, and this was demonstrated to have a
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substantial effect on the kinetics of NO release. Samples incorporating PEG with a molecular weight of 3000 g mol-1 had a much faster rate of release of NO from the matrix, despite
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having a lower total loaded mass of PEG. The authors attributed this to the effect of added PEG on the nanostructure of the particles, facilitating more rapid water infiltration and
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thereby faster NO release. Importantly, the authors presented evidence that the particles were stable of a period of many weeks, and that sustained release of NO was possible over a substantial time frame once the particles were hydrated. The particles were shown to exhibit minimal toxicity against fibroblast cells, paving the way for potential therapeutic applications of the particles. Moreover, the authors also demonstrated that it was possible to include chitosan in the matrix as an aid to mucodahesion and in order to enhance cell penetration and thereby bioavailability. The authors noted that the particulate system was “ideal for immediate development and deployment for applications requiring cutaneous delivery of exogenous NO.”
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Scheme 3. NO releasing nanoparticles as prepared by Friedman & co-workers.23
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Figure 1. (A) TEM micrograph of NO releasing sol-gel particles as prepared by Friedman and co-workers. (B) Evolution of fluorescence spectrum of diaminofluorescein following reaction with NO released from sol-gel/glass composites as prepared by Friedman and coworkers.
Reprinted from Nitric Oxide Biol. Chem. 19, A. J. Friedman, G. Han, M. S.
Navati, M. Chacko, L. Gunther, A. Alfieri, J. M. Friedman, Sustained release nitric oxide 10
ACCEPTED MANUSCRIPT releasing nanoparticles: Characterization of a novel delivery platform based on nitrite containing hydrogel/glass composites, 12-20. Copyright (2008), with permission from
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Elsevier.
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Hu and coworkers have recently reported the preparation of S-nitroso functional
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nanoparticles over a wide range of sizes via the condensation of nitrosated 3mercaptopropyltrimethoxysilane (MPTMS). In the first step the MPTMS was nitrosated by
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reaction with sodium nitrate in the presence of HCl (i.e., nitrous acid) overnight in dimethyl sulfoxide. An aliquot of this mixture was subsequently aspirated and injected with rapid
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stirring into water at room temperature. The authors demonstrated that the initial reaction time with nitrous acid had a profound impact on the final particle size, with smaller particles
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being formed with longer nitrosation times. Other factors, such as temperature, needle size,
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injection volume, ionic strength and pH were also shown to impact the ultimate particle size. The authors demonstrated that nitric oxide was released from the particles when exposed to
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aqueous solution at 25 °C or 37 °C. As is expected for S-nitrosothiols, light stimulated the release of NO, with near complete decomposition of the nitroso group over 24 h when
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exposed to light. The presented method has the advantage of providing relatively stable particles without added surfactant, although there is a lower limit to the particle size that could be obtained (100 nm). 2b. NO-releasing Surface Functionalised Metal / Metal Oxide Nanoparticles In addition to work on silica nanoparticles, studies have also been made into the use of gold nanoparticles as potential hosts for NO releasing groups. Rothrock et al examined the preparation of NO releasing gold nanoparticles by the simple modification of the gold surface using 11-bromo-1-undecanethiol.28 Gold nanoparticles were initially prepared by reduction of hydrogen tetrachloroaurate with sodium borohydride in the presence of hexanethiol. The
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ACCEPTED MANUSCRIPT gold nanoparticles (2 nm) were then surface functionalised with alkyl bromide groups through the place exchange method, using a 3:1 mixture of 11-bromo-1-undecanethiol and
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dodecanethiol. The resulting bromo-functional nanoparticles were then reacted with either
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ethylenediamine, butylamine, hexanedimanine or diethylenetriamine to give gold
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nanoparticles incorporating a secondary amine functionality. These particles were then purified by washing extensively with acetonitrile before resuspending in methanol and sodium methoxide.
Exposure to NO gas at 5 atm resulted in the formation of
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diazeniumdiolate functionalised gold nanoparticles.
For an equivalent % amine, the
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diazeniumdiolate containing particles prepared using ethylene diamine exhibited the longest half-life, while the particle functionalised with hexanediamine had the highest NO loading.
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While some release was noted at elevated temperature, exposure to an aqueous environment
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had the greatest impact on release, implying a combined thermal and proton driven mechanism for N-diazeniumdiolate dissociation.
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Gold monolayer protected clusters stabilised with Tiopronin have also been used as a substrate for preparing NO donor nanoparticles.29 In this case the terminal carboxylic acid
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group of the Tiopronin was activated by reaction with N-hydroxysucciniminde in the presence of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC), and was thereafter modified with either diethylenetriamine (DETA), tetraethylenepentamine (TEPA) or pentaethylenehexamine (PEHA) to provide secondary amine groups for conversion to Ndiazeniumdiolate. Ideally, conversion of the secondary amine groups to N-diazeniumdiolate would occur in the presence of a base such as sodium methoxide to ensure maximal conversion.
Unfortunately, these conditions induced destabilisation of the nanoparticle
dispersion, and so only limited conversion could be achieved. Nevertheless, exposure to NO gas at 5 atm yielded N-diazeniumdiolate functional gold nanoparticles, with those particles functionalised with PEHA provided the highest loading of NO. In order to abrogate the issue 12
ACCEPTED MANUSCRIPT of colloidal stability, particles were prepared that were directly stabilised by the DETA, TEPA and PEHA (i.e., without Tiopronin). Using this approach the authors were able to
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achieve substantially higher loading of NO, with substantially longer half-lives and sustained
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release over 16 hours.
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Superparamagnetic iron oxide nanoparticles with NO release capability have recently been reported by Haddad and coworkers.30 In this study, the nanoparticles were first prepared by
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coprecipiatation of iron (III) chloride hexahydrate and iron (II) chloride tetrahydrate with ammonia. While the particles were initially stabilised by the addition of oleic acid, this was
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subsequently exchanged with mercaptosuccinic acid to yield nanoparticles with thiol functionality. The thiol functional nanoparticles were then nitrosated by treatment with an
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acidified solution of sodium nitrite (i.e., nitrous acid), yielding S-nitrosothiol functionalised
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superparamagnetic iron oxide nanoparticles. Sustained release of NO over a period of hours was demonstrated by chemiluminescence measurements. These results demonstrate the
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relative ease with which NO releasing moieties can be conferred to a particle surface possessing thiol functionality.
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Veres and co-workers have prepared Fe3O4-silica core shell nanoparticles with S-nitrosothiol functionality.31 In this case superparamagnetic nanoparticles were first prepared by a thermal decomposition process, heating a solution of iron pentacarbonyl in the presence of oleylamine. After isolating the amine stabilised iron oxide nanoparticles, a silica shell was subsequently formed on the particle surface by condensation of tetraethoxysilane in the presence of ammonia. Addition of 3-mercaptopropyltrimethoxysilane and further reaction for 24 hours yielded thiol functionalised Fe3O4-silica core-shell particles. These were subsequently converted to S-nitrosothiol functionalised particles by reaction with t-butyl nitrite. The resulting particles were shown to have the expected superparamagnetic properties, and released NO in a controlled fashion over a number of hours, with the precise 13
ACCEPTED MANUSCRIPT rate dependent on temperature. Fluorescence studies indicated that the particles were taken up by epithelial cells over a 24 h time period, and that the NO release was effectively concluded
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with 48 hours. An MTT assay was conducted to examine particle toxicity, with S-nitrosothiol
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functionalised nanoparticles being generally less toxic to human alveolar epithelial cells than
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thiol functionalised particles. The authors attributed to this slight decrease in toxicity to the cytoprotective effect of low doses of NO.
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Voelcker and coworkers have recently reported on the preparation of thermally hydrocarbonized porous silica nanoparticles with NO release capability. 32 These particles
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were prepared by periodically etching p+ type silicon wafers at 50 mA/cm2 and 200 mA/cm2 in an aqueous HF/ethanol electrolyte, followed by film detachment by increasing the current
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density to 250 mA/cm2. The films were then hydrocarbonized by exposure to acetylene / N2
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at 500 ºC, and ball milled to provide the nanoparticles. NO release capability was imparted by exposure to a glucose / sodium nitrite solution, followed by drying at 65 ºC or
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lyophilisation. The authors monitored the release of NO using fluorescence and demonstrated that NO release predominantly occurred in the first two hours after exposure to water.
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Subsequent studies were undertaken to investigate the bacterical efficacy of these particles: these results are discussed in the “Therapeutic Applications” section below. 2c. NO releasing Polymer Coated Metal Nanoparticles Hybrid organic / inorganic nanoparticles with NO release capability have also been formed through the self-assembly of thiol terminated polymers onto the surface of gold nanoparticles.33 Specifically, poly((oligoethyelenglycol methyl ether) methacrylate)-blockvinylbenzyl chloride was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, and then reacted with hexylamine to provide materials with secondary amine groups. Treatment with hexylamine also resulted in conversion of the
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ACCEPTED MANUSCRIPT dithioester endgroups into thiol groups, thereby facilitating grafting onto the surface of the gold nanoparticles. Subsequent exposure to NO gas at elevated pressure (5 atm) led to
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conversion of the secondary amine groups to N-diazeniumdiolate groups. The resulting
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particles were shown to release NO slowly, with 90% release over 6 days at pH 6.8 and room
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temperature. Proof of principle experiments were subsequently conducted using these particles, with their utility in biofilm dispersion and cancer cell treatment being explored.
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2d. Nitric Oxide Delivery from Dendrimers
Dendrimers possess the considerable advantage of being highly monodisperse with wellMoreover, manipulating the generation of dendrimer
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defined molecular architecture.
synthesis allows precise control over the number of functional groups and particle size. The
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first report of using dendrimers as a scaffold for nitric oxide release described the
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functionalization of polypropyleneimine (PPI) dendrimers (Generation 3 or 5) with N-
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diazeniumdiolate moieites by exposure to NO gas at 5 atm (see Scheme 4).34 The inherent amino functionality of the PPI dendrimers made this a relatively straightforward procedure, although the dendrimers used included only primary or tertiary amino groups in the
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molecular structure, neither of which are ideal for N-diazeniumdiolate formation. While the authors were able to form some N-diazeniumdiolates using the primary amino groups, these were not effective in providing sustained release of NO. Moreover, it was demonstrated that the primary amine derived N-diazeniumdiolates tended to release nitroxyl (HNO) rather than NO at biologically relevant conditions (pH =7.4).
Only at lower pH (ca. 3) was an
appreciable amount of NO released. As such, the primary amine groups of the PPI dendrimers were modified by a two-step reaction to form secondary amines. Firstly, the primary amine was reacted with heptanoyl chloride to form an amide. Thereafter the amide was reduced using lithium aluminium hydride to form the secondary amine. The secondary amine functional dendrimers showed vastly improved loading compared to the primary 15
ACCEPTED MANUSCRIPT amines, provided longer half-lives and preferentially released NO rather than nitroxyl. As modification with heptanoyl chloride and LiAlH4 yielded a hydrophobic dendrimer, the
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authors applied an alternative strategy using propylene oxide to yield hydroxyl functional
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dendrimers. These dendrimers could host higher loadings of NO but had shorter half-lives
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than the hydrophobic version. The authors also demonstrated that reaction of the primary amines with acetic anhydride could be employed to form amide linkages which were inert to N-diazeniumdiolate formation. It was postulated that this might provide a facile means of
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tailoring the N-diazeniumdiolate loading by diluting out the secondary amine functionality.
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Scheme 4. NO releasing dendrimers as prepared by Schoenfisch and co-workers. A subsequent study from the same group examined attachment of S-nitrosothiol moieites to dendrimers to form NO donating polymer particles.35 Using generation 4 polyamidoamine dendrimers with terminal amine functionality as a starting material, the authors conjugated either N-acetyl-D,L-penicillamine or N-acetyl-L-cysteine to the outside of the dendrimer, 17
ACCEPTED MANUSCRIPT Near quantitative conversion of the terminal groups was achieved in the case of N-acetylD,L-penicillamine, while the yield with N-acetyl-L-cysteine was somewhat lower at 67%.
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Thereafter the sulfhydryl group in the penicillamine was modified by exposure to nitrous
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acid, while the sulfhydryl in the acetyl cysteine group was converted to an S-nitrosothiol
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using isopentyl nitrite. These S-nitrosothiol functional dendrimers were then shown to release NO when triggered by either Cu2+ or visible light, with the kinetics being influenced by the structure of the S-nitrosothiol group (primary being somewhat slower than tertiary). The S-
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nitroso-N-acetyl-D,L-penicillamine functional dendrimer was subsequently applied in a
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platelet aggregation assay, and was shown to inhibit thrombin mediated platelet aggregation by 62%, c.f. only 17% inhibition for a small molecule donor. These results demonstrated the
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potential utility of NO donating dendrimers prepared via nitrosation of an incorporated thiol
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moiety.
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2e. NO-Releasing Micelles
Micelles are considerably more straightforward to synthesize than dendrimers, and are dynamic assemblies which may undergo triggered disassembly in biological environments.
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Duong et al. explored a number of strategies for incorporating NO donor moieties into micellar structures. In their first report,36 the common NO donor S-nitrosoglutathione was conjugated into the hydrophobic domain of a block copolymer of oligo(ethylene glycol methyl ether) methacrylate and the functional monomer 2-vinyl-4,4-dimethyl-5-oxazolone. The conditions employed for the conjugation (basic aqueous solution) facilitated assembly of the GSNO functionalised polymer into micelles. Dynamic light scattering studies revealed a micelle size of 40 nm which increased to 50 nm upon incorporation of the GSNO. The authors observed that when incorporated into the core of a micelle the half-life of the GSNO was significantly lengthened when compared to free GSNO in aqueous solution (see Fig. 2). Intracellular release was also demonstrated using the fluorescent dye 4-amino-518
ACCEPTED MANUSCRIPT methylamino-2’,7’-difluorofluorescein diacetatate, which emits a fluorescent signal upon reaction with an NO molecule. Finally the authors demonstrated that application of NO
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releasing micelles with cisplatin improved the IC50 value for cisplatin against neuroblastoma
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by a factor of about 5, but interestingly did not have any appreciable impact on the IC 50 value
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for noncancerous fibroblast cells.
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ACCEPTED MANUSCRIPT Figure 2. Release of nitric oxide from NO releasing micelles (red line) compared with release from a small molecule NO donor (GSNO, blue line). Figure 2A shows the release
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profile in water and Figure 2B in an aqueous solution of ascorbic acid (5 mM). In each
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experiment the initial GSNO concentration was 0.4 mM. Adapted from Chem. Commun. 49
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(2013) 4190-4192 with permission of the Royal Society of Chemistry.
NO releasing micelles wherein the nitric oxide donor is an aliphatic nitrate moiety have also
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been prepared.37 In this case the block copolymer prepared was formed by chain extending poly[oligo(ethylene glycol methyl ether) methacrylate] with vinylbenzyl chloride and styrene
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to yield a block copolymer having segments of (i) hydrophilic polyethylene glycol brushes and (ii) hydrophobic styrene and vinyl benzyl chloride moieties. The ratio of styrene to
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vinylbenzyl chloride was manipulated to alter the number of sites for subsequent nitrate
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functionalization. To provide an NO donor moiety, the chloromethyl groups in the hydrophobic segment were converted to nitrate groups by reaction with silver nitrate in
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acetonitrile. These nitrate functional micelles were shown to have a diameter of approx..30 nm (by DLS and TEM). NO release at two temperatures (37 ºC and 60 ºC) was followed by H NMR (following the protons in the methylene unit adjacent the nitrate group). It was
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demonstrated that in the presence of glutathione approximately 99% of the NO was released over 21 h at 60 degrees, while only 36% was released at 37 degrees. Given the simplicity of synthesizing the organic nitrate polymer, these micelles have some promise for use as nitric oxide donor nanomaterials. 2f. NO Release from Core Cross-linked Star Polymers Core cross-linked star polymers have stability advantages over micelles and are more straightforward to synthesize than dendrimers. Duong et al prepared NO releasing star polymers incorporating N-diazeniumdiolate moieties for NO release.38 The star polymers
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ACCEPTED MANUSCRIPT were prepared by the living radical polymerization of polyethylene glycol methyl ether acrylate, followed by chain extension with 2-vinyl-4,4-dimethyl-5-oxazolone and the
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crosslinking agent N,N-methylenebisacrylamide (see Scheme 5). The incorporated oxazolone
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units provided a convenient locus for the incorporation of spermine, which in turn provided
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secondary amine sites for the formation of N-diazeniumdiolates by exposure to NO gas at 5 atm. The resulting polymers were applied in the dispersal of biofilm, as discussed in some
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detail below.
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Scheme 5. Core cross-linked star polymers functionalised for NO release, as prepared by Duong et al. Forrest and co-workers have recently reported on the preparation of four-arm, sugar-based polymeric nanocarriers for potential application against squamous cell carcinoma. 39 In this case a multifunctional pentaerthyritol based RAFT agent was first prepared as the starting material.
Using
this
RAFT
agent,
1,2,3,4-di-O-isopropylidene-6-O-acryloyl-α-d22
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acryloyl-D-galactose). Modification of this material with succinic anhydride gave an acid
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functional glycopolymer which could be conjugated to a hydroxyl functional N-
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diazeniumdiolate NO donor. The resulting NO releasing star polymer was examined for cytotoxicity, and it was demonstrated that the conjugated donor was substantially less toxic than were the small molecule NO donors conjugated to the polymeric nanocarrier. Moreover,
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conjugation to the glycopolymer slightly increased the half life of the donor compared
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withthe small molecule analogues.
3. Therapeutic Studies on NO-delivering Nanoparticles
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As noted above, exogenous delivery of nitric oxide offers potential benefits in a range of
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different clinical situations.40 To date, the controlled delivery of NO using nanoparticles has
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been explored in relation to wound healing, antimicrobial applications, cardiovascular treatments, anticancer agents, erectile dysfunction and novel dental materials. We explore
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each area in detail below. 3a. Wound healing
Wound healing is an important process in a variety of different contexts, but particularly in surgical and aged care situations.41 The development of chronic wounds can lead to further complications, which may in turn result in increased morbidity and mortality. Nanoparticle approaches to treating chronic wounds are not new, and there has been particular consideration of nanoparticulate silver in wound healing, although the utility of this material is the subject of some debate.42
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ACCEPTED MANUSCRIPT Nitric oxide is involved in all stages of the wound healing process, contributing through increasing vasodilation, inhibiting platelet aggregation, stimulating fibroblasts, keratinocytes
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and endothelial cells, increasing angiogenesis and stimulating the production and deposition
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of collagen. Blecher et al., having noted the critical role played by NO in wound healing,
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proposed that the application of nanoparticles capable of sustained release of nitric oxide may be advantageous for promoting wound healing.43 To this end, these authors applied sol-gel derived NO releasing nanoparticles to mice having a 5 mm excision wound. The mice used
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in the study were NOD-SCID mice, which have a number of different impairments resulting
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in substantial immune dysfunction. In particular the lack of CD4+ and CD8+ T lymphocytes and deficiencies in B lymphocytes are likely to significantly impair wound healing under
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ordinary conditions. Comparison was made with three sets of mice: (i) no treatment; (ii)
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treatment with nanoparticles without NO release capability; and (iii) treatment with the small molecule NO donor diethylenetriamine N-diazeniumdiolate (DETA-NONOate). The authors
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found that topical application of NO releasing nanoparticles to the wound area resulted in accelerated wound closure. Closure of the wounds treated with NO releasing nanoparticles
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remained ahead of the other treatment and control sets throughout the study duration, culminating in total wound closure at day 13. Histological studies demonstrated that there was an increase in the number of fibroblast like cells in both superficial and deep areas of the granulation tissue in wounds treated with NO releasing nanoparticles. Moreover, the authors also observed a decrease in the number of inflammatory cells in the wound compared to DETA-NONOate treated wounds. The DETA-NONOate treated wounds were observed to have minimal granulation tissue, and showed extensive inflammatory infiltrates and fibrinous debris. Further investigation demonstrated that wounds treated with NO releasing nanoparticles showed immature collagen fibers closer to the surface with more developed fibers in the deeper tissue. New vessel formation was also noted, with significantly more
24
ACCEPTED MANUSCRIPT neovascularization occurring in the mice treated with NO releasing nanoparticles than in the nanoparticle or control treatment groups.
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In a further study, Han et al. conducted a series of in vivo and in vitro examinations of the
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efficacy of NO releasing nanoparticles on wound healing.44 This study also utilised the sol-
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gel particles of Friedman and co-workers.23 It was observed that application of NO releasing nanoparticles to human dermal fibroblasts resulted in increased migration (observed using an
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in vitro scratch test) compared to control nanoparticle or untreated experiments. Moreover, it was also observed that application of NO releasing nanoparticles led to an increase in the
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expression of both collagen type I and type III. Interestingly, the application of nanoparticles without NO release capability increased the expression of Type I collagen to a similar extent
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as NO releasing particles, but was substantially less effective at stimulating production of
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Type III collagen. NO releasing nanoparticles were also able to modify the migration of leukocytes into wounded skin tissue.
Specifically, it was demonstrated that neutrophil
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migration was somewhat impaired by the application of NO releasing nanoparticles, while macrophage-like cell migration was significantly increased. In a further aspect of this study,
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the authors demonstrated that there was increased angiogenesis in wounds treated with NO releasing nanoparticles. This was demonstrated by observing an increase in CD34 expression and dense vascularization compared to either control nanoparticle or untreated samples. Clearly, NO releasing nanoparticles have a substantial impact on wound closure in normal and immunocompromised mice and are of considerable interest given the social and economic impact of chronic wounds worldwide. 3b. Antimicrobial Applications Pelgrift and Friedman have recently reviewed the potential use of nanoparticles for addressing the increasing problem of antimicrobial resistance.45 In particular, nanoparticles
25
ACCEPTED MANUSCRIPT capable of delivering a nitric oxide payload are considered to have some potential in overcoming this problem. Nitric oxide is considered to have three main antimicrobial effects.
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Firstly, NO has been shown to have a direct chemical effect on DNA, deaminating cytosine,
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guanine and adenine through the formation of nitrosating intermediates such as N2O3, and via
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radical induced scission or modification of the DNA strands. Secondly, NO can cause Snitrosation of the –SH moiety in the cysteine residues of DNA repair enzymes (such as DNA alkyl transferases), thereby inhibiting their function. Finally, elevated concentrations of NO
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can cause the formation of secondary species such as peroxynitrite and nitrogen dioxide,
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which can facilitate lipid peroxidation. The combined effect of these three mechanisms makes NO a potent antimicrobial agent which may be employed either alone or in concert
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with other antibiotic agents.
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Hetrick et al. have examined the antibacterial properties of NO releasing silica nanoparticles against both Staphylococcus aureus and Pseudomonas aeruginosa.46 In this particular study
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the particles were prepared by the sol-gel condensation of TEOS with the N-diazeniumdiolate derivative of N-(6-aminohexyl)aminopropyltrimethoxysilane. The half-life for NO release for
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the resulting particles was 18 min. The bactericidal efficacy of these particles was compared with a small molecule NO donor, the N-diazeniumdiolate derived from the amino acid proline (PROLI/NO). The NO releasing silica particles were substantially more effective at killing both S. aureus and P. aeruginosa than was the small molecule NO donor. Moreover, this trend was observed both in PBS buffer and in tryptic soy broth (i.e., under conditions were cell proliferation should be enhanced). Particle cytotoxicity was also examined against L929 mouse fibroblasts, with minimal cell toxicity observed at concentrations required for bactericidal activity. Interestingly, the small molecule donor was quite toxic to the mouse fibroblasts at bactericidal concentrations.
A subsequent study investigated the effect of
varying particle size on the antibacterial performance against P. aeruginosa.47 In this case 26
ACCEPTED MANUSCRIPT particles were prepared by the reverse microemulsion sol-gel condensation of TEOS with N(6-aminohexyl)aminopropyltrimethoxysilane, with small variations made to the synthetic
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protocol in order to yield particles of 50 nm, 100 nm and 200 nm. Thereafter the particles
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were functionalised with N-diazeniumdiolate moieties by exposing to NO gas at 10 bar. The
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NO release profiles, loading and half-lives were comparable for the three particle sizes. Antibacterial assays against P. aeruginosa demonstrated that the smallest particles were most effective after 2 h, with a two fold lower minimum bactericidal concentration (MBC) (0.8 mg
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mL-1 vs 1.5 mg mL-1). After 24 h the 100 nm particles were comparable in performance to
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the 50 nm particles (0.2 mg mL-1 vs 0.4 mg mL-1 for the 200 nm particles). Cytotoxicity tests against L929 mouse fibroblasts demonstrated that the NO releasing nanoparticles were non-
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toxic against normal mammalian cells at the concentrations required for bactericidal activity.
antibacterial applications.
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These results demonstrate the considerable promise which these particles offer for
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In an interesting variation on this work, Carpenter et al. have examined so-called “dual action antimicrobials” which have quaternary ammonium functionality as well as NO delivery
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capability.48 The particles applied were synthesized by the sol-gel condensation of N-(6aminohexyl)aminopropyltrimethoxysilane with TMOS. A library of quaternary ammonium functionalised epoxides was prepared in parallel by the reaction of epichlorhydrin with N,Ndimethylalkylamines These epoxides were then reacted with the particles to give silica nanoparticles incorporating: (i) two secondary amine groups for subsequent conversion to Ndiazeniumdiolates) and (ii) a quaternary ammonium moiety with two methyl substituents and a butyl, octyl or dodecyl substituent. The particles were converted to the diazeniumdiolate derivative by reacting with NO gas for three days at 10 bar. Following characterization of the NO release properties, antibacterial assays against S. aureus and P. aeruginosa demonstrated that those particles incorporating octyl or dodecyl groups in the quaternary ammonium 27
ACCEPTED MANUSCRIPT moiety were generally more potent than those with methyl or butyl groups. Moreover NO release improved the performance of the particles against P. aeruginosa more significantly
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than it did against S. aureus. The major limitation of the study was that cell toxicity against
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L929 mouse fibroblast cells was significant for all particles at the concentrations required for
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effective antimicrobial action, with the best result being ca. 65% viability after 24 h. Further investigation and optimization is required to ensure potency to bacteria with minimal toxicity
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to mammalian cells.
In addition to silica nanoparticles, silica nanorods have also been examined for their
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bactericidal efficacy against S. aureus and P. aeruginosa.49 In this case three different aspect ratios were examined (1, 4 and 8). The particles of differing geometry were prepared by
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subtle variations in the condensation conditions (temperature, surfactant concentration, silane
with
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concentration and base concentration). The resulting particles were surface functionalised either
N-(2-aminoethyl)-3-amino-isobutyl-dimethyl-methoxysilane
or
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aminopropyldimethylethoxysilane. The authors demonstrated that in general the higher aspect ratio nanorods were more effective as bactericides for both S. aureus and P. aeruginosa, and
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that nanorods with an aspect ratio of 8 were non-toxic to L929 mouse fibroblasts at the same concentrations necessary for bacterial killing. Further, the silica nanorods with aspect ratio of 4 were also modified with a structurally different secondary amine to give a different Ndiazeniumdiolate and appended PEG chain. This system was used to investigate the effect of NO flux, and it was shown that higher initial flux was important to bacterial killing. The results demonstrate the complex interplay between particle shape, size and surface functionality in applying NO releasing materials to bactericidal applications. Friedman and co-workers have previously reported the effect of sol gel derived NO releasing nanoparticles on a range of different bacteria.50 Specifically, the authors applied NO releasing nanoparticles either alone or in combination with glutathione (in order to generate S28
ACCEPTED MANUSCRIPT nitrosoglutathione in situ) to methicillin resistant S. aureus (MRSA), Escherichia coli, Klebsiella pneumoniae, and P. aeruginosa.
In each case application of NO releasing
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nanoparticles alone or with glutathione caused cell proliferation to be significantly inhibited
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(particularly over shorter time periods), and cell survival after 24 hours to be substantially
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decreased. Interestingly, the authors noted that application of S-nitrosoglutathione did not effect cell proliferation or survival to anywhere near the same extent as the nanoparticle / glutathione combination, giving clear evidence for usefulness of the nanoparticle delivery The authors attributed this difference to the ability of the NO releasing
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platform.
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nanoparticles to provide a sustained concentration of GSNO over the duration of the experiment through in situ nitrosation of the glutathione, as opposed to directly applying S-
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nitrosoglutsathione which has a relatively short half-life and decays completely in 6 h. The
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best results were achieved on P. aeruginosa, where coadministration of glutathione and NO releasing nanoparticles resulted in total inhibition for 24 h and cell survival well below 10%
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in the same period.
NO releasing dendrimers have also been explored for their antibacterial activity. In an early
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report, a suite of G2 and G5 polypropyleneimine (PPI) dendrimers were functionalised with epoxides to impart secondary amine functionality suitable for subsequent conversion to Ndiazeniumdiolate groups.51 Moreover, by appropriate choice of the epoxide the authors were able to modify the chemical periphery of the dendrimer with hydroxypropyl, hydroxybenzyl or oligoethylene glycol functional groups. In the case of P. aeruginosa, the addition of nitric oxide release capability typically improved the bactericidal efficacy, with the most potent formulation being the hydroxybenzyl terminated dendrimers of Generation 2. When the suite of dendrimers was assessed against S. aureus, the incorporation of N-diazeniumdiolate moieties again improved the bactericidal efficacy of all dendrimers with two exceptions: PPI dendrimers of generation 2 having terminal hydroxyl propyl groups (where the bactericidal 29
ACCEPTED MANUSCRIPT efficacy was reduced with addition of NO capability) and hydroxybenzyl terminated dendrimers of generation 8, (where the addition of N-diazeniumdiolate groups had no
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impact). Having identified hydroxybenzyl terminated dendrimers with NO release capability
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as the most effective for standard S. aureus, the same materials were tested against MRSA.
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Importantly, these dendrimers were equally effective for killing MRSA as they were for standard S. aureus. The authors also examined the toxicity of the dendrimers toward mammalian fibroblast cells (L929 mouse fibroblasts). While generation five dendrimers
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were quite toxic to the mammalian cells at the same concentrations necessary to kill bacteria,
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this could be substantially ameliorated by the inclusion of NO releasing N-diazeniumdiolate
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moieties, even at twice the minimum concentration required to kill the bacteria.
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In an interesting recent report, Schoenfisch and coworkers have explored the application of
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quaternary ammonium functionalised dendrimers with NO releasing capability for antibacterial application against S. aureus and P. aeruginosa.52 In this case polyamidoamine (PAMAM) dendrimers (G1 or G4) were modified using quaternary ammonium epoxides to
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give dendrimers having both quaternary ammonium groups and secondary amine groups at the dendrimer periphery. A range of quaternary ammonium epoxides were used so as to yield ammonium moieties having either three methyl or two methyl and a (i) butyl; (ii) octyl; or (iii) dodecyl substituent. These quaternized dendrimers were then exposed to nitric oxide at 10 bar to give N-diazeniumdiolate moieties via the secondary amine group. The resulting dendrimers were capable of releasing an NO payload of between 1.07 and 1.69 µmol mg-1, with a half-life of between 1.9 and 4.9 h. The N-diazeniumdiolate half-lives observed for dendrimers with longer quaternary ammonium substitutents (octyl, dodecyl) were typically lower than for the shorter substituents (methyl, butyl). This was attributed to the formation of dendrimer vesicles which presented the N-diazeniumdiolate moiety to the aqueous 30
ACCEPTED MANUSCRIPT environment, thereby facilitating faster NO release. The authors observed that longer chain functional dendrimers were more efficient at killing both S. aureus and P. aeruginosa, and
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that NO release capability made little difference to antibacterial activity in these cases.
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However, for the shorter chain quaternary ammonium groups the addition of NO release
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capability to the dendrimer significantly increased the bactericidal efficacy. Toxicity against mammalian cells was also evaluated by exposing the dendrimers to L929 mouse fibroblast cells. In stark contrast to the bacteria, the addition of NO release capability to the dendrimers
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generally improved cell viability for mammalian cells, even for the most toxic octyl and
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dodecyl substituted ammonium dendrimers. These results demonstrate the utility of NO release for both improving the antibacterial activity of quaternary ammonium functionalised
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dendrimers and reducing their toxicity to mammalian cells.
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Core cross-linked star polymers capable of NO delivery have recently been applied in the dispersion of biofilms of P. aeruginosa. Biofilm formation presents a particular problem as
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these highly structured adhered films tend to exhibit an increased resistance to biocidal agents.53 Biofilm formation can be a particular issue when patients are cannularized, such as
AC
with a urinary catheter, or in patients with longer term prostheses such as a tracheostomy.54 If such implants become colonised with resistant bacteria serious complications may arise. Moreover, the formation of a resistant extracellular matrix around the biofilm can reduce the effectiveness of biocidal agents and antibiotics. Importantly, nitric oxide has been shown to have a role in the later stages of biofilm development in order to induce biofilm dispersal. Specifically, concentrations of NO in the picomolar - nanomolar range can induce the dispersal of biofilms of a wide range of microbial species through a signalling pathway involving cyclic di-GMP.
31
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ACCEPTED MANUSCRIPT
Figure 3. Inhibition of biofilm formation in the presence of nitric oxide releasing star polymers. (A) Planktonic biomass as determined by measurement of OD600 of the cell culture supernatant. (B) Biofilm biomass as determined by crystal violet staining (OD550). Reprinted with permission from Biomacromolecules (2014) 2583-2589. Copyright 2014 American Chemical Society.
32
ACCEPTED MANUSCRIPT Duong et al demonstrated the application of polymer nanoparticles in aiding the dispersion of biofilm.38 Subsequent testing of these materials demonstrated that the star polymers were
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effective in inhibiting the formation of biofilm (see Fig. 3). Whereas small molecule NO
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donors were ineffective at inhibiting the formation of biofilms, star polymer vectors
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demonstrated near complete inhibition of biofilm formation. Moreover, exposure of existing P. aeruginosa films to the star polymer resulted in a decrease of film biomass of approximately 80% when the star polymers were inoculated at 400 μg/mL, with no
NU
corresponding reduction of planktonic growth. When the same star polymers were exposed to
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mutant strains of P. aeruginosa lacking the phosphodiesterases dipA and rdbA, the dispersal of biofilm was impeded. In the case of the rbdA mutant there was only a 50% reduction
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compared to 99% with wild type, while the dipA mutant was completely unaffected. These
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results indicated that the released NO was likely involved in stimulating phosphodiesterase activity, thereby maintaining low intracellular concentrations of cyclic di-GMP and
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maintaining the P. aeruginosa cells in planktonic form. Schoenfisch and coworkers have examined the impact of nanoparticle size and shape on
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biofilm eradication.55 In this case spherical nanoparticles were prepared at three different sizes: 14, 50 and 150 nm; and silica nanorods were prepared with three different aspect ratios:1, 4 and 8. Although slight synthetic variations were included in each case, the resulting particles were all silica materials carrying N-diazeniumdiolate groups as NO donors. The authors examined the bactericidal efficacy against both planktonic and biofilm variants of P. aeruginosa and S. aureus. In the case of P. aeruginosa, both the 14 nm and 50 nm particles were equally effective, and were close to double the efficacy of the 150 nm particles. In the case of S. aureus the 14 nm particles were generally more effective than the 50 or 150 nm particles. With regard to particle geometry, those particles with higher aspect ratio (4 or 8) were more effective against biofilm, while the highest ratio was comfortably the 33
ACCEPTED MANUSCRIPT best for planktonic S. aureus. Examination of the toxicity against L929 mouse fibroblasts at the concentration required for biofilm killing showed some toxicity, particularly at the
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concentration required for biofilm killing of S. aureus. The most promising results were
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obtained against P. aeruginosa biofilms with high aspect ratio silica nanorods. At
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concentrations required for biofilm killing these systems showed minimal killing of L929 fibroblasts.
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The application of NO releasing dendrimers in biofilm dispersion has also been investigated. PAMAM dendrimers with varying peripheral hydrophobicity were synthesized by reacting
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the terminal amino groups with (i) propylene oxide; (ii) 1,-2-epoxy-9-decene; or (iii) varying composition mixtures of propylene oxide and 1,-2-epoxy-9-decene.56 The reaction also yields
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a secondary amine moiety which can be subsequently reacted with NO gas to form N-
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diazeniumdiolate groups. The dendrimers formed had a similar NO loading capacity (ca. 1 μmol / mg) and comparable half-life (ca. 1 hr). Minimum bactericidal concentration was
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measured against both planktonic and biofilm-forming P. aeruginosa. The more hydrophilic dendrimer (with only propylene oxide derived groups at the dendrimer periphery) were
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generally less effective at killing the planktonic P. aeruginosa than were the hydrophobic or amphiphilic dendrimers. Moreover, the incorporation of NO releasing N-diazeniumdiolate moieties improved the bactericidal efficacy in all cases. When the materials were applied to P. aeruginosa biofilms the authors again observed the most effective eradication with hydrophobic or amphiphilic dendrimers, albeit with slightly higher concentrations required. Inclusion of NO releasing moieties again improved the potency. Toxicity studies on mammalian cells revealed that the optimum formulation was a ratio of 7:3 or 5:5 of propylene to decene moieties at the dendrimer periphery. With this ratio the dendrimers were effective at eradicating P. aeruginosa biofilm but, at the concentration required for biofilm dispersal, had minimal toxicity toward L929 mouse fibroblasts. 34
ACCEPTED MANUSCRIPT Thermally hydrocarbonized porous silica nanoparticles (THCPSi NPs) with NO release capability have also been investigated for their bactericidal efficacy against both planktonic
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and biofilm forming bacteria.32 These materials were able to inhibit planktonic growth of S.
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aureus, P. aeruginosa, and E. coli, and cause some reduction in biofilm mass of
little toxicity toward NIH 3T3 fibroblast cells.
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Staphylococcus epidermis. Moreover, at the concentrations studied the authors observed
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The ability of NO to inhibit biofilm formation is suggestive of its potential application in dental materials, where plaque formation is a significant issue. However, significant
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challenges exist in this area, as N-diazeniumdiolate NO donors have half-lives which are typically on the scale of minutes to hours, and therefore are unlikely to confer any protective
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effect over the time scales required for useful dental materials. Schoenfisch and co-workers
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have recently addressed this issue by preparing NO releasing mesoporous silica nanoparticles which incorporate O2 substituted N-diazeniumdiolates to increase the half-life of the NO
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donor substantially.57 By preparing an O2 substituted N-diazeniumdiolate with a condensable silane group, the authors were able to prepare mesoporous silica nanoparticles which possess
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the protected N-diazeniumdiolate functionality. The resulting nanoparticles had a Ndiazeniumdiolate half-life of 23 days, which is substantially longer than that typically observed for nanomaterials functionalised with this particular class of NO donor. When incorporated into composites the particles provide a low NO flux (0.1 – 0.4 pmol cm-2 s-1) which the authors demonstrate is sufficient to inhibit adhesion of Streptococcus mutans. Interestingly, the use of a conventional NO releasing particle proves ineffective for inhibiting S. mutans, even over the relatively short time scale of the experiment (24 h). These results are significant not only in that they provide evidence for the applicability of NO releasing materials in dental composites, but also because they provide a platform (protected Ndiazeniumdiolates) for sustained picomolar delivery of NO over the time scale of weeks. 35
ACCEPTED MANUSCRIPT In addition to in vitro studies of antibacterial activity, there has also been substantial investigation of the in vivo effect of NO releasing nanoparticles on dermal infections.
These authors codelivered the sol-gel particles synthesised by
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aeruginosa infection.58
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Chouake et al explored the use of NO generating nanoparticles in the treatment of P.
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Friedman et al.23 with glutathione, leading to the in situ generation of S-nitrosoglutathione. By appropriate selection of the nanoparticle and glutathione concentration, the authors were able to deliver a constant concentration of GSNO of ca. 8.7 nM over a 24 hr period.
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Application of the NO nanoparticles and glutathione completely inhibited P.aeruginosa for
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24 hr, which was longer than application of the nanoparticles alone (complete inhibition for 8 hours). Moreover, cell viability at 24 hours was also lower for cells treated with both
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nanoparticles and glutathione (8.3%, c.f. 36.1% for nanoparticles alone, 96.4% with
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glutathione alone and 84.3% for nanoparticles without NO delivery capability. Interestingly, these authors also demonstrated that application of NO releasing nanoparticles and
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glutathione was effective in accelerating wound closure at 3, 5 and 7 days post excision. At day 7 nanoparticle / glutathione treated wounds were at 25% ± 15% of the initial wound
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width, compared with 67% ±10% for wounds treated with nanoparticles only or 83% ± 9% for wounds without any treatment. Importantly, the use of NO nanoparticles and glutathione was shown to accelerate wound healing in lymphocyte deficient non-obese diabetic / severe combined immunodeficient mice. These promising results suggest that nitric oxide delivering nanoparticles may find application in treating patients with topical infections, or in situations where wound healing would be otherwise somewhat retarded. Martinez et al. have examined the application of sol gel based NO releasing nanoparticles for the treatment of wounds infected with MRSA.59 The authors demonstrated that, in addition to effectively killing both methicillin resistant and methicillin sensitive S. aureus, application of the nanoparticles also increased the rate of wound healing in Balb/c mice, with the size of 36
ACCEPTED MANUSCRIPT eschar being substantially reduced after 3 days. The microbial burden of the infected wounds was substantially decreased, as demonstrated by swabbing the wounds and culturing on
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tryptic soy agar. Additionally, it was shown that the use of NO releasing nanoparticles also
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reduced tissue damage, preserving the dermal architecture and maintaining high collagen
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content. Given that MRSA is becoming an increasing issue in clinical settings, these results demonstrate the potential utility of NO releasing nanoparticles in dealing with resistant
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bacteria.
Topical and intradermal application of sol-gel based NO releasing nanoparticles for the
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treatment of MRSA infection in Balb/c mice has been explored by Martinez, Nosanchuk and co-workers.60 The authors found that topical administration of the NO releasing nanoparticles
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led to less inflammation, increased fibrin deposition and reduced bacterial number compared
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to application of control nanoparticles or unchecked MRSA infection. Moreover, injection of the NO releasing nanoparticles directly into MRSA infected abscesses led to a reduction in
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abscess area of about 25% over four days, and complete abscess resolution in 7±2 days (c.f. 14-21 days in untreated or control nanoparticle groups). Topical application of NO releasing
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nanoparticles also led to a reduction in abscess area of about 15%. Interestingly, the application of NO releasing nanoparticles appeared to reduce collagen degradation by MRSA, leading to a commensurate improvement in overall wound healing. Cytokine expression was also enhanced by the NO releasing particles, leading to elevated levels of TNF-α, IFN-γ, IL-12, IL-1β, MCP-1 and TGF-β. The release of nitric oxide also reduced angiogenesis and generally preserved the histological architecture of the infected area. These results provide compelling evidence for the usefulness of NO releasing particle therapies in dealing with antibiotic resistant strains of bacteria, and may be useful either on their own or in tandem with surgical drainage.
37
ACCEPTED MANUSCRIPT One of the clinical areas in which effective treatment of MRSA is important is in the management of pyomyositis.
Characterised by the formation of abscesses in the large
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skeletal muscles, pyomyositis is often associated with S. aureus, and with the increasing
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prevalence of MRSA is therefore becoming a more challenging clinical problem. To this end,
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Schairer et al. examined both the topical and intralesional application of sol gel based NO releasing nanoparticles to intramuscular abscesses infected with MRSA in Balb/c mice.61 The authors observed that application of NO releasing nanoparticles led to significant
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changes in the tissue morphology. At day 4, those animals which were either untreated or
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treated with control (non-NO releasing) nanoparticles had significant inflammation, significant amounts of neutrophil rich infiltrate and a commensurate loss of normal tissue
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architecture. While treatment with vancomycin led to a lessening of inflammation and
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neutrophil infiltration, the most pronounced improvement was observed with either topical or intralesional treatment with NO releasing nanoparticles. Intralesional treatment in particular
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was very effective, resulting in no obvious abscess after 4 days. Tissue cultures taken at day 4 and day 7 demonstrated that there was a reduction in the number of colony forming units
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when NO releasing nanoparticles were applied, this reduction being more significant than could be achieved with application of vancomycin. The authors attributed the rapid resolution of the intramuscular abscesses to the numerous functions of NO. In particular, the authors proposed that NO is able to block microbial physiologic processes and damage microbial DNA, while also down regulating ICAM-1 to reduce neutrophil infiltration. Clearly there is some potential for NO releasing nanoparticles in the treatment of intramuscular abscesses resulting from S. aureus infection. Clearly, one of the areas where delivery of exogenous NO is thought to be of possible utility is in the treatment of resistant bacteria.62 Nevertheless, the possibility exists that bacteria may develop resistance to exogenous NO delivery. To this end, Schoenfisch and co-workers have 38
ACCEPTED MANUSCRIPT conducted a series of spontaneous and serial passage mutagenesis assays with a range of different bacteria (S. aureus, MRSA, S.epidermis, E. coli and P. aeruginosa. The particles
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applied in the study were nitrosated sol-gel particles formed from the reaction of
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mercaptopropyltrimethoxysilane and TEOS. Both the spontaneous mutagenesis and serial
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passage assays did not provide any evidence for the development of resistance. The authors attributed this result to the multiple mechanisms by which NO exerts its antimicrobial action, thereby rendering the development of resistance too complex to be evident in a relatively
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short experiment. Nevertheless, the authors do caution that the development of resistance to
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exogenous NO could occur given the ability of bacteria to adapt, and that this should be taken into account if exogenous NO delivery is ever applied clinically in antimicrobial applications.
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Nitric oxide releasing nanoparticles have also been demonstrated to have some efficacy in the
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treatment of fungal infections. Specifically, Macherla et al. have examined the use of sol-gel based particles in the treatment of Candida albicans infection in mice having a burn injury.63
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C. albicans has been implicated in burn wound infections, and may contribute to the development of sepsis. Invasive candidiasis has been shown to contribute to ca. 23% of
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severe infections, and mortality rates are in the order of 14-70%. As such, novel treatments are of considerable interest. Macherla et al. have demonstrated the effectiveness of NO releasing nanoparticles in inhibiting the proliferation of C. albicans when compared to untreated fungi, or fungi treated with nanoparticles lacking NO release capability. Interestingly, while treatment with NO releasing nanoparticles had the most profound effect, the application of nanoparticles without NO capability still inhibited the proliferation to some extent. NO releasing nanoparticles were also demonstrated to accelerate the burn healing process (similar to the wound healing results discussed elsewhere in this review), and also to prevent spreading of the fungal infection to the paws of the test mice. In order to examine the effect of NO nanoparticles on fungal proliferation in the wound, samples were excised on day 39
ACCEPTED MANUSCRIPT 3 and day 7 and were cultured on YPD agar plates. Samples from the wounds treated with NO releasing nanoparticles showed lower fungal burden than those from control or non-
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functional nanoparticle samples. Moreover histological examination demonstrated an absence
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of hyphal or pseudohyphal structures in the wounds treated with NO releasing nanoparticles,
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as compared with the control nanoparticle and untreated experiments. Time lapse microscopy was used to demonstrate that C. albicans cell division was substantially inhibited by NO releasing nanoparticles over the time course studied (60 min). Control nanoparticles also led
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to some inhibition (48 min) while untreated samples were shown to undergo budding and
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morphogenesis on a relatively short time scale (12 min). As observed in the wound healing studies, the addition of NO nanoparticles to the wound was shown to increase collagen
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deposition during burn healing, and to increase the migration of macrophage-like cells into
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the wound tissue. The NO releasing nanoparticles were also shown to increase the infiltration of neutrophils into the wound tissue, which is in contrast to findings on uninfected wounds
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where neutrophil migration was impaired. Altogether these results indicate that NO releasing nanoparticles have considerable potential for reducing and reversing C. albicans infection in
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burn victims.
3c. Cardiovascular Applications One of the most studied aspects of nitric oxide function is its role in vasodilation, and this gives rise to substantial therapeutic potential in the treatment of acute pulmonary hypertension and other disease of the vasculature. At present, therapeutic delivery of nitric oxide must be done by employing either gaseous NO or an NO precursor such as a Ndiazeniumdiolate, S-nitrosothiol, organic nitrate or sodium nitroprusside (see Scheme 1). Gaseous NO has substantial limitations in that it requires the use of pressurized gas tanks, which is costly and inconvenient. Precursor molecules have some benefit, and typically have half-lives in the order of minutes - hours and that are dependent on pH, temperature and 40
ACCEPTED MANUSCRIPT molecular structure. The ability to give sustained delivery of NO from a long-circulating particle may be particularly advantageous in the treatment of cardiovascular disease, and this
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has motivated a number of investigations into the approach.
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In an early study on the potential use of NO releasing nanoparticles in the cardiovascular
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system, Cabrales et al. studied the effect of infusing NO releasing nanoparticles formed by the sol gel approach into 50-65 g Golden Syrian Hamsters.64 The infusion solution was given
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via the jugular vein at a rate of 100 µL/min (total volume 50 µL, less than 2% of the total blood volume). The NO releasing nanoparticles and control nanoparticles lacking NO release
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capability were infused at two different concentrations: 10 mg / kg and 20 mg / kg. For further comparison, experiments were also conducted using infusions of diethylenetriamine
The authors found that for an equivalent mass, NONOates delivered a
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NONOate).
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N-diazeniumdiolate (DETA-NONOate) or dipropylenetriamine N-diazeniumdiolate (DPTA-
substantially higher payload of NO, exceeding the NO releasing nanoparticles by a factor of
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thirty. The authors observed some significant changes in the blood gas parameters in the hamsters treated with NO releasing nanoparticles. Specifically, Methaemoglobin (MetHb)
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levels increased from the baseline reading after 2 h, reaching 9±2% for a 10 mg/kg infusion and 14±3% for a 20 mg/kg infusion after 4 h. As might be expected, there was also an increase in plasma nitrate and nitrite, with the measured values being proportional to the infused dose. An increase in arterial pO2 was also observed, along with a decrease in arterial pH.
Importantly, no significant changes were observed with infusion of the non-NO
releasing nanoparticles. As might be expected, the authors observed that the infusion of NO releasing nanoparticles resulted in a decrease in the mean arterial blood pressure which reached a minimum value at 90 min and then returned to the initial level at 3 h, thereafter increasing slightly at 3.5 h. Infusions of DETA-NONOate and DPTA-NONOate at the same concentrations produced similar effects on the blood pressure, but led to increased MetHb 41
ACCEPTED MANUSCRIPT levels (19-26%) and thus a decrease in the oxygen carrying capability of the haemoglobin. The authors also measured the exhaled NO, and observed a maximum 1 h after treatment
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with the NO releasing nanoparticles. Fluorescent studies were undertaken to follow the
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circulation time of the NO releasing nanoparticles, and it was observed that the particles were
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still circulating 6 h after the infusion but had been completely cleared from the system by 24 h. Interestingly, nanoparticles without NO release capability were not observed in the circulation after 4 h which is suggestive of NO release somehow increasing the
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biopersistence of the particles. Perhaps unsurprisingly, the particles were shown to cause
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microvascular vasodilation and increased blood flow, while the control particles had no effect on arteriolar diameter or blood flow. On the venular side neither the NO releasing or control
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particles had any effect on the vessel diameter, with a slight reduction of blood flow observed
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at 20 mg/kg infusion. As for a direct effect on the blood cells, NO releasing nanoparticles did not increase the proportion of immobilised leukocytes, in contrast to the non-functional
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nanoparticles where a slight increase was observed. Further, NO releasing nanoparticles did not lead to an increase in rolling leukocytes above the baseline reading, whereas an increase
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was observed with the control nanoparticles. Studies were also undertaken to probe the effect of the nanoparticles on subjects with reduced endogenous NO. When endogenous production of NO was inhibited using N-nitro-L-arginine methyl ester (an NO synthase inhibitor), infusion of the NO releasing nanoparticles partially reversed the observed vasoconstriction and increase in mean arterial blood pressure. When heme site soluble guanylyl cyclase was inhibited with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, infusion of NO releasing nanoparticles led to a modest but statistically significant reduction in vasoconstriction and hypertension. Moreover, co-administration of NO releasing nanoparticles with a phosphodiesterase inhibitor (Zaprinast) led to a reduction in mean arterial pressure and a corresponding increase in arteriolar diameter. Altogether these results suggest that NO
42
ACCEPTED MANUSCRIPT releasing nanoparticles behave in the blood stream as one might expect, and may have some utility in the treatment of hypertensive disorders.
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In an extension of this work, Cabrales and coworkers examined the application of NO
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releasing nanoparticles for the reversal of induced vasoconstriction.65 The study utilised 50-
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65 g male Golden Syrian hamsters in which vasoconstriction was induced by infusing polymerised bovine haemoglobin (PBH). PBH causes vasoconstriction by scavenging NO
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through two reactions: an oxidative reaction of NO with NO dioxygenase and NO binding to deoxyHb. There may also be some reaction of NO with the cysteine moiety at position 93 in
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the Hb. These side reactions consume sufficient NO to result in observable vasoconstriction. As in the initial study, NO releasing nanoparticles were infused at 10 mg / kg and 20 mg / kg
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although this time the control nanoparticles were only infused at one concentration (20 mg /
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kg). Infusion of PBH led to increased total and acellular haemoglobin in all groups, with the addition of NO releasing nanoparticles also leading to increased total and acellular
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methemoglobin (metHb). Elevated metHb was measured up to 4 h post influsion, with a steady increase between time of infusion and 3 h. Blood nitrate and nitrite were also elevated
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by the infusion of NO releasing nanoparticles. The primary finding of the study was that infusion of NO releasing nanoparticles was effective in reducing the mean arterial blood pressure of hamsters with induced vasoconstriction, relative to infusion of non-NO releasing nanoparticles and infusion vehicle (see Fig 4). While the reduction was more pronounced with a higher infusion concentration, the higher dose of NO releasing nanoparticles (20 mg/kg) also resulted in an increase in heart rate. Infusion of 10 mg/kg of NO releasing nanoparticles led to a reduction in mean arterial blood pressure and no measureable impact on heart rate. The effect of nitric oxide releasing nanoparticles was also observed on arteriolar vessel diameter and blood flow. The infusion of PBH led to immediate vasoconstriction and a commensurate decrease in flow, which was reversed over time by the infusion of NO 43
ACCEPTED MANUSCRIPT releasing nanoparticles. Infusion of control nanoparticles or infusion vehicle alone had no impact on the arteriolar diameter and blood flow. Functional capillary density was also
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improved by the infusion of NO releasing nanoparticles. Taken altogether these results
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indicate that NO releasing nanoparticles may be useful for inducing vasodilation in a subject
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in need thereof, and may thus be useful in treating hypertensive disorders.
Figure 4. Change in mean arterial pressure (MAP) and heart rate (HR) relative to baseline after (i) inducing vasoconstriction with polymerised bovine haemoglobin and (ii) treatment with NO releasing nanoparticles. †P < 0.05 compared with baseline; ‡P < 0.05 compared with control nanoparticles at the same time point; ¶P < 0.05 compared with 10 mg/kg NO 44
ACCEPTED MANUSCRIPT releasing nanoparticles; §P < 0.05 compared with vehicle. Note that symbols for each experimental group have been artificially displaced at each time point for clarity. Control
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nanoparticles -4 min; NO releasing nanoparticles at 20 mg/kg - 1 min; NO releasing
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nanoparticles at 10 mg/kg +1 min; and vehicle + 4 min. Reproduced from Am. J. Physiol.
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Heart Circ. Physiol. 300, P. Cabrales, G. Han, P. Nacharaju, A. J. Friedman, J. M. Friedman, Reversal of hemoglobin-induced vasoconstriction with sustained release of nitric oxide, H49-
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H56 Copyright (2011), with permission from The American Physiological Society. Cabrales and co-workers have also investigated the potential application of nitric oxide
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releasing nanoparticles prepared via the sol-gel approach for maintaining systemic and microvascular function during haemorrhagic shock.66 The authors postulated that during
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haemorrhagic shock NO production is reduced via three main routes: (i) reduction of
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endothelial shear stress leading to reduced endothelial nitric oxide synthase (eNOS) activity; (ii) free radical formation and accumulation interfering with eNOS function; and (iii) reduced
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O2 concentration (hypoxia), removing the necessary substrate for NO production. To explore the effect of replenishing NO using NO releasing nanoparticles, 50-65 g male Golden Syrian
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hamsters were subjected to haemorrhagic shock by the removal of 50% of blood volume (estimated assuming blood mass is 7% of body mass) over a five minute period. The hamsters were then treated with NO releasing nanoparticles, control nanoparticles lacking NO release capability and infusion vehicle. In all cases haemorrhage led to a substantial and expected decrease in mean arterial blood pressure (MAP). Treatment with nitric oxide releasing nanoparticles led to some improvement in MAP over the study duration (90 min), with a more noticeable improvement than that observed with infusion of control nanoparticles or vehicle alone. That said, at 90 min blood pressure only reached approx. 50% of the baseline pressure. In all cases heart rate increased slightly after haemorrhage, thereafter falling away substantially when control nanoparticles or vehicle were infused. However, infusion of NO 45
ACCEPTED MANUSCRIPT releasing nanoparticles proved effective in supporting heart rate close to the baseline level. Infusion of NO releasing nanoparticles also led to some changes in the blood gas chemistry.
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Nitrate and nitrite levels were elevated wrt baseline and control or vehicle infusion, and pO 2
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levels were also elevated, though to a lesser extent than was observed with the control
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nanoparticle and vehicle infusions. Methaemoglobin increased in the animals treated with NO releasing nanoparticles (as was observed in Cabrales’ earlier study), and this is indicative of slightly reduced O2 carrying capacity. The authors also examined arteriolar and venular
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diameters and corresponding blood flow, and observed that after haemorrhage all diameters
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were significantly constricted, with a commensurate decrease in microvascular flow. Application of NO releasing nanoparticles led to a recovery in both arteriolar and venular
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diameter, as well as some improvement in the microvascular flow (although not returning to
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baseline levels). Functional capillary density was also decreased substantially by haemorrhage, with NO releasing nanoparticles giving a modest improvement over the study
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duration and significantly more improvement than was obtained with control nanoparticles or vehicle alone. Peripheral vascular resistance was observed to increase considerably after
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haemorrhage. Nitric oxide releasing nanoparticles were able to give some reduction in peripheral vascular resistance, though the level did not completely return to baseline in the study duration. These results indicate that NO releasing nanoparticles were effective in mitigating the effects of haemorrhagic shock and as such may have some utility in supporting an individual until appropriate volume can be returned to the cardiovascular system. Moreover, the results underscore the profound role played by nitric oxide in maintaining function of the cardiovascular system. Nacharaju et al. have recently investigated the effect of three different nanoparticulate NO delivery systems on the vascular system.67 Specifically, the authors investigated the standard NO releasing nanoparticles as initially synthesised by Friedman and co-workers,23 along with 46
ACCEPTED MANUSCRIPT sol-gel particles with physically entrapped S-nitrosoacetylcysteine and sol-gel particles having chemically linked S-nitrosothiol groups (formed by the co-condensation of TMOS
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with nitrosated 3-mercaptopropyltrimethoxysilane). In order to examine the efficiency with
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which the particle could nitrosate thiol groups, the particles were mixed with solutions of
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glutathione and the formation of S-nitrosoglutathione was followed with HPLC. The particles which were most effective at forming S-nitrosoglutathione were those with encapsulated Snitroso-N-acetylcysteine. The effect of particle infusion on mean arterial pressure, heart rate
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and vessel diameter in Golden Syrian hamsters was also examined.
Although all
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formulations gave the anticipated results (decrease in mean arterial pressure, increase in heart rate and increase in vessel diameter), particles incorporating the S-nitroso-N-acetylcysteine
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had a longer duration of action than did the particles incorporating reduced NO in a sol-gel
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matrix. A dose dependent relationship was also observed, with particles infused at 20 mg/kg giving greater reduction in mean arterial pressure, increase in heart rate and increase in
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microvessel diameter. These results shows that the chemistry of NO release can have a significant impact on the duration of the therapeutic effect, and provide evidence for the
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ability to tune NO release to specific applications. There is considerable evidence to suggest that following myocardial infarction immediate reperfusion of the ischemic myocardium reduces the extent of infarction, and preserves the mechanical functioning of the heart. Maintaining appropriate, low NO concentrations during reperfusion may be critical in reducing the likelihood of ischemia/reperfusion injury. With this in mind, Schoenfisch and co-workers have examined application of S-nitroso-N-acetylD,L-penicillamine (SNAP) functional dendrimers (discussed above) to prevent ischemic damage in an isolated perfused rat heart.68 The hearts examined were excised from Sprague Dawley rats and retrograde-perfused using the Langendorff technique. Perfusion was suspended for 20 min in order to cause global ischemia, after which reperfusion was 47
ACCEPTED MANUSCRIPT performed for 20 min. The authors observed that reperfusion with the addition of SNAP either as a free molecule or conjugated to a dendrimer resulted in a reduction of infarcted
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tissue, compared to reperfusion with Krebs-Heinseleit solution alone or control solutions
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incorporating N-acetyl-D,L-penicillamine either free or conjugated to the dendrimer.
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Moreover, infusion of SNAP functional dendrimer with glutathione led to a further reduction in the %infarct, with an optimum concentration of 230 pM of dendrimer determined (corresponding to a SNAP concentration of 15 nM).
These results demonstrated that
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conjugation of small molecule NO donors to nanoparticulate (dendrimer) scaffolds led to no
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reduction in the efficacy of the drug, and may have some utility in reducing ischemia reperfusion injury following myocardial infarction.
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3d. Erectile Dysfunction
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It is well known that nitric oxide is involved in the onset of erection. As such Han et al.
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hypothesised that the transdermal application of NO releasing nanoparticles may be effective in the treatment of erectile dysfunction.69 To this end, studies were undertaken using retired breeder male Sprague-Dawley rats weighing >650 g. A suspension of nanoparticles capable
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of sustained NO release was applied to the glans and shaft of the penis of the rats, after which systemic blood pressure and intracorporal pressure were followed. Control rats were treated with a similar suspension wherein the nanoparticles were not capable of NO release. The authors observed that for the control rats the ICP/BP remained less than 0.1 for the entire duration of the experiment. However, where NO releasing nanoparticles were applied five out of seven rats showed an erectile response in the ICP/BP ratio, with visile erections occurring approx. 4.5 minutes after application of the particles. A typical erection induced by the NO releasing nanoparticles lasted less than two minutes, with an average duration of 1.42 minutes. In the same study, the authors applied nanoparticles loaded with sialorphin (a neutral endopeptidase inhibitor) which led to an unstimulated erection of longer duration than 48
ACCEPTED MANUSCRIPT that obtained using the NO releasing nanoparticles. A further element of the study was to incorporate tadalafil (a phosphodiesterase type 5 inhibitor) into nanoparticles.
This
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compound was effective in inducing erection when combined with cavernous nerve
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stimulation using a stainless steel bipolar hook electrode, although only when stimulation
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occurred 60 min after nanoparticle application. These results demonstrated that NO releasing nanoparticles have some promise as a topical treatment for erectile dysfunction, although the precise system studied did have some limitations when compared to nanoparticles loaded
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with sialorphin of tadalafil. That said, the previously demonstrated ability to control the
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release of NO from sol gel nanoparticles through variation of the matrix composition does provide some further avenues for potential optimisation of the treatment methodology.
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3e. Anticancer Activity
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The first investigation of NO releasing nanoparticles for potential use in cancer therapy was
diazeniumdiolate
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reported by Stevens et al.70 In this case the particles were prepared by condensing either Nmodified
N-(6-aminohexyl)aminopropyltrimethoxysilane
methylaminopropyltrimethoxysilane
with
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By
varying
the
or
amount
3of
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aminoalkoxysilane the authors were able to vary the particle size between 90 and 160 nm. Variation of solvent enabled formation of even larger particles (350 nm). The authors examined the toxicity of the particles to ovarian carcinoma cells using an MTT assay, and demonstrated that the particles were more effective against the carcinoma cells than a small molecule NO donor (PYRRO/NO). An interesting relationship to particle size was observed, with the largest NO releasing particles being effective against carcinoma cells while having much lower toxicity to normal ovarian cells. The particles were shown to cause inhibition through apoptosis, and also to reduce anchorage independent growth. Subcellular localisation of the NO releasing particles was investigated by using confocal microscopy, and it was shown that the particles were co-localised with the late endosomes and lysosomes. Although 49
ACCEPTED MANUSCRIPT potential cancer applications of NO releasing nanoparticles have not received anywhere near the volume of attention that antimicrobial applications have, there is clearly considerable
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scope for further work in the area.
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The glycopolymer based polymer nanocarriers prepared by Forrest and co-workers were
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examined for their activity against human head and neck squamous cell carcinomas.39 A xenograft model was used, with subcutaneous injection of the NO releasing nanocarrier local
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to the tumour being shown to significantly reduce tumour volume. Tumour survival was also improved, with two animals having a complete response. These results were extremely
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favourable, as the outcomes in the control groups (either untreated or treated with a small molecule NO donor) were poor, with 100% of animals being euthanized within seven weeks
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due to proliferation of the cancer. In contrast to the localised subcutaneous delivery of the
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polymeric nanocarrier, intravenous infusion of the small molecule donor was not effective for
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reducing tumour volume or extending the lifetime of the treated mice. 3f. Treatment of liver fibrosis
Previous studies of liver fibrosis have demonstrated that treatment with gaseous nitric oxide
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can result in the down regulation of certain pro-fibrogenic mediators such as collagen I and transforming growth factor β-1 (TGF-β1), and thereby reduce production of fibrotic tissue. As such, Davis and co-workers hypothesized that nitric oxide releasing polymer nanoparticles may have some utility in reducing and/or reversing liver fibrosis. 71 To this end studies were conducted to examine the impact of NO releasing nanoparticles on hepatic stellate cells (HSCs). HSCs have been shown to have a significant role in liver fibrosis, as following hepatic injury or damage HSCs secrete and accumulate extracellular matrix in the liver. Since part of the normal function of HSCs is to store vitamin A, the polymer nanoparticles prepared were decorated with vitamin A moieties so as to facilitate specific cell uptake of the NO releasing nanoparticles into the HSCs. The particular particles applied in this study were 50
ACCEPTED MANUSCRIPT similar to those reported previously by Duong et al.36, and were prepared though selfassembly of block copolymers incorporating hydrophilic and hydrophobic domains. The
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block copolymers were synthesized by polymerizing oligoethylene glycol methyl ether
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methacrylate in the presence of the chain transfer agent 4-cyanopentanoic acid-4-
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dithiobenzoate and initiator azobisisbutyronitrile (AIBN), followed by chain extension with 2-vinyl-4,4,-dimethyl-5-oxazolone. The resulting polymer possessed a carboxylic acid endgroup which was exploited for subsequent attachment of vitamin A through formation of
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an ester linkage with the terminal hydroxyl of the vitamin A. Moreover, the oxazolone
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moieties were used for subsequent attachment of S-nitrosoglutathione via formation of amide linkages. The resulting micelles possessed a hydrophilic PEG corona around a hydrophobic
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core wherein the NO donor moieties were housed. The authors demonstrated that the particles
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were taken up by the HSCs and human hepatic cells, while control particles without the vitamin A functionality were not taken up to any great extent. Biodistribution studies were
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also conducted, and it was clearly demonstrated that the particles accumulated in the liver (as expected) as well as in the kidneys. The vitamin A decorated particles were shown to
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accumulate with substantial specificity to the HSCs, with significantly lower uptake into Kuppfer cells, hepatocytes and sinusoidal liver endothelial cells. Along with the specific targeting of the liver and selective uptake into HSCs, the authors also demonstrated that the particles were effective for reducing the expression of mRNA for collagen type I, α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF), and therefore might be expected to reduce the extent of fibrosis. Importantly, not all profibrogenic species were inhibited, with the levels of mRNA for TIMP-1 metallopeptidase inhibitor and transforming growth factor β (TGF-β) remaining relative unchanged. Similar results were obtained using unconjugated S-nitrosoglutathione. In vitro experiments demonstrated that some toxicity was observed with particle doses equivalent to 1000 µM, and so 500 µM was chosen as the ideal
51
ACCEPTED MANUSCRIPT concentration. In addition to the potential use of the particles in reducing liver fibrosis, the authors also observed a reduction in portal hypertension (which often accompanies extensive
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fibrosis) when NO releasing nanoparticles were administered to BDL rats. Importantly, the
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targeted nature of the nanoparticles seemed to minimise the effect on mean arterial pressure,
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which remained largely unperturbed. These results were supported by in vitro studies which showed that NO releasing nanoparticles were effective in countering the contractile effect of endothelin-1 (ET-1) in a collagen gel assay. Taken altogether, these results demonstrate that
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vitamin A decorated nanoparticles were effective for reducing portal hypertension by Importantly, the targeted nature of the
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reducing the contractile response to ET-1.
nanoparticle therapy reduced the systemic effects of NO (i.e., the effect on mean arterial
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pressure) which is a significant limitation of non-specific agents such as sodium
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nitroprusside.
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5. Conclusions and Further Outlook The level of interest in nitric oxide releasing nanoparticles has exploded in the last decade. From the initial investigations of NO releasing silica nanoparticles, further application of the
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technology to gold nanoparticles, dendrimers, polymeric micelles, star polymers and polymer coated nanoparticles have demonstrated the multiple platforms that can be employed for exogenous delivery of NO.
Of course, each of these platforms has advantages and
disadvantages. Dendrimers, while highly defined and very monodisperse are costly and timeconsuming to produce. Particles which incorporate metal or inorganic nanoparticles are highly stable, but may exhibit significant toxicity when administered through certain routes (e.g., via the lung). As such, the use of polymeric platforms is particularly attractive due to the low cost of production, the relative ease with which different synthetic handles can be incorporated and the potential for preparing truly biocompatible systems. For instance, polymeric particles can be readily functionalised with a poly(ethylene glycol) corona, thereby 52
ACCEPTED MANUSCRIPT imparting so-called “stealth” properties which significantly increase circulation time and improve accumulation in tumours by the enhanced permeability and retention (EPR) effect.
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Given the wide array of synthetic approaches that can be applied to the production of
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polymer nanoparticles, the preparation of truly biocompatible and biodegradable
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nanoparticles capable of NO delivery is within reach. Indeed, for the technology to find ultimate clinical application such biocompatible systems will be required.
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As noted above, an increasing number of studies have demonstrated potential clinical applications for NO-releasing nanoparticles, in areas as diverse as dental materials and sexual
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health. Without doubt the most promising area for application of NO releasing nanoparticles is in the area of antimicrobial agents. The increasing problem of antibiotic resistance requires
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considerable research activity toward the development of new agents capable of combatting
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so-called “superbugs”. Nanoparticles designed for exogenous delivery of NO may well be part of the armoury that mankind applies in addressing this pressing clinical problem.
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Moreover, there is already compelling evidence for the utility of NO-releasing nanoparticles in dispersing bacterial biofilms. Given the clinical implications of biofilm formation and the
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difficulties which arise in managing such films, NO-releasing nanoparticles may well have a readymade niche within the broader sweep of antimicrobial research. While there are a considerable number of nanoparticle systems available for the delivery of exogenous NO, there has been comparatively little investigation of materials which respond to endogenous NO. This relatively new field of NO capturing materials may also provide hitherto unexplored areas for clinical investigation. A recent report from Hu et al. discusses the synthesis and application of polymers which incorporate o-phenylene diamine groups to capture NO through the formation of benzotriazole rings.72 By varying the structure of the monomer to control hydrolytic stability, the authors showed that exposure to NO could lead to charge reversal. Moreover, by incorporating NO and temperature responsive units into a 53
ACCEPTED MANUSCRIPT polymer the formation of micelles could be triggered by exposure to NO (through the formation of hydrophobic benzotriazoles and a commensurate decrease in the lower critical
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solubility temperature). Incorporating a polarity sensitive fluorescent dye into such polymers
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means that the formation of micelles and aggregates could effectively “turn on” fluorescence.
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Importantly, these effects could be observed inside a cell, presumably as NO was captured and micelles / aggregates formed. These results demonstrate the possibility of nanoparticle formation being induced by endogenous NO, opening new possibilities in delivery and / or
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imaging applications. Altogether, there is a strong mandate for continued research into
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materials which not only deliver, but respond to NO. Acknowledgements
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This research was funded by the Australian Research Council Centre of Excellence in
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Convergent Bio-Nano Science and Technology (project number CE140100036). TPD wishes
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to acknowledge the award of an Australian Laureate Fellowship (FL140100052). The authors acknowledge significant financial and infrastructure support from the Monash
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Institute of Pharmaceutical Sciences.
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H. T. T. Duong, Z. M. Kamarudin, R. B. Erlich, Y. Li, M. W. Jones, M. Kavallaris,
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C. K. Sen, G. M. Gordillo, S. Roy, R. Kirsner, L. Lambert, T. K. Hunt, F. Gottrup, G.
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K. Blecher, L. R. Martinez, C. Tuckman-Vernon, P. Nacharaju, D. Schairer, J.
Chouake, J. M Friedman, A. Alfieri, C. Guha, J. D. Nosanchuk, A. J. Friedman, Nitric oxide-
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releasing nanoparticles accelerate wound healing in NOD-SCID mice, Nanomed. Nanotech.
G. Han, L. N. Nguyen, C. Macherla, Y. L. Chi, J. M. Friedman, J. D. Nosanchuk, L.
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Scaffold Size on Bactericidal Activity of Nitric Oxide-Releasing Silica Nanoparticles, ACS Nano 5 (2011) 7235-7244
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Antimicrobials: Nitric Oxide Release from Quaternary Ammonium-Functionalized Silica Nanoparticles, Biomacromolecules 13 (2012) 3334-3342 49.
Y. Lu, D. L. Slomberg, B. Sun, M. H. Schoenfisch, Shape- and Nitric Oxide Flux-
Dependent Bactericidal Activity of Nitric Oxide-Releasing Silica Nanorods, Small 9 (2013) 2189-2198 50.
A. J. Friedman, K. Blecher, D. Schairer, C. Tuckman-Vernon, P. Nacharaju, D.
Sanchez, P. Gialanella, L. R. Martinez, J. M. Friedman, J. D. Nosanchuk, Improved
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B. Sun, D. L. Slomberg, S. L. Chudasama, Y. Lu, M. H. Schoenfisch, Nitric Oxide-
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Releasing Dendrimers as Antibacterial Agents, Biomacromolecules 13 (2012) 3343-3354. B. V. Worley, D. L. Slomberg, M. H. Schoenfisch, Nitric Oxide-Releasing
Quaternary
Ammonium-Modified
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Poly(amidoamine)
(PAMAM)
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Agents,
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A. W. Carpenter, K. P. Reighard, J. E. Saavedra, M. H. Schoenfisch, O-2-Protected
diazeniumdiolate-modified silica nanoparticles for extended nitric oxide release from dental composites, Biomaterials Sci. 1 (2013) 456-459 58.
J. Chouake, D. Schairer, A. Kutner, D. A. Sanchez, J. Makdisi, K. Blecher-Paz, P.
Nacharaju, C. Tuckman-Vernon, P. Gialanella, J. M. Friedman, J. D. Nosanchuk, A. J. Friedman, Nitrosoglutathione Generating Nitric Oxide Nanoparticles as an Improved Strategy 61
ACCEPTED MANUSCRIPT for Combating Pseudomonas aeruginosa-Infected Wounds, J. Drugs. Derm. 11 (2012) 14711477.
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L. R. Martinez, G. Han, M. Chacko, M. R. Mihu, M. Jacobson, P. Gialanella, A. J.
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G. Han, L. R. Martinez, M. R. Mihu, A. J. Friedman, J. M. Friedman, J. D.
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Nosanchuk, Nitric Oxide Releasing Nanoparticles Are Therapeutic for Staphylococcus aureus Abscesses in a Murine Model of Infection, Plos One 4 (2009)Art e7804 D. Schairer, L.R. Martinez, K. Blecher, J. Chouake, P. Nacharaju, P. Gialanella, J. M.
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Friedman, J. D. Nosanchuk, A. J. Friedman, Nitric oxide nanoparticles Pre-clinical utility as a therapeutic for intramuscular abscesses, Virulence 3 (2012) 62-67 B. J. Privett, A. D. Broadnax, S. J. Bauman, D. A. Riccio, M. H. Schoenfisch,
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Nosanchuk, L. R. Martinez, Nitric oxide releasing nanoparticles for treatment of Candida albicans burn infections, Front. Microbiol. 3 (2012) Art. 193 64.
P. Cabrales, G. Han, C. Roche, P. Nacharaju, A. J. Friedman, J. M. Friedman,
Sustained release nitric oxide from long-lived circulating nanoparticles Free Rad. Biol. Med. 49 (2010) 530-538
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P. Cabrales, G. Han, P. Nacharaju, A. J. Friedman, J. M. Friedman, Reversal of
hemoglobin-induced vasoconstriction with sustained release of nitric oxide, Am. J. Physiol.
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P. Nachuraju, A. J. Friedman, J. M. Friedman, P. Cabrales, Exogenous nitric oxide
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nitric oxide-releasing dendrimers, Nitric Oxide Biol. Chem. 22 (2010) 30-36. G. Han, M. Tar, D. S. R. Kuppam, A. Friedman, A. Melman, J. Friedman, K. P.
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Davies, Nanoparticles as a Novel Delivery Vehicle for Therapeutics Targeting Erectile Dysfunction, J. Sexual. Med. 7 (2010) 224-233 E. V. Stevens, A. W. Carpenter, J. H. Shin, J. S. Liu, C. J. Der, M. H. Schoenfisch,
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Graphical abstract
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S0168-3659(15)00097-8 doi: 10.1016/j.jconrel.2015.02.007 COREL 7556
To appear in:
Journal of Controlled Release
Received date: Revised date: Accepted date:
23 October 2014 30 January 2015 4 February 2015
Please cite this article as: John F. Quinn, Michael R. Whittaker, Thomas P. Davis, Delivering Nitric Oxide with Nanoparticles, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.02.007
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Delivering Nitric Oxide with Nanoparticles John F. Quinn 1, Michael R. Whittaker 1 and Thomas P. Davis*1,2
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ARC Centre of Excellence in Convergent Bio-Nano Science and Technology. Monash
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Institute of Pharmaceutical Sciences, Monash University (Parkville Campus), 399 Royal Parade, Parkville, Victoria 3152, Australia.
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK. correspondence author: [email protected]; tel +61 3 9903 9260
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Abstract
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While best known for its important signalling functions in human physiology, nitric oxide is also of considerable therapeutic interest. As such, nanoparticle-based systems which enable
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the sustained exogenous delivery of nitric oxide have been the subject of considerable investigation in recent years. Herein we review the various nanoparticle systems that have been used to date for nitric oxide delivery, and explore the array of potential therapeutic applications that have been reported. Specifically, we discuss the modification of sol-gel based silica particles, functionalised metal / metal oxide nanoparticles, polymer-coated metal nanoparticles, dendrimers, micelles and star polymers to impart nitric oxide release capability. We also consider the various areas in which therapeutic applications are envisaged: wound healing, antimicrobial applications, cardiovascular treatments, sexual medicine and cancer treatment.
Finally, we discuss possible future directions for this
versatile and potentially important technology. .
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ACCEPTED MANUSCRIPT 1. Introduction The gasotransmitters (nitric oxide, carbon monoxide and hydrogen sulphide) are a small
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family of otherwise gaseous molecules with an important role in intra- and extracellular
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signalling.1 The role of gaseous molecules in cell signalling was first identified in the 1980s,2
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though the term “gasotransmitter” only came into common usage in 2001.3 Of the three molecules so far identified, nitric oxide (NO) has been the subject of the most considerable
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investigation, and has been shown to have a role in many different physiological events.4 For instance, nitric oxide signalling is important in pain perception,5 sleep control and
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regulation6, vasodilation7, mucus production8, sphincter contraction and relaxation9 regulation of erection10 and the proper functioning of the immune system11.
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Deficiency or overproduction of NO is associated with a number of pathologies.
For
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instance, reduced NO is associated with hypertension in preeclampsia12 and Prinzmetal’s
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angina.13 Elevated levels of NO have also been associated with autoimmune diseases such as rheumatoid arthritis14, systemic lupus erythematosus15 and multiple sclerosis.16 High levels of NO are also related to the processes in transplant rejection17 and in septic shock.18 Clearly
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appropriate regulation of NO in the body is important in maintaining good overall health. In addition to its role in biological signalling processes and in various pathologies, NO has also demonstrated some potential as a therapeutic agent. In particular, exogenous delivery of NO has been demonstrated to have some activity against tumour cells,19 and to be potentially useful in antimicrobial applications.20 For instance, exogenous NO has been applied successfully in the dissipation of biofilms.21 With increased antibiotic resistance becoming a major clinical problem, new antimicrobials which release NO may be of clinical benefit either alone or when applied alongside traditional antibiotics.
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description. Although there are a number of different chemistries available, organic nitrates,
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S-nitrosothiols, metal complexes and N-diazeniumdiolates have received much attention as
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potential small molecule agents for the delivery of NO in a therapeutic context (see Scheme 1).22 One of the principle ways to alter the biodistribution and pharmacokinetic properties of NO donors is to incorporate them into a nanoparticulate form. Friedman and co-workers have
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previously noted that the most important features of an NO delivery system are that the
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material should be easily applied, and be capable of delivering NO over a therapeutically meaningful time interval.23 Issues of cost, storage stability and transportability also need to be
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considered (it is these issues that render the use of gaseous NO particularly impractical).
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Herein we review the current state of the art for nitric oxide delivery using nanoparticles, looking at both the preparation of the particles, post assembly modification to incorporate the
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NO delivering moiety, and in vitro and in vivo studies of the particles. We also explore possible future strategies for the preparation of NO delivering nanoparticles, and examine
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some potential future directions for this important technology.
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ACCEPTED MANUSCRIPT Scheme 1. Common small molecule nitric oxide donors. 2. Preparation of NO-Releasing Nanoparticles
These may be broadly categorized as (i) sol-gel derived silica
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delivery of NO.24
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To date, studies have been conducted on a number of different vehicles for the exogenous
nanoparticles; (ii) surface functionalised metal / metal oxide nanoparticles; (iii) polymer coated metal nanoparticles; (iv) dendrimers; (v) micelles; and (vi) core cross-linked star
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polymers (see Scheme 2).
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Scheme 2. Main classes of nanomaterials applied in the exogenous delivery of nitric oxide. 2a. Nitric Oxide Delivery from Sol-gel Derived Silica Nanoparticles In important preliminary work Meyerhoff and coworkers investigated the functionalization of fumed slilica particles with N-diazeniumdiolate groups as a method for preparing NO releasing particles.25 Although with a size range of 0.2-0.3 µm the particles prepared were
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ACCEPTED MANUSCRIPT not strictly nanoparticles, the study provides an important proof of principle and serves as a starting point for the numerous nanoparticle investigations that have followed. The silica
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particles in question were first surface modified with amine containing silylation reagents to
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provide particles having either primary or secondary amine functionality (or both) on the
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surface. The particles were then exposed to nitric oxide gas to form N-diazeniumdiolates, with secondary amines proving to be the best precursor for stable N-diazeniumdiolate formation. The authors found the N-diazeniumdiolate conversion was maximised in the
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presence of a low concentration of base to maintain the secondary amine in a deprotonated
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state while causing minimal cleavage of the silyl ether groups. These particular particles were applied in thromboresistant tubing, and led to a decrease in the number of thrombi and a
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lower degree of platelet activation when imbedded in a polyurethane coating on the inner
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wall of tubing used in a rabbit extracorporeal blood circulation system. The approaches in
nanoparticles.
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this work have been revisited in numerous subsequent studies to form NO releasing
Substantial early work in the preparation nanoparticles capable of delivering an NO payload
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was conducted by the groups of Schoenfisch and Friedman. In their initial study, Schoenfisch and co-workers applied a similar approach to that which was used by Meyerhoff on fumed silica, this being the formation of NO releasing N-diazeniumdiolate groups on the particles.26 Sol-gel based silica particles with secondary amine functionality were first synthesized by the condensation of tetraethoxysilane (TEOS) or tetramethoxysilane (TMOS) in the presence of N-(6-aminohexyl)aminopropyltrimethoxysilane
(AHAP3), or
(aminoethylamino-
methyl)phenethyltrimethoxysilane
(AEMP3),
N-(2-aminoethyl)-3-
aminopropyltrimethoxysilane (AEAP3).
The authors observed that by variation of the
proportion of amino functional monomer they were also able to control the diameter of the resulting silica nanoparticles. For instance, using TEOS alone resulted in particles with 6
ACCEPTED MANUSCRIPT diameter of 250 nm. By the addition of 10% AHAP3 to the reaction mixture the particle diameter was substantially reduced to 20 nm. Conversely, addition of 10% AEAP3 resulted
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in particles of 500 nm diameter. The particles prepared were essentially non-porous, as
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determined using surface area and pore volume analysis. It is worth noting that there was an
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upper limit to the amount of secondary amine that could readily be incorporated in the systems based on TEOS (20%), as at higher amounts of secondary amine functional monomer significant particle aggregation occurred. The authors attributed this phenomenon
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to hydrogen bonding between the amino groups and silanols. In order to abrogate this issue,
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the authors applied the faster condensing TMOS to decrease the likelihood of aggregation during synthesis.
The strategy was successful with particles having up to 87% amino
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functional monomer being successfully prepared.
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Having prepared sol gel particles with secondary amino groups, the authors then converted these secondary amino groups to N-diazeniumdiolate groups by exposing to elevated
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pressures (5 atm) of NO gas for three days. When the resulting particles were exposed to aqueous conditions the N-diazeniumdiolate moieties were capable of releasing NO, with the
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half-life depending on the specific amine used and the particle size. The total amount of NO released was, as expected, dependent on the total amount of amino functional monomer used in the particle synthesis. The authors demonstrated that the particles could be incorporated into tecophilic polyurethane and silicone rubber, and that the hydrophilicity of the host polymer influenced the kinetics of the NO release. These results demonstrated that particle size, chemical structure and the localised chemical environment all influenced the capability of the particles to release NO, and provide tunability of NO release. For instance, a subsequent study from the same group has examined incorporating these particles into electrospun polyurethane fibers as a potential method for preparing porous coatings with NO release capability.27 7
ACCEPTED MANUSCRIPT Friedman et al. employed an approach wherein nitric oxide was effectively frozen inside a glassy sol-gel matrix, from where NO could be released through the thermal degradation of
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nitrite ions in the presence of reducing sugars such as glucose and tagatose. (see Scheme 3).23
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The particular sol gel matrix employed was again based on either TMOS or TEOS. The
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particles prepared were characterised using transmission electron microscopy, and were relatively uniform in both size and shape (diameter = 10 nm) (see Fig 1 a). Sustained release of NO from the particles was characterised by the use of an NO specific probe and the NO
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specific fluorescence dye 4,5-diaminofluorescein (see Fig 1b). By judicious selection of the
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matrix components and reaction protocol, the authors were able to demonstrate sustained delivery of NO over a variety of different time frames. Varying molecular weight
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polyethylene glycol (PEG) was added to the matrix, and this was demonstrated to have a
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substantial effect on the kinetics of NO release. Samples incorporating PEG with a molecular weight of 3000 g mol-1 had a much faster rate of release of NO from the matrix, despite
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having a lower total loaded mass of PEG. The authors attributed this to the effect of added PEG on the nanostructure of the particles, facilitating more rapid water infiltration and
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thereby faster NO release. Importantly, the authors presented evidence that the particles were stable of a period of many weeks, and that sustained release of NO was possible over a substantial time frame once the particles were hydrated. The particles were shown to exhibit minimal toxicity against fibroblast cells, paving the way for potential therapeutic applications of the particles. Moreover, the authors also demonstrated that it was possible to include chitosan in the matrix as an aid to mucodahesion and in order to enhance cell penetration and thereby bioavailability. The authors noted that the particulate system was “ideal for immediate development and deployment for applications requiring cutaneous delivery of exogenous NO.”
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Scheme 3. NO releasing nanoparticles as prepared by Friedman & co-workers.23
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a
Figure 1. (A) TEM micrograph of NO releasing sol-gel particles as prepared by Friedman and co-workers. (B) Evolution of fluorescence spectrum of diaminofluorescein following reaction with NO released from sol-gel/glass composites as prepared by Friedman and coworkers.
Reprinted from Nitric Oxide Biol. Chem. 19, A. J. Friedman, G. Han, M. S.
Navati, M. Chacko, L. Gunther, A. Alfieri, J. M. Friedman, Sustained release nitric oxide 10
ACCEPTED MANUSCRIPT releasing nanoparticles: Characterization of a novel delivery platform based on nitrite containing hydrogel/glass composites, 12-20. Copyright (2008), with permission from
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Elsevier.
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Hu and coworkers have recently reported the preparation of S-nitroso functional
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nanoparticles over a wide range of sizes via the condensation of nitrosated 3mercaptopropyltrimethoxysilane (MPTMS). In the first step the MPTMS was nitrosated by
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reaction with sodium nitrate in the presence of HCl (i.e., nitrous acid) overnight in dimethyl sulfoxide. An aliquot of this mixture was subsequently aspirated and injected with rapid
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stirring into water at room temperature. The authors demonstrated that the initial reaction time with nitrous acid had a profound impact on the final particle size, with smaller particles
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being formed with longer nitrosation times. Other factors, such as temperature, needle size,
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injection volume, ionic strength and pH were also shown to impact the ultimate particle size. The authors demonstrated that nitric oxide was released from the particles when exposed to
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aqueous solution at 25 °C or 37 °C. As is expected for S-nitrosothiols, light stimulated the release of NO, with near complete decomposition of the nitroso group over 24 h when
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exposed to light. The presented method has the advantage of providing relatively stable particles without added surfactant, although there is a lower limit to the particle size that could be obtained (100 nm). 2b. NO-releasing Surface Functionalised Metal / Metal Oxide Nanoparticles In addition to work on silica nanoparticles, studies have also been made into the use of gold nanoparticles as potential hosts for NO releasing groups. Rothrock et al examined the preparation of NO releasing gold nanoparticles by the simple modification of the gold surface using 11-bromo-1-undecanethiol.28 Gold nanoparticles were initially prepared by reduction of hydrogen tetrachloroaurate with sodium borohydride in the presence of hexanethiol. The
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ACCEPTED MANUSCRIPT gold nanoparticles (2 nm) were then surface functionalised with alkyl bromide groups through the place exchange method, using a 3:1 mixture of 11-bromo-1-undecanethiol and
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dodecanethiol. The resulting bromo-functional nanoparticles were then reacted with either
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ethylenediamine, butylamine, hexanedimanine or diethylenetriamine to give gold
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nanoparticles incorporating a secondary amine functionality. These particles were then purified by washing extensively with acetonitrile before resuspending in methanol and sodium methoxide.
Exposure to NO gas at 5 atm resulted in the formation of
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diazeniumdiolate functionalised gold nanoparticles.
For an equivalent % amine, the
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diazeniumdiolate containing particles prepared using ethylene diamine exhibited the longest half-life, while the particle functionalised with hexanediamine had the highest NO loading.
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While some release was noted at elevated temperature, exposure to an aqueous environment
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had the greatest impact on release, implying a combined thermal and proton driven mechanism for N-diazeniumdiolate dissociation.
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Gold monolayer protected clusters stabilised with Tiopronin have also been used as a substrate for preparing NO donor nanoparticles.29 In this case the terminal carboxylic acid
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group of the Tiopronin was activated by reaction with N-hydroxysucciniminde in the presence of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC), and was thereafter modified with either diethylenetriamine (DETA), tetraethylenepentamine (TEPA) or pentaethylenehexamine (PEHA) to provide secondary amine groups for conversion to Ndiazeniumdiolate. Ideally, conversion of the secondary amine groups to N-diazeniumdiolate would occur in the presence of a base such as sodium methoxide to ensure maximal conversion.
Unfortunately, these conditions induced destabilisation of the nanoparticle
dispersion, and so only limited conversion could be achieved. Nevertheless, exposure to NO gas at 5 atm yielded N-diazeniumdiolate functional gold nanoparticles, with those particles functionalised with PEHA provided the highest loading of NO. In order to abrogate the issue 12
ACCEPTED MANUSCRIPT of colloidal stability, particles were prepared that were directly stabilised by the DETA, TEPA and PEHA (i.e., without Tiopronin). Using this approach the authors were able to
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achieve substantially higher loading of NO, with substantially longer half-lives and sustained
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release over 16 hours.
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Superparamagnetic iron oxide nanoparticles with NO release capability have recently been reported by Haddad and coworkers.30 In this study, the nanoparticles were first prepared by
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coprecipiatation of iron (III) chloride hexahydrate and iron (II) chloride tetrahydrate with ammonia. While the particles were initially stabilised by the addition of oleic acid, this was
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subsequently exchanged with mercaptosuccinic acid to yield nanoparticles with thiol functionality. The thiol functional nanoparticles were then nitrosated by treatment with an
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acidified solution of sodium nitrite (i.e., nitrous acid), yielding S-nitrosothiol functionalised
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superparamagnetic iron oxide nanoparticles. Sustained release of NO over a period of hours was demonstrated by chemiluminescence measurements. These results demonstrate the
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relative ease with which NO releasing moieties can be conferred to a particle surface possessing thiol functionality.
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Veres and co-workers have prepared Fe3O4-silica core shell nanoparticles with S-nitrosothiol functionality.31 In this case superparamagnetic nanoparticles were first prepared by a thermal decomposition process, heating a solution of iron pentacarbonyl in the presence of oleylamine. After isolating the amine stabilised iron oxide nanoparticles, a silica shell was subsequently formed on the particle surface by condensation of tetraethoxysilane in the presence of ammonia. Addition of 3-mercaptopropyltrimethoxysilane and further reaction for 24 hours yielded thiol functionalised Fe3O4-silica core-shell particles. These were subsequently converted to S-nitrosothiol functionalised particles by reaction with t-butyl nitrite. The resulting particles were shown to have the expected superparamagnetic properties, and released NO in a controlled fashion over a number of hours, with the precise 13
ACCEPTED MANUSCRIPT rate dependent on temperature. Fluorescence studies indicated that the particles were taken up by epithelial cells over a 24 h time period, and that the NO release was effectively concluded
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with 48 hours. An MTT assay was conducted to examine particle toxicity, with S-nitrosothiol
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functionalised nanoparticles being generally less toxic to human alveolar epithelial cells than
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thiol functionalised particles. The authors attributed to this slight decrease in toxicity to the cytoprotective effect of low doses of NO.
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Voelcker and coworkers have recently reported on the preparation of thermally hydrocarbonized porous silica nanoparticles with NO release capability. 32 These particles
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were prepared by periodically etching p+ type silicon wafers at 50 mA/cm2 and 200 mA/cm2 in an aqueous HF/ethanol electrolyte, followed by film detachment by increasing the current
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density to 250 mA/cm2. The films were then hydrocarbonized by exposure to acetylene / N2
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at 500 ºC, and ball milled to provide the nanoparticles. NO release capability was imparted by exposure to a glucose / sodium nitrite solution, followed by drying at 65 ºC or
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lyophilisation. The authors monitored the release of NO using fluorescence and demonstrated that NO release predominantly occurred in the first two hours after exposure to water.
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Subsequent studies were undertaken to investigate the bacterical efficacy of these particles: these results are discussed in the “Therapeutic Applications” section below. 2c. NO releasing Polymer Coated Metal Nanoparticles Hybrid organic / inorganic nanoparticles with NO release capability have also been formed through the self-assembly of thiol terminated polymers onto the surface of gold nanoparticles.33 Specifically, poly((oligoethyelenglycol methyl ether) methacrylate)-blockvinylbenzyl chloride was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, and then reacted with hexylamine to provide materials with secondary amine groups. Treatment with hexylamine also resulted in conversion of the
14
ACCEPTED MANUSCRIPT dithioester endgroups into thiol groups, thereby facilitating grafting onto the surface of the gold nanoparticles. Subsequent exposure to NO gas at elevated pressure (5 atm) led to
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conversion of the secondary amine groups to N-diazeniumdiolate groups. The resulting
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particles were shown to release NO slowly, with 90% release over 6 days at pH 6.8 and room
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temperature. Proof of principle experiments were subsequently conducted using these particles, with their utility in biofilm dispersion and cancer cell treatment being explored.
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2d. Nitric Oxide Delivery from Dendrimers
Dendrimers possess the considerable advantage of being highly monodisperse with wellMoreover, manipulating the generation of dendrimer
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defined molecular architecture.
synthesis allows precise control over the number of functional groups and particle size. The
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first report of using dendrimers as a scaffold for nitric oxide release described the
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functionalization of polypropyleneimine (PPI) dendrimers (Generation 3 or 5) with N-
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diazeniumdiolate moieites by exposure to NO gas at 5 atm (see Scheme 4).34 The inherent amino functionality of the PPI dendrimers made this a relatively straightforward procedure, although the dendrimers used included only primary or tertiary amino groups in the
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molecular structure, neither of which are ideal for N-diazeniumdiolate formation. While the authors were able to form some N-diazeniumdiolates using the primary amino groups, these were not effective in providing sustained release of NO. Moreover, it was demonstrated that the primary amine derived N-diazeniumdiolates tended to release nitroxyl (HNO) rather than NO at biologically relevant conditions (pH =7.4).
Only at lower pH (ca. 3) was an
appreciable amount of NO released. As such, the primary amine groups of the PPI dendrimers were modified by a two-step reaction to form secondary amines. Firstly, the primary amine was reacted with heptanoyl chloride to form an amide. Thereafter the amide was reduced using lithium aluminium hydride to form the secondary amine. The secondary amine functional dendrimers showed vastly improved loading compared to the primary 15
ACCEPTED MANUSCRIPT amines, provided longer half-lives and preferentially released NO rather than nitroxyl. As modification with heptanoyl chloride and LiAlH4 yielded a hydrophobic dendrimer, the
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authors applied an alternative strategy using propylene oxide to yield hydroxyl functional
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dendrimers. These dendrimers could host higher loadings of NO but had shorter half-lives
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than the hydrophobic version. The authors also demonstrated that reaction of the primary amines with acetic anhydride could be employed to form amide linkages which were inert to N-diazeniumdiolate formation. It was postulated that this might provide a facile means of
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tailoring the N-diazeniumdiolate loading by diluting out the secondary amine functionality.
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Scheme 4. NO releasing dendrimers as prepared by Schoenfisch and co-workers. A subsequent study from the same group examined attachment of S-nitrosothiol moieites to dendrimers to form NO donating polymer particles.35 Using generation 4 polyamidoamine dendrimers with terminal amine functionality as a starting material, the authors conjugated either N-acetyl-D,L-penicillamine or N-acetyl-L-cysteine to the outside of the dendrimer, 17
ACCEPTED MANUSCRIPT Near quantitative conversion of the terminal groups was achieved in the case of N-acetylD,L-penicillamine, while the yield with N-acetyl-L-cysteine was somewhat lower at 67%.
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Thereafter the sulfhydryl group in the penicillamine was modified by exposure to nitrous
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acid, while the sulfhydryl in the acetyl cysteine group was converted to an S-nitrosothiol
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using isopentyl nitrite. These S-nitrosothiol functional dendrimers were then shown to release NO when triggered by either Cu2+ or visible light, with the kinetics being influenced by the structure of the S-nitrosothiol group (primary being somewhat slower than tertiary). The S-
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nitroso-N-acetyl-D,L-penicillamine functional dendrimer was subsequently applied in a
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platelet aggregation assay, and was shown to inhibit thrombin mediated platelet aggregation by 62%, c.f. only 17% inhibition for a small molecule donor. These results demonstrated the
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potential utility of NO donating dendrimers prepared via nitrosation of an incorporated thiol
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moiety.
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2e. NO-Releasing Micelles
Micelles are considerably more straightforward to synthesize than dendrimers, and are dynamic assemblies which may undergo triggered disassembly in biological environments.
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Duong et al. explored a number of strategies for incorporating NO donor moieties into micellar structures. In their first report,36 the common NO donor S-nitrosoglutathione was conjugated into the hydrophobic domain of a block copolymer of oligo(ethylene glycol methyl ether) methacrylate and the functional monomer 2-vinyl-4,4-dimethyl-5-oxazolone. The conditions employed for the conjugation (basic aqueous solution) facilitated assembly of the GSNO functionalised polymer into micelles. Dynamic light scattering studies revealed a micelle size of 40 nm which increased to 50 nm upon incorporation of the GSNO. The authors observed that when incorporated into the core of a micelle the half-life of the GSNO was significantly lengthened when compared to free GSNO in aqueous solution (see Fig. 2). Intracellular release was also demonstrated using the fluorescent dye 4-amino-518
ACCEPTED MANUSCRIPT methylamino-2’,7’-difluorofluorescein diacetatate, which emits a fluorescent signal upon reaction with an NO molecule. Finally the authors demonstrated that application of NO
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releasing micelles with cisplatin improved the IC50 value for cisplatin against neuroblastoma
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by a factor of about 5, but interestingly did not have any appreciable impact on the IC 50 value
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for noncancerous fibroblast cells.
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ACCEPTED MANUSCRIPT Figure 2. Release of nitric oxide from NO releasing micelles (red line) compared with release from a small molecule NO donor (GSNO, blue line). Figure 2A shows the release
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profile in water and Figure 2B in an aqueous solution of ascorbic acid (5 mM). In each
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experiment the initial GSNO concentration was 0.4 mM. Adapted from Chem. Commun. 49
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(2013) 4190-4192 with permission of the Royal Society of Chemistry.
NO releasing micelles wherein the nitric oxide donor is an aliphatic nitrate moiety have also
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been prepared.37 In this case the block copolymer prepared was formed by chain extending poly[oligo(ethylene glycol methyl ether) methacrylate] with vinylbenzyl chloride and styrene
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to yield a block copolymer having segments of (i) hydrophilic polyethylene glycol brushes and (ii) hydrophobic styrene and vinyl benzyl chloride moieties. The ratio of styrene to
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vinylbenzyl chloride was manipulated to alter the number of sites for subsequent nitrate
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functionalization. To provide an NO donor moiety, the chloromethyl groups in the hydrophobic segment were converted to nitrate groups by reaction with silver nitrate in
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acetonitrile. These nitrate functional micelles were shown to have a diameter of approx..30 nm (by DLS and TEM). NO release at two temperatures (37 ºC and 60 ºC) was followed by H NMR (following the protons in the methylene unit adjacent the nitrate group). It was
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demonstrated that in the presence of glutathione approximately 99% of the NO was released over 21 h at 60 degrees, while only 36% was released at 37 degrees. Given the simplicity of synthesizing the organic nitrate polymer, these micelles have some promise for use as nitric oxide donor nanomaterials. 2f. NO Release from Core Cross-linked Star Polymers Core cross-linked star polymers have stability advantages over micelles and are more straightforward to synthesize than dendrimers. Duong et al prepared NO releasing star polymers incorporating N-diazeniumdiolate moieties for NO release.38 The star polymers
20
ACCEPTED MANUSCRIPT were prepared by the living radical polymerization of polyethylene glycol methyl ether acrylate, followed by chain extension with 2-vinyl-4,4-dimethyl-5-oxazolone and the
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crosslinking agent N,N-methylenebisacrylamide (see Scheme 5). The incorporated oxazolone
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units provided a convenient locus for the incorporation of spermine, which in turn provided
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secondary amine sites for the formation of N-diazeniumdiolates by exposure to NO gas at 5 atm. The resulting polymers were applied in the dispersal of biofilm, as discussed in some
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detail below.
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Scheme 5. Core cross-linked star polymers functionalised for NO release, as prepared by Duong et al. Forrest and co-workers have recently reported on the preparation of four-arm, sugar-based polymeric nanocarriers for potential application against squamous cell carcinoma. 39 In this case a multifunctional pentaerthyritol based RAFT agent was first prepared as the starting material.
Using
this
RAFT
agent,
1,2,3,4-di-O-isopropylidene-6-O-acryloyl-α-d22
ACCEPTED MANUSCRIPT galactopyranose was polymerised to give a four arm star polymer with protected galactose groups. The isopropylidene protecting groups were subsequently removed to give poly(6-O-
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acryloyl-D-galactose). Modification of this material with succinic anhydride gave an acid
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functional glycopolymer which could be conjugated to a hydroxyl functional N-
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diazeniumdiolate NO donor. The resulting NO releasing star polymer was examined for cytotoxicity, and it was demonstrated that the conjugated donor was substantially less toxic than were the small molecule NO donors conjugated to the polymeric nanocarrier. Moreover,
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conjugation to the glycopolymer slightly increased the half life of the donor compared
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withthe small molecule analogues.
3. Therapeutic Studies on NO-delivering Nanoparticles
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As noted above, exogenous delivery of nitric oxide offers potential benefits in a range of
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different clinical situations.40 To date, the controlled delivery of NO using nanoparticles has
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been explored in relation to wound healing, antimicrobial applications, cardiovascular treatments, anticancer agents, erectile dysfunction and novel dental materials. We explore
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each area in detail below. 3a. Wound healing
Wound healing is an important process in a variety of different contexts, but particularly in surgical and aged care situations.41 The development of chronic wounds can lead to further complications, which may in turn result in increased morbidity and mortality. Nanoparticle approaches to treating chronic wounds are not new, and there has been particular consideration of nanoparticulate silver in wound healing, although the utility of this material is the subject of some debate.42
23
ACCEPTED MANUSCRIPT Nitric oxide is involved in all stages of the wound healing process, contributing through increasing vasodilation, inhibiting platelet aggregation, stimulating fibroblasts, keratinocytes
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and endothelial cells, increasing angiogenesis and stimulating the production and deposition
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of collagen. Blecher et al., having noted the critical role played by NO in wound healing,
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proposed that the application of nanoparticles capable of sustained release of nitric oxide may be advantageous for promoting wound healing.43 To this end, these authors applied sol-gel derived NO releasing nanoparticles to mice having a 5 mm excision wound. The mice used
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in the study were NOD-SCID mice, which have a number of different impairments resulting
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in substantial immune dysfunction. In particular the lack of CD4+ and CD8+ T lymphocytes and deficiencies in B lymphocytes are likely to significantly impair wound healing under
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ordinary conditions. Comparison was made with three sets of mice: (i) no treatment; (ii)
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treatment with nanoparticles without NO release capability; and (iii) treatment with the small molecule NO donor diethylenetriamine N-diazeniumdiolate (DETA-NONOate). The authors
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found that topical application of NO releasing nanoparticles to the wound area resulted in accelerated wound closure. Closure of the wounds treated with NO releasing nanoparticles
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remained ahead of the other treatment and control sets throughout the study duration, culminating in total wound closure at day 13. Histological studies demonstrated that there was an increase in the number of fibroblast like cells in both superficial and deep areas of the granulation tissue in wounds treated with NO releasing nanoparticles. Moreover, the authors also observed a decrease in the number of inflammatory cells in the wound compared to DETA-NONOate treated wounds. The DETA-NONOate treated wounds were observed to have minimal granulation tissue, and showed extensive inflammatory infiltrates and fibrinous debris. Further investigation demonstrated that wounds treated with NO releasing nanoparticles showed immature collagen fibers closer to the surface with more developed fibers in the deeper tissue. New vessel formation was also noted, with significantly more
24
ACCEPTED MANUSCRIPT neovascularization occurring in the mice treated with NO releasing nanoparticles than in the nanoparticle or control treatment groups.
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In a further study, Han et al. conducted a series of in vivo and in vitro examinations of the
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efficacy of NO releasing nanoparticles on wound healing.44 This study also utilised the sol-
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gel particles of Friedman and co-workers.23 It was observed that application of NO releasing nanoparticles to human dermal fibroblasts resulted in increased migration (observed using an
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in vitro scratch test) compared to control nanoparticle or untreated experiments. Moreover, it was also observed that application of NO releasing nanoparticles led to an increase in the
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expression of both collagen type I and type III. Interestingly, the application of nanoparticles without NO release capability increased the expression of Type I collagen to a similar extent
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as NO releasing particles, but was substantially less effective at stimulating production of
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Type III collagen. NO releasing nanoparticles were also able to modify the migration of leukocytes into wounded skin tissue.
Specifically, it was demonstrated that neutrophil
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migration was somewhat impaired by the application of NO releasing nanoparticles, while macrophage-like cell migration was significantly increased. In a further aspect of this study,
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the authors demonstrated that there was increased angiogenesis in wounds treated with NO releasing nanoparticles. This was demonstrated by observing an increase in CD34 expression and dense vascularization compared to either control nanoparticle or untreated samples. Clearly, NO releasing nanoparticles have a substantial impact on wound closure in normal and immunocompromised mice and are of considerable interest given the social and economic impact of chronic wounds worldwide. 3b. Antimicrobial Applications Pelgrift and Friedman have recently reviewed the potential use of nanoparticles for addressing the increasing problem of antimicrobial resistance.45 In particular, nanoparticles
25
ACCEPTED MANUSCRIPT capable of delivering a nitric oxide payload are considered to have some potential in overcoming this problem. Nitric oxide is considered to have three main antimicrobial effects.
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Firstly, NO has been shown to have a direct chemical effect on DNA, deaminating cytosine,
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guanine and adenine through the formation of nitrosating intermediates such as N2O3, and via
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radical induced scission or modification of the DNA strands. Secondly, NO can cause Snitrosation of the –SH moiety in the cysteine residues of DNA repair enzymes (such as DNA alkyl transferases), thereby inhibiting their function. Finally, elevated concentrations of NO
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can cause the formation of secondary species such as peroxynitrite and nitrogen dioxide,
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which can facilitate lipid peroxidation. The combined effect of these three mechanisms makes NO a potent antimicrobial agent which may be employed either alone or in concert
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with other antibiotic agents.
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Hetrick et al. have examined the antibacterial properties of NO releasing silica nanoparticles against both Staphylococcus aureus and Pseudomonas aeruginosa.46 In this particular study
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the particles were prepared by the sol-gel condensation of TEOS with the N-diazeniumdiolate derivative of N-(6-aminohexyl)aminopropyltrimethoxysilane. The half-life for NO release for
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the resulting particles was 18 min. The bactericidal efficacy of these particles was compared with a small molecule NO donor, the N-diazeniumdiolate derived from the amino acid proline (PROLI/NO). The NO releasing silica particles were substantially more effective at killing both S. aureus and P. aeruginosa than was the small molecule NO donor. Moreover, this trend was observed both in PBS buffer and in tryptic soy broth (i.e., under conditions were cell proliferation should be enhanced). Particle cytotoxicity was also examined against L929 mouse fibroblasts, with minimal cell toxicity observed at concentrations required for bactericidal activity. Interestingly, the small molecule donor was quite toxic to the mouse fibroblasts at bactericidal concentrations.
A subsequent study investigated the effect of
varying particle size on the antibacterial performance against P. aeruginosa.47 In this case 26
ACCEPTED MANUSCRIPT particles were prepared by the reverse microemulsion sol-gel condensation of TEOS with N(6-aminohexyl)aminopropyltrimethoxysilane, with small variations made to the synthetic
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protocol in order to yield particles of 50 nm, 100 nm and 200 nm. Thereafter the particles
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were functionalised with N-diazeniumdiolate moieties by exposing to NO gas at 10 bar. The
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NO release profiles, loading and half-lives were comparable for the three particle sizes. Antibacterial assays against P. aeruginosa demonstrated that the smallest particles were most effective after 2 h, with a two fold lower minimum bactericidal concentration (MBC) (0.8 mg
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mL-1 vs 1.5 mg mL-1). After 24 h the 100 nm particles were comparable in performance to
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the 50 nm particles (0.2 mg mL-1 vs 0.4 mg mL-1 for the 200 nm particles). Cytotoxicity tests against L929 mouse fibroblasts demonstrated that the NO releasing nanoparticles were non-
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toxic against normal mammalian cells at the concentrations required for bactericidal activity.
antibacterial applications.
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These results demonstrate the considerable promise which these particles offer for
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In an interesting variation on this work, Carpenter et al. have examined so-called “dual action antimicrobials” which have quaternary ammonium functionality as well as NO delivery
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capability.48 The particles applied were synthesized by the sol-gel condensation of N-(6aminohexyl)aminopropyltrimethoxysilane with TMOS. A library of quaternary ammonium functionalised epoxides was prepared in parallel by the reaction of epichlorhydrin with N,Ndimethylalkylamines These epoxides were then reacted with the particles to give silica nanoparticles incorporating: (i) two secondary amine groups for subsequent conversion to Ndiazeniumdiolates) and (ii) a quaternary ammonium moiety with two methyl substituents and a butyl, octyl or dodecyl substituent. The particles were converted to the diazeniumdiolate derivative by reacting with NO gas for three days at 10 bar. Following characterization of the NO release properties, antibacterial assays against S. aureus and P. aeruginosa demonstrated that those particles incorporating octyl or dodecyl groups in the quaternary ammonium 27
ACCEPTED MANUSCRIPT moiety were generally more potent than those with methyl or butyl groups. Moreover NO release improved the performance of the particles against P. aeruginosa more significantly
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than it did against S. aureus. The major limitation of the study was that cell toxicity against
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L929 mouse fibroblast cells was significant for all particles at the concentrations required for
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effective antimicrobial action, with the best result being ca. 65% viability after 24 h. Further investigation and optimization is required to ensure potency to bacteria with minimal toxicity
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to mammalian cells.
In addition to silica nanoparticles, silica nanorods have also been examined for their
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bactericidal efficacy against S. aureus and P. aeruginosa.49 In this case three different aspect ratios were examined (1, 4 and 8). The particles of differing geometry were prepared by
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subtle variations in the condensation conditions (temperature, surfactant concentration, silane
with
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concentration and base concentration). The resulting particles were surface functionalised either
N-(2-aminoethyl)-3-amino-isobutyl-dimethyl-methoxysilane
or
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aminopropyldimethylethoxysilane. The authors demonstrated that in general the higher aspect ratio nanorods were more effective as bactericides for both S. aureus and P. aeruginosa, and
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that nanorods with an aspect ratio of 8 were non-toxic to L929 mouse fibroblasts at the same concentrations necessary for bacterial killing. Further, the silica nanorods with aspect ratio of 4 were also modified with a structurally different secondary amine to give a different Ndiazeniumdiolate and appended PEG chain. This system was used to investigate the effect of NO flux, and it was shown that higher initial flux was important to bacterial killing. The results demonstrate the complex interplay between particle shape, size and surface functionality in applying NO releasing materials to bactericidal applications. Friedman and co-workers have previously reported the effect of sol gel derived NO releasing nanoparticles on a range of different bacteria.50 Specifically, the authors applied NO releasing nanoparticles either alone or in combination with glutathione (in order to generate S28
ACCEPTED MANUSCRIPT nitrosoglutathione in situ) to methicillin resistant S. aureus (MRSA), Escherichia coli, Klebsiella pneumoniae, and P. aeruginosa.
In each case application of NO releasing
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nanoparticles alone or with glutathione caused cell proliferation to be significantly inhibited
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(particularly over shorter time periods), and cell survival after 24 hours to be substantially
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decreased. Interestingly, the authors noted that application of S-nitrosoglutathione did not effect cell proliferation or survival to anywhere near the same extent as the nanoparticle / glutathione combination, giving clear evidence for usefulness of the nanoparticle delivery The authors attributed this difference to the ability of the NO releasing
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platform.
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nanoparticles to provide a sustained concentration of GSNO over the duration of the experiment through in situ nitrosation of the glutathione, as opposed to directly applying S-
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nitrosoglutsathione which has a relatively short half-life and decays completely in 6 h. The
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best results were achieved on P. aeruginosa, where coadministration of glutathione and NO releasing nanoparticles resulted in total inhibition for 24 h and cell survival well below 10%
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in the same period.
NO releasing dendrimers have also been explored for their antibacterial activity. In an early
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report, a suite of G2 and G5 polypropyleneimine (PPI) dendrimers were functionalised with epoxides to impart secondary amine functionality suitable for subsequent conversion to Ndiazeniumdiolate groups.51 Moreover, by appropriate choice of the epoxide the authors were able to modify the chemical periphery of the dendrimer with hydroxypropyl, hydroxybenzyl or oligoethylene glycol functional groups. In the case of P. aeruginosa, the addition of nitric oxide release capability typically improved the bactericidal efficacy, with the most potent formulation being the hydroxybenzyl terminated dendrimers of Generation 2. When the suite of dendrimers was assessed against S. aureus, the incorporation of N-diazeniumdiolate moieties again improved the bactericidal efficacy of all dendrimers with two exceptions: PPI dendrimers of generation 2 having terminal hydroxyl propyl groups (where the bactericidal 29
ACCEPTED MANUSCRIPT efficacy was reduced with addition of NO capability) and hydroxybenzyl terminated dendrimers of generation 8, (where the addition of N-diazeniumdiolate groups had no
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impact). Having identified hydroxybenzyl terminated dendrimers with NO release capability
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as the most effective for standard S. aureus, the same materials were tested against MRSA.
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Importantly, these dendrimers were equally effective for killing MRSA as they were for standard S. aureus. The authors also examined the toxicity of the dendrimers toward mammalian fibroblast cells (L929 mouse fibroblasts). While generation five dendrimers
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were quite toxic to the mammalian cells at the same concentrations necessary to kill bacteria,
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this could be substantially ameliorated by the inclusion of NO releasing N-diazeniumdiolate
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moieties, even at twice the minimum concentration required to kill the bacteria.
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In an interesting recent report, Schoenfisch and coworkers have explored the application of
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quaternary ammonium functionalised dendrimers with NO releasing capability for antibacterial application against S. aureus and P. aeruginosa.52 In this case polyamidoamine (PAMAM) dendrimers (G1 or G4) were modified using quaternary ammonium epoxides to
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give dendrimers having both quaternary ammonium groups and secondary amine groups at the dendrimer periphery. A range of quaternary ammonium epoxides were used so as to yield ammonium moieties having either three methyl or two methyl and a (i) butyl; (ii) octyl; or (iii) dodecyl substituent. These quaternized dendrimers were then exposed to nitric oxide at 10 bar to give N-diazeniumdiolate moieties via the secondary amine group. The resulting dendrimers were capable of releasing an NO payload of between 1.07 and 1.69 µmol mg-1, with a half-life of between 1.9 and 4.9 h. The N-diazeniumdiolate half-lives observed for dendrimers with longer quaternary ammonium substitutents (octyl, dodecyl) were typically lower than for the shorter substituents (methyl, butyl). This was attributed to the formation of dendrimer vesicles which presented the N-diazeniumdiolate moiety to the aqueous 30
ACCEPTED MANUSCRIPT environment, thereby facilitating faster NO release. The authors observed that longer chain functional dendrimers were more efficient at killing both S. aureus and P. aeruginosa, and
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that NO release capability made little difference to antibacterial activity in these cases.
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However, for the shorter chain quaternary ammonium groups the addition of NO release
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capability to the dendrimer significantly increased the bactericidal efficacy. Toxicity against mammalian cells was also evaluated by exposing the dendrimers to L929 mouse fibroblast cells. In stark contrast to the bacteria, the addition of NO release capability to the dendrimers
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generally improved cell viability for mammalian cells, even for the most toxic octyl and
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dodecyl substituted ammonium dendrimers. These results demonstrate the utility of NO release for both improving the antibacterial activity of quaternary ammonium functionalised
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dendrimers and reducing their toxicity to mammalian cells.
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Core cross-linked star polymers capable of NO delivery have recently been applied in the dispersion of biofilms of P. aeruginosa. Biofilm formation presents a particular problem as
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these highly structured adhered films tend to exhibit an increased resistance to biocidal agents.53 Biofilm formation can be a particular issue when patients are cannularized, such as
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with a urinary catheter, or in patients with longer term prostheses such as a tracheostomy.54 If such implants become colonised with resistant bacteria serious complications may arise. Moreover, the formation of a resistant extracellular matrix around the biofilm can reduce the effectiveness of biocidal agents and antibiotics. Importantly, nitric oxide has been shown to have a role in the later stages of biofilm development in order to induce biofilm dispersal. Specifically, concentrations of NO in the picomolar - nanomolar range can induce the dispersal of biofilms of a wide range of microbial species through a signalling pathway involving cyclic di-GMP.
31
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Figure 3. Inhibition of biofilm formation in the presence of nitric oxide releasing star polymers. (A) Planktonic biomass as determined by measurement of OD600 of the cell culture supernatant. (B) Biofilm biomass as determined by crystal violet staining (OD550). Reprinted with permission from Biomacromolecules (2014) 2583-2589. Copyright 2014 American Chemical Society.
32
ACCEPTED MANUSCRIPT Duong et al demonstrated the application of polymer nanoparticles in aiding the dispersion of biofilm.38 Subsequent testing of these materials demonstrated that the star polymers were
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effective in inhibiting the formation of biofilm (see Fig. 3). Whereas small molecule NO
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donors were ineffective at inhibiting the formation of biofilms, star polymer vectors
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demonstrated near complete inhibition of biofilm formation. Moreover, exposure of existing P. aeruginosa films to the star polymer resulted in a decrease of film biomass of approximately 80% when the star polymers were inoculated at 400 μg/mL, with no
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corresponding reduction of planktonic growth. When the same star polymers were exposed to
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mutant strains of P. aeruginosa lacking the phosphodiesterases dipA and rdbA, the dispersal of biofilm was impeded. In the case of the rbdA mutant there was only a 50% reduction
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compared to 99% with wild type, while the dipA mutant was completely unaffected. These
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results indicated that the released NO was likely involved in stimulating phosphodiesterase activity, thereby maintaining low intracellular concentrations of cyclic di-GMP and
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maintaining the P. aeruginosa cells in planktonic form. Schoenfisch and coworkers have examined the impact of nanoparticle size and shape on
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biofilm eradication.55 In this case spherical nanoparticles were prepared at three different sizes: 14, 50 and 150 nm; and silica nanorods were prepared with three different aspect ratios:1, 4 and 8. Although slight synthetic variations were included in each case, the resulting particles were all silica materials carrying N-diazeniumdiolate groups as NO donors. The authors examined the bactericidal efficacy against both planktonic and biofilm variants of P. aeruginosa and S. aureus. In the case of P. aeruginosa, both the 14 nm and 50 nm particles were equally effective, and were close to double the efficacy of the 150 nm particles. In the case of S. aureus the 14 nm particles were generally more effective than the 50 or 150 nm particles. With regard to particle geometry, those particles with higher aspect ratio (4 or 8) were more effective against biofilm, while the highest ratio was comfortably the 33
ACCEPTED MANUSCRIPT best for planktonic S. aureus. Examination of the toxicity against L929 mouse fibroblasts at the concentration required for biofilm killing showed some toxicity, particularly at the
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concentration required for biofilm killing of S. aureus. The most promising results were
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obtained against P. aeruginosa biofilms with high aspect ratio silica nanorods. At
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concentrations required for biofilm killing these systems showed minimal killing of L929 fibroblasts.
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The application of NO releasing dendrimers in biofilm dispersion has also been investigated. PAMAM dendrimers with varying peripheral hydrophobicity were synthesized by reacting
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the terminal amino groups with (i) propylene oxide; (ii) 1,-2-epoxy-9-decene; or (iii) varying composition mixtures of propylene oxide and 1,-2-epoxy-9-decene.56 The reaction also yields
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a secondary amine moiety which can be subsequently reacted with NO gas to form N-
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diazeniumdiolate groups. The dendrimers formed had a similar NO loading capacity (ca. 1 μmol / mg) and comparable half-life (ca. 1 hr). Minimum bactericidal concentration was
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measured against both planktonic and biofilm-forming P. aeruginosa. The more hydrophilic dendrimer (with only propylene oxide derived groups at the dendrimer periphery) were
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generally less effective at killing the planktonic P. aeruginosa than were the hydrophobic or amphiphilic dendrimers. Moreover, the incorporation of NO releasing N-diazeniumdiolate moieties improved the bactericidal efficacy in all cases. When the materials were applied to P. aeruginosa biofilms the authors again observed the most effective eradication with hydrophobic or amphiphilic dendrimers, albeit with slightly higher concentrations required. Inclusion of NO releasing moieties again improved the potency. Toxicity studies on mammalian cells revealed that the optimum formulation was a ratio of 7:3 or 5:5 of propylene to decene moieties at the dendrimer periphery. With this ratio the dendrimers were effective at eradicating P. aeruginosa biofilm but, at the concentration required for biofilm dispersal, had minimal toxicity toward L929 mouse fibroblasts. 34
ACCEPTED MANUSCRIPT Thermally hydrocarbonized porous silica nanoparticles (THCPSi NPs) with NO release capability have also been investigated for their bactericidal efficacy against both planktonic
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and biofilm forming bacteria.32 These materials were able to inhibit planktonic growth of S.
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aureus, P. aeruginosa, and E. coli, and cause some reduction in biofilm mass of
little toxicity toward NIH 3T3 fibroblast cells.
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Staphylococcus epidermis. Moreover, at the concentrations studied the authors observed
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The ability of NO to inhibit biofilm formation is suggestive of its potential application in dental materials, where plaque formation is a significant issue. However, significant
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challenges exist in this area, as N-diazeniumdiolate NO donors have half-lives which are typically on the scale of minutes to hours, and therefore are unlikely to confer any protective
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effect over the time scales required for useful dental materials. Schoenfisch and co-workers
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have recently addressed this issue by preparing NO releasing mesoporous silica nanoparticles which incorporate O2 substituted N-diazeniumdiolates to increase the half-life of the NO
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donor substantially.57 By preparing an O2 substituted N-diazeniumdiolate with a condensable silane group, the authors were able to prepare mesoporous silica nanoparticles which possess
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the protected N-diazeniumdiolate functionality. The resulting nanoparticles had a Ndiazeniumdiolate half-life of 23 days, which is substantially longer than that typically observed for nanomaterials functionalised with this particular class of NO donor. When incorporated into composites the particles provide a low NO flux (0.1 – 0.4 pmol cm-2 s-1) which the authors demonstrate is sufficient to inhibit adhesion of Streptococcus mutans. Interestingly, the use of a conventional NO releasing particle proves ineffective for inhibiting S. mutans, even over the relatively short time scale of the experiment (24 h). These results are significant not only in that they provide evidence for the applicability of NO releasing materials in dental composites, but also because they provide a platform (protected Ndiazeniumdiolates) for sustained picomolar delivery of NO over the time scale of weeks. 35
ACCEPTED MANUSCRIPT In addition to in vitro studies of antibacterial activity, there has also been substantial investigation of the in vivo effect of NO releasing nanoparticles on dermal infections.
These authors codelivered the sol-gel particles synthesised by
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aeruginosa infection.58
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Chouake et al explored the use of NO generating nanoparticles in the treatment of P.
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Friedman et al.23 with glutathione, leading to the in situ generation of S-nitrosoglutathione. By appropriate selection of the nanoparticle and glutathione concentration, the authors were able to deliver a constant concentration of GSNO of ca. 8.7 nM over a 24 hr period.
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Application of the NO nanoparticles and glutathione completely inhibited P.aeruginosa for
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24 hr, which was longer than application of the nanoparticles alone (complete inhibition for 8 hours). Moreover, cell viability at 24 hours was also lower for cells treated with both
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nanoparticles and glutathione (8.3%, c.f. 36.1% for nanoparticles alone, 96.4% with
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glutathione alone and 84.3% for nanoparticles without NO delivery capability. Interestingly, these authors also demonstrated that application of NO releasing nanoparticles and
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glutathione was effective in accelerating wound closure at 3, 5 and 7 days post excision. At day 7 nanoparticle / glutathione treated wounds were at 25% ± 15% of the initial wound
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width, compared with 67% ±10% for wounds treated with nanoparticles only or 83% ± 9% for wounds without any treatment. Importantly, the use of NO nanoparticles and glutathione was shown to accelerate wound healing in lymphocyte deficient non-obese diabetic / severe combined immunodeficient mice. These promising results suggest that nitric oxide delivering nanoparticles may find application in treating patients with topical infections, or in situations where wound healing would be otherwise somewhat retarded. Martinez et al. have examined the application of sol gel based NO releasing nanoparticles for the treatment of wounds infected with MRSA.59 The authors demonstrated that, in addition to effectively killing both methicillin resistant and methicillin sensitive S. aureus, application of the nanoparticles also increased the rate of wound healing in Balb/c mice, with the size of 36
ACCEPTED MANUSCRIPT eschar being substantially reduced after 3 days. The microbial burden of the infected wounds was substantially decreased, as demonstrated by swabbing the wounds and culturing on
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tryptic soy agar. Additionally, it was shown that the use of NO releasing nanoparticles also
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reduced tissue damage, preserving the dermal architecture and maintaining high collagen
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content. Given that MRSA is becoming an increasing issue in clinical settings, these results demonstrate the potential utility of NO releasing nanoparticles in dealing with resistant
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bacteria.
Topical and intradermal application of sol-gel based NO releasing nanoparticles for the
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treatment of MRSA infection in Balb/c mice has been explored by Martinez, Nosanchuk and co-workers.60 The authors found that topical administration of the NO releasing nanoparticles
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led to less inflammation, increased fibrin deposition and reduced bacterial number compared
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to application of control nanoparticles or unchecked MRSA infection. Moreover, injection of the NO releasing nanoparticles directly into MRSA infected abscesses led to a reduction in
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abscess area of about 25% over four days, and complete abscess resolution in 7±2 days (c.f. 14-21 days in untreated or control nanoparticle groups). Topical application of NO releasing
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nanoparticles also led to a reduction in abscess area of about 15%. Interestingly, the application of NO releasing nanoparticles appeared to reduce collagen degradation by MRSA, leading to a commensurate improvement in overall wound healing. Cytokine expression was also enhanced by the NO releasing particles, leading to elevated levels of TNF-α, IFN-γ, IL-12, IL-1β, MCP-1 and TGF-β. The release of nitric oxide also reduced angiogenesis and generally preserved the histological architecture of the infected area. These results provide compelling evidence for the usefulness of NO releasing particle therapies in dealing with antibiotic resistant strains of bacteria, and may be useful either on their own or in tandem with surgical drainage.
37
ACCEPTED MANUSCRIPT One of the clinical areas in which effective treatment of MRSA is important is in the management of pyomyositis.
Characterised by the formation of abscesses in the large
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skeletal muscles, pyomyositis is often associated with S. aureus, and with the increasing
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prevalence of MRSA is therefore becoming a more challenging clinical problem. To this end,
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Schairer et al. examined both the topical and intralesional application of sol gel based NO releasing nanoparticles to intramuscular abscesses infected with MRSA in Balb/c mice.61 The authors observed that application of NO releasing nanoparticles led to significant
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changes in the tissue morphology. At day 4, those animals which were either untreated or
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treated with control (non-NO releasing) nanoparticles had significant inflammation, significant amounts of neutrophil rich infiltrate and a commensurate loss of normal tissue
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architecture. While treatment with vancomycin led to a lessening of inflammation and
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neutrophil infiltration, the most pronounced improvement was observed with either topical or intralesional treatment with NO releasing nanoparticles. Intralesional treatment in particular
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was very effective, resulting in no obvious abscess after 4 days. Tissue cultures taken at day 4 and day 7 demonstrated that there was a reduction in the number of colony forming units
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when NO releasing nanoparticles were applied, this reduction being more significant than could be achieved with application of vancomycin. The authors attributed the rapid resolution of the intramuscular abscesses to the numerous functions of NO. In particular, the authors proposed that NO is able to block microbial physiologic processes and damage microbial DNA, while also down regulating ICAM-1 to reduce neutrophil infiltration. Clearly there is some potential for NO releasing nanoparticles in the treatment of intramuscular abscesses resulting from S. aureus infection. Clearly, one of the areas where delivery of exogenous NO is thought to be of possible utility is in the treatment of resistant bacteria.62 Nevertheless, the possibility exists that bacteria may develop resistance to exogenous NO delivery. To this end, Schoenfisch and co-workers have 38
ACCEPTED MANUSCRIPT conducted a series of spontaneous and serial passage mutagenesis assays with a range of different bacteria (S. aureus, MRSA, S.epidermis, E. coli and P. aeruginosa. The particles
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applied in the study were nitrosated sol-gel particles formed from the reaction of
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mercaptopropyltrimethoxysilane and TEOS. Both the spontaneous mutagenesis and serial
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passage assays did not provide any evidence for the development of resistance. The authors attributed this result to the multiple mechanisms by which NO exerts its antimicrobial action, thereby rendering the development of resistance too complex to be evident in a relatively
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short experiment. Nevertheless, the authors do caution that the development of resistance to
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exogenous NO could occur given the ability of bacteria to adapt, and that this should be taken into account if exogenous NO delivery is ever applied clinically in antimicrobial applications.
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Nitric oxide releasing nanoparticles have also been demonstrated to have some efficacy in the
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treatment of fungal infections. Specifically, Macherla et al. have examined the use of sol-gel based particles in the treatment of Candida albicans infection in mice having a burn injury.63
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C. albicans has been implicated in burn wound infections, and may contribute to the development of sepsis. Invasive candidiasis has been shown to contribute to ca. 23% of
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severe infections, and mortality rates are in the order of 14-70%. As such, novel treatments are of considerable interest. Macherla et al. have demonstrated the effectiveness of NO releasing nanoparticles in inhibiting the proliferation of C. albicans when compared to untreated fungi, or fungi treated with nanoparticles lacking NO release capability. Interestingly, while treatment with NO releasing nanoparticles had the most profound effect, the application of nanoparticles without NO capability still inhibited the proliferation to some extent. NO releasing nanoparticles were also demonstrated to accelerate the burn healing process (similar to the wound healing results discussed elsewhere in this review), and also to prevent spreading of the fungal infection to the paws of the test mice. In order to examine the effect of NO nanoparticles on fungal proliferation in the wound, samples were excised on day 39
ACCEPTED MANUSCRIPT 3 and day 7 and were cultured on YPD agar plates. Samples from the wounds treated with NO releasing nanoparticles showed lower fungal burden than those from control or non-
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functional nanoparticle samples. Moreover histological examination demonstrated an absence
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of hyphal or pseudohyphal structures in the wounds treated with NO releasing nanoparticles,
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as compared with the control nanoparticle and untreated experiments. Time lapse microscopy was used to demonstrate that C. albicans cell division was substantially inhibited by NO releasing nanoparticles over the time course studied (60 min). Control nanoparticles also led
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to some inhibition (48 min) while untreated samples were shown to undergo budding and
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morphogenesis on a relatively short time scale (12 min). As observed in the wound healing studies, the addition of NO nanoparticles to the wound was shown to increase collagen
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deposition during burn healing, and to increase the migration of macrophage-like cells into
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the wound tissue. The NO releasing nanoparticles were also shown to increase the infiltration of neutrophils into the wound tissue, which is in contrast to findings on uninfected wounds
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where neutrophil migration was impaired. Altogether these results indicate that NO releasing nanoparticles have considerable potential for reducing and reversing C. albicans infection in
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burn victims.
3c. Cardiovascular Applications One of the most studied aspects of nitric oxide function is its role in vasodilation, and this gives rise to substantial therapeutic potential in the treatment of acute pulmonary hypertension and other disease of the vasculature. At present, therapeutic delivery of nitric oxide must be done by employing either gaseous NO or an NO precursor such as a Ndiazeniumdiolate, S-nitrosothiol, organic nitrate or sodium nitroprusside (see Scheme 1). Gaseous NO has substantial limitations in that it requires the use of pressurized gas tanks, which is costly and inconvenient. Precursor molecules have some benefit, and typically have half-lives in the order of minutes - hours and that are dependent on pH, temperature and 40
ACCEPTED MANUSCRIPT molecular structure. The ability to give sustained delivery of NO from a long-circulating particle may be particularly advantageous in the treatment of cardiovascular disease, and this
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has motivated a number of investigations into the approach.
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In an early study on the potential use of NO releasing nanoparticles in the cardiovascular
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system, Cabrales et al. studied the effect of infusing NO releasing nanoparticles formed by the sol gel approach into 50-65 g Golden Syrian Hamsters.64 The infusion solution was given
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via the jugular vein at a rate of 100 µL/min (total volume 50 µL, less than 2% of the total blood volume). The NO releasing nanoparticles and control nanoparticles lacking NO release
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capability were infused at two different concentrations: 10 mg / kg and 20 mg / kg. For further comparison, experiments were also conducted using infusions of diethylenetriamine
The authors found that for an equivalent mass, NONOates delivered a
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NONOate).
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N-diazeniumdiolate (DETA-NONOate) or dipropylenetriamine N-diazeniumdiolate (DPTA-
substantially higher payload of NO, exceeding the NO releasing nanoparticles by a factor of
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thirty. The authors observed some significant changes in the blood gas parameters in the hamsters treated with NO releasing nanoparticles. Specifically, Methaemoglobin (MetHb)
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levels increased from the baseline reading after 2 h, reaching 9±2% for a 10 mg/kg infusion and 14±3% for a 20 mg/kg infusion after 4 h. As might be expected, there was also an increase in plasma nitrate and nitrite, with the measured values being proportional to the infused dose. An increase in arterial pO2 was also observed, along with a decrease in arterial pH.
Importantly, no significant changes were observed with infusion of the non-NO
releasing nanoparticles. As might be expected, the authors observed that the infusion of NO releasing nanoparticles resulted in a decrease in the mean arterial blood pressure which reached a minimum value at 90 min and then returned to the initial level at 3 h, thereafter increasing slightly at 3.5 h. Infusions of DETA-NONOate and DPTA-NONOate at the same concentrations produced similar effects on the blood pressure, but led to increased MetHb 41
ACCEPTED MANUSCRIPT levels (19-26%) and thus a decrease in the oxygen carrying capability of the haemoglobin. The authors also measured the exhaled NO, and observed a maximum 1 h after treatment
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with the NO releasing nanoparticles. Fluorescent studies were undertaken to follow the
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circulation time of the NO releasing nanoparticles, and it was observed that the particles were
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still circulating 6 h after the infusion but had been completely cleared from the system by 24 h. Interestingly, nanoparticles without NO release capability were not observed in the circulation after 4 h which is suggestive of NO release somehow increasing the
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biopersistence of the particles. Perhaps unsurprisingly, the particles were shown to cause
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microvascular vasodilation and increased blood flow, while the control particles had no effect on arteriolar diameter or blood flow. On the venular side neither the NO releasing or control
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particles had any effect on the vessel diameter, with a slight reduction of blood flow observed
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at 20 mg/kg infusion. As for a direct effect on the blood cells, NO releasing nanoparticles did not increase the proportion of immobilised leukocytes, in contrast to the non-functional
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nanoparticles where a slight increase was observed. Further, NO releasing nanoparticles did not lead to an increase in rolling leukocytes above the baseline reading, whereas an increase
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was observed with the control nanoparticles. Studies were also undertaken to probe the effect of the nanoparticles on subjects with reduced endogenous NO. When endogenous production of NO was inhibited using N-nitro-L-arginine methyl ester (an NO synthase inhibitor), infusion of the NO releasing nanoparticles partially reversed the observed vasoconstriction and increase in mean arterial blood pressure. When heme site soluble guanylyl cyclase was inhibited with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, infusion of NO releasing nanoparticles led to a modest but statistically significant reduction in vasoconstriction and hypertension. Moreover, co-administration of NO releasing nanoparticles with a phosphodiesterase inhibitor (Zaprinast) led to a reduction in mean arterial pressure and a corresponding increase in arteriolar diameter. Altogether these results suggest that NO
42
ACCEPTED MANUSCRIPT releasing nanoparticles behave in the blood stream as one might expect, and may have some utility in the treatment of hypertensive disorders.
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In an extension of this work, Cabrales and coworkers examined the application of NO
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releasing nanoparticles for the reversal of induced vasoconstriction.65 The study utilised 50-
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65 g male Golden Syrian hamsters in which vasoconstriction was induced by infusing polymerised bovine haemoglobin (PBH). PBH causes vasoconstriction by scavenging NO
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through two reactions: an oxidative reaction of NO with NO dioxygenase and NO binding to deoxyHb. There may also be some reaction of NO with the cysteine moiety at position 93 in
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the Hb. These side reactions consume sufficient NO to result in observable vasoconstriction. As in the initial study, NO releasing nanoparticles were infused at 10 mg / kg and 20 mg / kg
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although this time the control nanoparticles were only infused at one concentration (20 mg /
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kg). Infusion of PBH led to increased total and acellular haemoglobin in all groups, with the addition of NO releasing nanoparticles also leading to increased total and acellular
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methemoglobin (metHb). Elevated metHb was measured up to 4 h post influsion, with a steady increase between time of infusion and 3 h. Blood nitrate and nitrite were also elevated
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by the infusion of NO releasing nanoparticles. The primary finding of the study was that infusion of NO releasing nanoparticles was effective in reducing the mean arterial blood pressure of hamsters with induced vasoconstriction, relative to infusion of non-NO releasing nanoparticles and infusion vehicle (see Fig 4). While the reduction was more pronounced with a higher infusion concentration, the higher dose of NO releasing nanoparticles (20 mg/kg) also resulted in an increase in heart rate. Infusion of 10 mg/kg of NO releasing nanoparticles led to a reduction in mean arterial blood pressure and no measureable impact on heart rate. The effect of nitric oxide releasing nanoparticles was also observed on arteriolar vessel diameter and blood flow. The infusion of PBH led to immediate vasoconstriction and a commensurate decrease in flow, which was reversed over time by the infusion of NO 43
ACCEPTED MANUSCRIPT releasing nanoparticles. Infusion of control nanoparticles or infusion vehicle alone had no impact on the arteriolar diameter and blood flow. Functional capillary density was also
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improved by the infusion of NO releasing nanoparticles. Taken altogether these results
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indicate that NO releasing nanoparticles may be useful for inducing vasodilation in a subject
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D
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NU
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in need thereof, and may thus be useful in treating hypertensive disorders.
Figure 4. Change in mean arterial pressure (MAP) and heart rate (HR) relative to baseline after (i) inducing vasoconstriction with polymerised bovine haemoglobin and (ii) treatment with NO releasing nanoparticles. †P < 0.05 compared with baseline; ‡P < 0.05 compared with control nanoparticles at the same time point; ¶P < 0.05 compared with 10 mg/kg NO 44
ACCEPTED MANUSCRIPT releasing nanoparticles; §P < 0.05 compared with vehicle. Note that symbols for each experimental group have been artificially displaced at each time point for clarity. Control
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nanoparticles -4 min; NO releasing nanoparticles at 20 mg/kg - 1 min; NO releasing
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nanoparticles at 10 mg/kg +1 min; and vehicle + 4 min. Reproduced from Am. J. Physiol.
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Heart Circ. Physiol. 300, P. Cabrales, G. Han, P. Nacharaju, A. J. Friedman, J. M. Friedman, Reversal of hemoglobin-induced vasoconstriction with sustained release of nitric oxide, H49-
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H56 Copyright (2011), with permission from The American Physiological Society. Cabrales and co-workers have also investigated the potential application of nitric oxide
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releasing nanoparticles prepared via the sol-gel approach for maintaining systemic and microvascular function during haemorrhagic shock.66 The authors postulated that during
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haemorrhagic shock NO production is reduced via three main routes: (i) reduction of
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endothelial shear stress leading to reduced endothelial nitric oxide synthase (eNOS) activity; (ii) free radical formation and accumulation interfering with eNOS function; and (iii) reduced
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O2 concentration (hypoxia), removing the necessary substrate for NO production. To explore the effect of replenishing NO using NO releasing nanoparticles, 50-65 g male Golden Syrian
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hamsters were subjected to haemorrhagic shock by the removal of 50% of blood volume (estimated assuming blood mass is 7% of body mass) over a five minute period. The hamsters were then treated with NO releasing nanoparticles, control nanoparticles lacking NO release capability and infusion vehicle. In all cases haemorrhage led to a substantial and expected decrease in mean arterial blood pressure (MAP). Treatment with nitric oxide releasing nanoparticles led to some improvement in MAP over the study duration (90 min), with a more noticeable improvement than that observed with infusion of control nanoparticles or vehicle alone. That said, at 90 min blood pressure only reached approx. 50% of the baseline pressure. In all cases heart rate increased slightly after haemorrhage, thereafter falling away substantially when control nanoparticles or vehicle were infused. However, infusion of NO 45
ACCEPTED MANUSCRIPT releasing nanoparticles proved effective in supporting heart rate close to the baseline level. Infusion of NO releasing nanoparticles also led to some changes in the blood gas chemistry.
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Nitrate and nitrite levels were elevated wrt baseline and control or vehicle infusion, and pO 2
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levels were also elevated, though to a lesser extent than was observed with the control
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nanoparticle and vehicle infusions. Methaemoglobin increased in the animals treated with NO releasing nanoparticles (as was observed in Cabrales’ earlier study), and this is indicative of slightly reduced O2 carrying capacity. The authors also examined arteriolar and venular
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diameters and corresponding blood flow, and observed that after haemorrhage all diameters
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were significantly constricted, with a commensurate decrease in microvascular flow. Application of NO releasing nanoparticles led to a recovery in both arteriolar and venular
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diameter, as well as some improvement in the microvascular flow (although not returning to
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baseline levels). Functional capillary density was also decreased substantially by haemorrhage, with NO releasing nanoparticles giving a modest improvement over the study
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duration and significantly more improvement than was obtained with control nanoparticles or vehicle alone. Peripheral vascular resistance was observed to increase considerably after
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haemorrhage. Nitric oxide releasing nanoparticles were able to give some reduction in peripheral vascular resistance, though the level did not completely return to baseline in the study duration. These results indicate that NO releasing nanoparticles were effective in mitigating the effects of haemorrhagic shock and as such may have some utility in supporting an individual until appropriate volume can be returned to the cardiovascular system. Moreover, the results underscore the profound role played by nitric oxide in maintaining function of the cardiovascular system. Nacharaju et al. have recently investigated the effect of three different nanoparticulate NO delivery systems on the vascular system.67 Specifically, the authors investigated the standard NO releasing nanoparticles as initially synthesised by Friedman and co-workers,23 along with 46
ACCEPTED MANUSCRIPT sol-gel particles with physically entrapped S-nitrosoacetylcysteine and sol-gel particles having chemically linked S-nitrosothiol groups (formed by the co-condensation of TMOS
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with nitrosated 3-mercaptopropyltrimethoxysilane). In order to examine the efficiency with
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which the particle could nitrosate thiol groups, the particles were mixed with solutions of
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glutathione and the formation of S-nitrosoglutathione was followed with HPLC. The particles which were most effective at forming S-nitrosoglutathione were those with encapsulated Snitroso-N-acetylcysteine. The effect of particle infusion on mean arterial pressure, heart rate
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and vessel diameter in Golden Syrian hamsters was also examined.
Although all
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formulations gave the anticipated results (decrease in mean arterial pressure, increase in heart rate and increase in vessel diameter), particles incorporating the S-nitroso-N-acetylcysteine
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had a longer duration of action than did the particles incorporating reduced NO in a sol-gel
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matrix. A dose dependent relationship was also observed, with particles infused at 20 mg/kg giving greater reduction in mean arterial pressure, increase in heart rate and increase in
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microvessel diameter. These results shows that the chemistry of NO release can have a significant impact on the duration of the therapeutic effect, and provide evidence for the
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ability to tune NO release to specific applications. There is considerable evidence to suggest that following myocardial infarction immediate reperfusion of the ischemic myocardium reduces the extent of infarction, and preserves the mechanical functioning of the heart. Maintaining appropriate, low NO concentrations during reperfusion may be critical in reducing the likelihood of ischemia/reperfusion injury. With this in mind, Schoenfisch and co-workers have examined application of S-nitroso-N-acetylD,L-penicillamine (SNAP) functional dendrimers (discussed above) to prevent ischemic damage in an isolated perfused rat heart.68 The hearts examined were excised from Sprague Dawley rats and retrograde-perfused using the Langendorff technique. Perfusion was suspended for 20 min in order to cause global ischemia, after which reperfusion was 47
ACCEPTED MANUSCRIPT performed for 20 min. The authors observed that reperfusion with the addition of SNAP either as a free molecule or conjugated to a dendrimer resulted in a reduction of infarcted
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tissue, compared to reperfusion with Krebs-Heinseleit solution alone or control solutions
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incorporating N-acetyl-D,L-penicillamine either free or conjugated to the dendrimer.
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Moreover, infusion of SNAP functional dendrimer with glutathione led to a further reduction in the %infarct, with an optimum concentration of 230 pM of dendrimer determined (corresponding to a SNAP concentration of 15 nM).
These results demonstrated that
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conjugation of small molecule NO donors to nanoparticulate (dendrimer) scaffolds led to no
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reduction in the efficacy of the drug, and may have some utility in reducing ischemia reperfusion injury following myocardial infarction.
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3d. Erectile Dysfunction
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It is well known that nitric oxide is involved in the onset of erection. As such Han et al.
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hypothesised that the transdermal application of NO releasing nanoparticles may be effective in the treatment of erectile dysfunction.69 To this end, studies were undertaken using retired breeder male Sprague-Dawley rats weighing >650 g. A suspension of nanoparticles capable
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of sustained NO release was applied to the glans and shaft of the penis of the rats, after which systemic blood pressure and intracorporal pressure were followed. Control rats were treated with a similar suspension wherein the nanoparticles were not capable of NO release. The authors observed that for the control rats the ICP/BP remained less than 0.1 for the entire duration of the experiment. However, where NO releasing nanoparticles were applied five out of seven rats showed an erectile response in the ICP/BP ratio, with visile erections occurring approx. 4.5 minutes after application of the particles. A typical erection induced by the NO releasing nanoparticles lasted less than two minutes, with an average duration of 1.42 minutes. In the same study, the authors applied nanoparticles loaded with sialorphin (a neutral endopeptidase inhibitor) which led to an unstimulated erection of longer duration than 48
ACCEPTED MANUSCRIPT that obtained using the NO releasing nanoparticles. A further element of the study was to incorporate tadalafil (a phosphodiesterase type 5 inhibitor) into nanoparticles.
This
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compound was effective in inducing erection when combined with cavernous nerve
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stimulation using a stainless steel bipolar hook electrode, although only when stimulation
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occurred 60 min after nanoparticle application. These results demonstrated that NO releasing nanoparticles have some promise as a topical treatment for erectile dysfunction, although the precise system studied did have some limitations when compared to nanoparticles loaded
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with sialorphin of tadalafil. That said, the previously demonstrated ability to control the
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release of NO from sol gel nanoparticles through variation of the matrix composition does provide some further avenues for potential optimisation of the treatment methodology.
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3e. Anticancer Activity
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The first investigation of NO releasing nanoparticles for potential use in cancer therapy was
diazeniumdiolate
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reported by Stevens et al.70 In this case the particles were prepared by condensing either Nmodified
N-(6-aminohexyl)aminopropyltrimethoxysilane
methylaminopropyltrimethoxysilane
with
TEOS.
By
varying
the
or
amount
3of
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aminoalkoxysilane the authors were able to vary the particle size between 90 and 160 nm. Variation of solvent enabled formation of even larger particles (350 nm). The authors examined the toxicity of the particles to ovarian carcinoma cells using an MTT assay, and demonstrated that the particles were more effective against the carcinoma cells than a small molecule NO donor (PYRRO/NO). An interesting relationship to particle size was observed, with the largest NO releasing particles being effective against carcinoma cells while having much lower toxicity to normal ovarian cells. The particles were shown to cause inhibition through apoptosis, and also to reduce anchorage independent growth. Subcellular localisation of the NO releasing particles was investigated by using confocal microscopy, and it was shown that the particles were co-localised with the late endosomes and lysosomes. Although 49
ACCEPTED MANUSCRIPT potential cancer applications of NO releasing nanoparticles have not received anywhere near the volume of attention that antimicrobial applications have, there is clearly considerable
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scope for further work in the area.
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The glycopolymer based polymer nanocarriers prepared by Forrest and co-workers were
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examined for their activity against human head and neck squamous cell carcinomas.39 A xenograft model was used, with subcutaneous injection of the NO releasing nanocarrier local
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to the tumour being shown to significantly reduce tumour volume. Tumour survival was also improved, with two animals having a complete response. These results were extremely
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favourable, as the outcomes in the control groups (either untreated or treated with a small molecule NO donor) were poor, with 100% of animals being euthanized within seven weeks
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due to proliferation of the cancer. In contrast to the localised subcutaneous delivery of the
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polymeric nanocarrier, intravenous infusion of the small molecule donor was not effective for
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reducing tumour volume or extending the lifetime of the treated mice. 3f. Treatment of liver fibrosis
Previous studies of liver fibrosis have demonstrated that treatment with gaseous nitric oxide
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can result in the down regulation of certain pro-fibrogenic mediators such as collagen I and transforming growth factor β-1 (TGF-β1), and thereby reduce production of fibrotic tissue. As such, Davis and co-workers hypothesized that nitric oxide releasing polymer nanoparticles may have some utility in reducing and/or reversing liver fibrosis. 71 To this end studies were conducted to examine the impact of NO releasing nanoparticles on hepatic stellate cells (HSCs). HSCs have been shown to have a significant role in liver fibrosis, as following hepatic injury or damage HSCs secrete and accumulate extracellular matrix in the liver. Since part of the normal function of HSCs is to store vitamin A, the polymer nanoparticles prepared were decorated with vitamin A moieties so as to facilitate specific cell uptake of the NO releasing nanoparticles into the HSCs. The particular particles applied in this study were 50
ACCEPTED MANUSCRIPT similar to those reported previously by Duong et al.36, and were prepared though selfassembly of block copolymers incorporating hydrophilic and hydrophobic domains. The
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block copolymers were synthesized by polymerizing oligoethylene glycol methyl ether
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methacrylate in the presence of the chain transfer agent 4-cyanopentanoic acid-4-
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dithiobenzoate and initiator azobisisbutyronitrile (AIBN), followed by chain extension with 2-vinyl-4,4,-dimethyl-5-oxazolone. The resulting polymer possessed a carboxylic acid endgroup which was exploited for subsequent attachment of vitamin A through formation of
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an ester linkage with the terminal hydroxyl of the vitamin A. Moreover, the oxazolone
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moieties were used for subsequent attachment of S-nitrosoglutathione via formation of amide linkages. The resulting micelles possessed a hydrophilic PEG corona around a hydrophobic
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core wherein the NO donor moieties were housed. The authors demonstrated that the particles
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were taken up by the HSCs and human hepatic cells, while control particles without the vitamin A functionality were not taken up to any great extent. Biodistribution studies were
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also conducted, and it was clearly demonstrated that the particles accumulated in the liver (as expected) as well as in the kidneys. The vitamin A decorated particles were shown to
AC
accumulate with substantial specificity to the HSCs, with significantly lower uptake into Kuppfer cells, hepatocytes and sinusoidal liver endothelial cells. Along with the specific targeting of the liver and selective uptake into HSCs, the authors also demonstrated that the particles were effective for reducing the expression of mRNA for collagen type I, α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF), and therefore might be expected to reduce the extent of fibrosis. Importantly, not all profibrogenic species were inhibited, with the levels of mRNA for TIMP-1 metallopeptidase inhibitor and transforming growth factor β (TGF-β) remaining relative unchanged. Similar results were obtained using unconjugated S-nitrosoglutathione. In vitro experiments demonstrated that some toxicity was observed with particle doses equivalent to 1000 µM, and so 500 µM was chosen as the ideal
51
ACCEPTED MANUSCRIPT concentration. In addition to the potential use of the particles in reducing liver fibrosis, the authors also observed a reduction in portal hypertension (which often accompanies extensive
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fibrosis) when NO releasing nanoparticles were administered to BDL rats. Importantly, the
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targeted nature of the nanoparticles seemed to minimise the effect on mean arterial pressure,
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which remained largely unperturbed. These results were supported by in vitro studies which showed that NO releasing nanoparticles were effective in countering the contractile effect of endothelin-1 (ET-1) in a collagen gel assay. Taken altogether, these results demonstrate that
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vitamin A decorated nanoparticles were effective for reducing portal hypertension by Importantly, the targeted nature of the
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reducing the contractile response to ET-1.
nanoparticle therapy reduced the systemic effects of NO (i.e., the effect on mean arterial
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pressure) which is a significant limitation of non-specific agents such as sodium
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nitroprusside.
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5. Conclusions and Further Outlook The level of interest in nitric oxide releasing nanoparticles has exploded in the last decade. From the initial investigations of NO releasing silica nanoparticles, further application of the
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technology to gold nanoparticles, dendrimers, polymeric micelles, star polymers and polymer coated nanoparticles have demonstrated the multiple platforms that can be employed for exogenous delivery of NO.
Of course, each of these platforms has advantages and
disadvantages. Dendrimers, while highly defined and very monodisperse are costly and timeconsuming to produce. Particles which incorporate metal or inorganic nanoparticles are highly stable, but may exhibit significant toxicity when administered through certain routes (e.g., via the lung). As such, the use of polymeric platforms is particularly attractive due to the low cost of production, the relative ease with which different synthetic handles can be incorporated and the potential for preparing truly biocompatible systems. For instance, polymeric particles can be readily functionalised with a poly(ethylene glycol) corona, thereby 52
ACCEPTED MANUSCRIPT imparting so-called “stealth” properties which significantly increase circulation time and improve accumulation in tumours by the enhanced permeability and retention (EPR) effect.
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Given the wide array of synthetic approaches that can be applied to the production of
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polymer nanoparticles, the preparation of truly biocompatible and biodegradable
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nanoparticles capable of NO delivery is within reach. Indeed, for the technology to find ultimate clinical application such biocompatible systems will be required.
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As noted above, an increasing number of studies have demonstrated potential clinical applications for NO-releasing nanoparticles, in areas as diverse as dental materials and sexual
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health. Without doubt the most promising area for application of NO releasing nanoparticles is in the area of antimicrobial agents. The increasing problem of antibiotic resistance requires
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considerable research activity toward the development of new agents capable of combatting
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so-called “superbugs”. Nanoparticles designed for exogenous delivery of NO may well be part of the armoury that mankind applies in addressing this pressing clinical problem.
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Moreover, there is already compelling evidence for the utility of NO-releasing nanoparticles in dispersing bacterial biofilms. Given the clinical implications of biofilm formation and the
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difficulties which arise in managing such films, NO-releasing nanoparticles may well have a readymade niche within the broader sweep of antimicrobial research. While there are a considerable number of nanoparticle systems available for the delivery of exogenous NO, there has been comparatively little investigation of materials which respond to endogenous NO. This relatively new field of NO capturing materials may also provide hitherto unexplored areas for clinical investigation. A recent report from Hu et al. discusses the synthesis and application of polymers which incorporate o-phenylene diamine groups to capture NO through the formation of benzotriazole rings.72 By varying the structure of the monomer to control hydrolytic stability, the authors showed that exposure to NO could lead to charge reversal. Moreover, by incorporating NO and temperature responsive units into a 53
ACCEPTED MANUSCRIPT polymer the formation of micelles could be triggered by exposure to NO (through the formation of hydrophobic benzotriazoles and a commensurate decrease in the lower critical
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solubility temperature). Incorporating a polarity sensitive fluorescent dye into such polymers
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means that the formation of micelles and aggregates could effectively “turn on” fluorescence.
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Importantly, these effects could be observed inside a cell, presumably as NO was captured and micelles / aggregates formed. These results demonstrate the possibility of nanoparticle formation being induced by endogenous NO, opening new possibilities in delivery and / or
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imaging applications. Altogether, there is a strong mandate for continued research into
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materials which not only deliver, but respond to NO. Acknowledgements
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This research was funded by the Australian Research Council Centre of Excellence in
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Convergent Bio-Nano Science and Technology (project number CE140100036). TPD wishes
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to acknowledge the award of an Australian Laureate Fellowship (FL140100052). The authors acknowledge significant financial and infrastructure support from the Monash
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Institute of Pharmaceutical Sciences.
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