Chemistry for oncotheranostic gold nanoparticles.

European Journal of Medicinal Chemistry 99 (2015) 92e112

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Review article

Chemistry for oncotheranostic gold nanoparticles b, Fatima el Bahhaj a, Teko W. Napporn b, Anne Juliette Trouiller a, Seydou Hebie a, * Philippe Bertrand a University of Poitiers, UMR CNRS 7285, Institut de Chimie des Milieux et des Mat eriaux de Poitiers, Equipe Synth ese Organique, 4 rue Michel Brunet, B28, 86073 Poitiers, France b University of Poitiers, UMR CNRS 7285, Institut de Chimie des Milieux et des Mat eriaux de Poitiers, Equipe SAMCat, 4 rue Michel Brunet, B27, 86073 Poitiers, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 April 2015 Received in revised form 13 May 2015 Accepted 14 May 2015 Available online 20 May 2015

This review presents in a comprehensive ways the chemical methods used to functionalize gold nanoparticles with focus on anti-cancer applications. The review covers the parameters required for the synthesis gold nanoparticles with defined shapes and sizes, method for targeted delivery in tumours, and selected examples of anti-cancers compounds delivered with gold nanoparticles. A short survey of bioassays for oncology based on gold nanoparticles is also presented. © 2015 Elsevier Masson SAS. All rights reserved.

Keywords: Gold Nanoparticles Cancer Therapy Diagnostic

1. Introduction Applications of gold nanoparticles (GNPs) have been documented in several reviews [1] describing their uses as drug delivery systems (DDS) combined with their optical properties. In this review we selected their anticancer applications from the therapeutic or diagnostic point of view and tried to organize this manuscript for medicinal chemists not familiar to this topic. Indeed a first part is dedicated to explain how GNPs can be obtained in various sizes and shapes and which parameters govern this. We then will briefly recall some standard anticancer compounds that were delivered with GNPs. In a third part we will present the fundamental aspects of delivery in cancers. With these several elements in hand, we then present key applications, selected to illustrate the various methods used to obtain functional GNPs as anticancer nanomedicines. The review is organized from the different types of chemicals used to cover or functionalize GNPs. The last part of the review describes some principle for designing GNP-based bioassay used in oncology. This comprehensive approach should help medicinal chemists not involved in the field to understanding how

* Corresponding author. E-mail address: [email protected] (P. Bertrand). http://dx.doi.org/10.1016/j.ejmech.2015.05.024 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

covalent or non-covalent binding to GNPs or already coated GNPs are possible to deliver bioactive compounds. 2. Gold nanoparticles synthesis Over the last three decades, considerable interest has focused on gold nanoparticles synthesis both because of their unique properties and promising applications in different interdisciplinary fields of physics, chemistry, biology, medicine and material science [2]. These nanoscale objects of a size in the range of 1e100 nm have physicochemical properties depending on the size and shape including the surface plasmon resonance and fluorescence enhancement which substantially differ from the behaviour of the bulk material. To date, shaped-controlled synthesis techniques have been reported for fabricating GNPs with various morphologies e rods, cubes, prisms, disks, octahedrons and polyhedrons e nanoparticles [3]. Various methods such as chemical reduction, photochemical, the template method, the electrochemical method, the microwave rapid heating, the laser ablation, UV irradiation, sonochemical, sonoelectrochemical and the seed-mediated growth methods have been undertaken to yield gold nanoparticles with uniform sizes and morphologies [3bef,4]. Many factors are governing the size and shape during GNP synthesis: the method, e.g. chemical or physical, the reducing

A.J. Trouiller et al. / European Journal of Medicinal Chemistry 99 (2015) 92e112

agent, the surfactant, the temperature, the time and the solvent (Table 1). In the chemical reduction method, quasi-spherical and mono-dispersed spherical gold nanoparticles (GNSs) in water can be obtained by reduction of a small amount of chloroauric acid with sodium citrate [5]. The nanoparticles size can be controlled by varying the amount of citrate ions, stabilizing and preventing the nanoparticles from agglomerating by providing a sufficient electrostatic repulsion, by adsorption on the surface of the particles and creating a negatively charged layer. GNSs can be synthesized in organic medium [6] with sodium borohydride in the presence of tetraoctylammonium bromide (TOAB) and toluene or using alternatively HCl and NaOH [7]. In these synthesis conditions, the gold nanoparticles are very small with sizes in the range 2e6 nm. The reduction in the presence of citrate tannic acid as stabilizer allows obtaining spherical GNPs of monodisperse size ranging from 3 to 17 nm [8]. Fig. 1 shows the synthetic process and images of transmission electron microscopy and size distributions of spherical GNPs with an average diameter of 4.2 nm and a very narrow size distribution. In this synthesis method sodium citrate reduced the gold salt while tannic acid acts as a stabilizer to control the shape and structure (surfactant). Thus, one can easily control the particle size by varying the amount of tannic acid. TEM images show homogenous size distribution. The synthesized nanoparticles are faceted and the (111) facets are the majority. In fact, the nanoparticles obtained by tannic acid method are mainly decahedron. The kinetics of the particle growth in the {100} direction is superior to that in the {111} direction promoting the development of decahedral delimited facets (111). The seed-mediated growth was developed to synthesize a wide variety of size, shape and structure controlled nanoparticles [9] with in general two steps (nucleation and growth) performed in the same solution or in separate medium. The precursor generates intermediate atoms to form a nanoparticle with an unknown mechanism. During the nucleation [10], the concentration of metal atoms increases as regularly as the precursor is decomposed under the temperature or by ultrasonic agitation [11]. At supersaturation point atoms aggregate into small clusters and once the nuclei are formed, they grow quickly decreasing the concentration of metal atoms. Equilibrium between available atom concentration and number of nuclei formed will control the final size. During nucleation, the clusters reach a critical size corresponding to the formation of the seeds. The key to synthesis nanoparticle with controlled-shape is to ensure close monitoring of the population of seeds with different internal structures. Thermodynamic and kinetic factors are important in controlling the nucleation process. In Fig. 2, the sample noted GNSs-1 is the seeds solution prepared after one day. The GNSs-20 sample corresponds to the seeds solution having aged for 20 days. Two types of populations of GNPs are observed in GNSs-20. Small particles are probably derived from seeds that have not undergone growth after formation. The formation of large nanoparticles in GNSs-20 sample would probably linked to the overgrowth of seeds. The observations made on this sample allow making some remark to the effect of ageing due to overgrowth of sprouts. This effect overgrowth is therefore a very important parameter affecting the form of long term GNSs. In the growth stage of NPs, the reaction conditions for the control of their shape are less severe as the seeds are often preformed into a synthesis step, separated and then added to the growth medium. Control of shape can be considered a process of proliferation (overgrowth) of seeds, with a well-defined crystalline structure. The overall growth of the crystal is controlled by a competition between a decrease of apparent energy (favouring growth) and an increase of the surface energy (which promotes dissolution). Obtaining a particular shape is determined by the rate

93

of addition of metal atoms on different crystal faces. The general strategy for controlling the shape anisotropy in the growth of nanoparticles is to stabilize a particular facet or through a molecular interaction. Surfactants are used for their preferential affinity for a particular surface orientation [12]. When using seeds with truncated octahedrons with crystallographic faces (111) and (100), the selective adsorption of a surfactant on the faces (100) will cause the decrease in the rate of growth of these faces and block their access. In this way, the atoms formed during nucleation preferably are deposited on the facets (111) and cause the enlargement of the areas (100). A wide variety of molecules facilitate the control of the shape as in rods [13e15], cuboids [16] or cubes [17,18]: bromide or chloride hexadecylammonium (CTAB or CTAC), polymers and biomolecules, small molecules such as adsorbed gas and even halide salts (KI or KBr). Different seeds solutions may evolve upon storage. The freshly prepared seeds (Fig. 3, blue curve (in web version)), initially shows a single absorption band located at 519 nm. This band is becoming more intense and well defined when the ageing time becomes longer. Moreover, it shifts to the red (in web version) when the solution aged (see insert Fig. 3). The shift towards higher lengths means that the size of the seeds increases [19]. The absorbance increases linearly until the tenth day it stabilizes. The growth in presence of silver ions is illustrated in Fig. 4. It was issued that in the presence of CTAB, the bromide anion precipitates with Ag(I) which adsorb to the seeds surface during the grow process, leading to crystal faces masking and therefore restricted growth. Under the experimental conditions (acidic pH), the adsorption capacity of Ag(I) is much easier and outweighs the reduction of Ag(I). By adjusting the amount of Ag(I) in the growth solution, the aspect ratio can be controlled. Sau et al. showed that the aspect ratio and the diameter of GNRs decrease when the amount of seeds growth in the solution increases [13a]. The mechanism proposed involves a rigid structure of CTAB monomers that helps to maintain a unidirectional growth by “zipping” mechanism, the basis of one-dimensional growth of monocrystalline nanocylinder. GNPs with cuboid shape can be synthesized by seed-mediated growth process in presence of copper ions. It consists to add seeds in a growth solution containing Cu(II) ions. TEM and HRTEM images showed a homogenous size and shape distribution of anisotropic nanoparticles synthesized by seed-mediated growth approach (Fig. 4). GNCs exhibit preferentially (100) facets and (110) while (111) facets are observed for GNPoly. In contrast, GNRs present some stairs at the lateral sides. Spherical, cubic and rods gold nanoparticles were synthesized by the electrochemical method in the presence of surfactant by a redox reaction in a single cell with two electrodes [4a,20]. The gold plate is used as an anode while the cathode is made of a platinum plate. Both electrodes are immersed in the electrochemical cell containing an electrolytic solution. During the synthesis, the electrode is dissolved and gold ions pass into solution, reduced at the cathode to form the GNPs, and growth takes place in the interfacial region of the cathode and within the electrolytic solution. Laser ablation method consists in focussing a pulsed through a lens on a block of metal called a target immersed in a liquid laser beam [20]. The laser will then eject particles from the metal surface thereby forming a colloidal solution of NPs. An important advantage of this physical synthesis method is its independence on colloidal chemistry that avoids the use of toxic substances. Indeed, the nanomaterials are produced directly from the solid material, while the manufacturing process may be performed in a clean environment (water or surfactant containing solutions). Laser ablation allows the formation of two different populations of nanoparticles (small and large particle size). The average particle size and the relative contribution are strongly affected by the intensity or power of the radiation. Kabashin et al. [20b] have shown

94

Table 1 Selected parameters influencing size and shape controlled gold nanoparticles synthesis. Experimental conditions Shape

Chemical or physical approach

Spherical Chemical reduction

Surfactant

Heat temperature

Aqueous trisodium citrate

Trisodium citrate/water

Boiling

Aqueous NaBH4

Tetraoctyl-ammonium bromide/ toluene/dodecanethiol HCl and NaOH

Aqueous NaBH4 Aqueous trisodium citrate

Rod

Cuboids Cubic

Particles size (nm)

References

5 to 200

[5]

1 to 3

[6,7]

3.2 to 5.2

[7]

3 to 17

[8]

Intensity of the radiation (fluence of the laser) Intensity of the beam and the concentration of the ion in solution

1 to 300

[4,20]

2 to 3

[21]

Appropriate amount of seeds, surfactant or Microorganisms and silver ion are used to control the shape Electrolysis current, ultrasound, temperature and a small amount of acetone

[2b, 2e, 3, Aspect ratio (R ¼ L/d) depends on the reaction 3d, 20] conditions Aspects of ratios [14] from 1.3 to 5.1

pH

[15] Average length 34.27 ± 7.28 and aspect ratio 4.90 ± 1.06

Structural nature of the seeds, Cu(II) ions Small amount of NaBr

29 ± 3

[14b,16]

44 and 72

[17]

Parameters influencing the size and shape distribution

% of sodium citrate, auric salt and heat temperature Room temperature Ratio of thiol to gold BH 4 /OH

(III)

Room temperature % solution and the [Au cations in water 60 C pH value

Tannic acid plus some drops of NaOH to control the pH value between 7.5 and 8 Laser ablation e Pure H2O, organics solvent (THF, DMSO, Room temperature ACN), NaSO4C12, cyclodextrins Room temperature Radiolysis and the Ionic precursor reduction with electromagnetic irradiation of the solution. photochemical methods The solvated electrons or the excited state induced by light or ionizing radiation are used to reduce the metal precursor which undergoes subsequently nucleation and growth. Pure H2O, isopropanol and polyacrylic acid, The reverse micelles (2-ethylhexyl) sulfosuccinate or a mixture of divalent: di(2-ethyl hexyl) and sodium sulfosuccinate Chemical reduction Seeds preparation (solution 1) / particles growth (solution 2) NaBH4, trisodium citrate or metals seeds CTAB or CTAC, polymers, room temperature biomolecules and halide salts (KI, KBr) Electrochemical method In a single cell, a gold plate is used as an anode and the cathode is made of a platinum plate e C16TAB Controlled temperature of 38 C Chemical reduction One-step (gold salt is reduced in the same solution) acetylacetone CTAB, Ag(I) ions plus some drops of Room temperature carbonate buffer to control the pH value Chemical reduction NaBH4, trisodium citrate, CTAB, Cu(II) ions Room temperature ascorbic acid metals seeds CTAC, small amount of NaBr Room temperature Chemical reduction NaBH4, trisodium citrate, ascorbic acid metals seeds controlled Electrochemical method e C16TAB, cosurfactant and an amount of acetone temperature

]

30 appropriate amount of cosurfactant powder is placed on top of the solution, the rate of addition of acetone and controlled temperature

[18]


Reduction agent


95

Fig. 1. TEM, HRTEM images and corresponding size distributions of spherical GNPs.

two different mechanisms: associated with the free thermal femtosecond ablation which occurs at relatively low laser fluences F < 400 J/cm2 and leads to monodisperse GNPs with very small sizes (3e10 nm), or attributed to induced heating and plasma ablation of the target. The laser ablation in pure water generally gives large particles (20e300 nm) and finely divided (50e300 nm). Radiolysis method and the photochemical method consist in the reduction of metal ions in solution by incipient atoms undergoing controlled nucleation [21]. The reduction of ionic precursor occurs under irradiation of the solution with electromagnetic radiation. These methods have the advantage to be realized in simple physicochemical conditions (ambient temperature and in the absence of contaminants) to yield a uniform reduction. The particle size can be controlled by optimizing the intensity of the beam and the ion concentration in solution. When the rate of irradiation increases, the particles formed are small. By this method, GNPs smaller than 5 nm were synthesized. In both methods the reducing radical, the solvated electrons or the excited state induced by light or ionizing radiation are used to reduce the metal precursor which undergoes subsequently nucleation and growth. In the case of radiolysis,

solvated electrons and reducing radicals are generated by excitation of a solvent. In the photochemical approach, metal precursors (salts or complexes) may be directly excited by the light and then reduced. They also generated photochemical intermediates as excited molecules and radicals that will reduce the metal precursors. The GNRs were synthesized by the method of radiolysis. If currently GNPs are essentially obtained by chemical or physical ways, few synthetic green chemistry strategies are available. Green chemistry uses biological agents from living organisms as reductive agents and ligands for the synthesis of GNPs, thereby obtaining biosynthesized and biofunctionalized GNPs. These biological agents can be extracted from enzymes, proteins, amino acids, polysaccharides or vitamin. Bhat and co-workers [22] proposed a way of green chemistry for the synthesis of GNPs obtained by photo-biosynthesis in the presence of Pleurotus florida fungi extract. The biochemical mechanism involved is not yet clear, but the most probable hypothesis is due to the photosensitivity of riboflavin. Riboflavin is a coenzyme of the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and catalyses redox reactions. These bio-GNPs display anti-proliferative activity

96


Fig. 2. Microscopy images of gold spherical particles prepared by the reduction of gold salt in the presence of NaBH4 and CTAB as surfactant. TEM images and corresponding size distributions of gold seeds obtained with samples GNSs-1 and GNSs-20. Average size of GNSs-1 is 6.2 ± 1.8 nm while that of GNSs-20 is 14.7 ± 6.9 nm.

towards tumour cells A-549, HeLa, K-562 and MDA-MB in concentration-dependant manner. However, this biosynthetic pathway has limits, the GNPs exhibit shape anisotropy with spherical or triangular particles, and size dispersion range from 10 to 50 nm in diameter.

Fig. 3. UVevisible spectra of the gold seeds solutions measured between 1 and 3 months after their synthesis.

3. Anticancer compounds Many molecules have been evaluated as anticancer agents. Many have several side effects with dose limiting toxicities (DLT) in clinic and sometimes they are very poorly soluble, requiring specific formulations. Such molecules, with efficient activities but facing solubility and side effects troubles, are good candidates for drug delivery systems (DDS) strategies. Fig. 5 depicts the compounds that were used to prepare GNP-based DDS presented in this review. Many other reviews can be accessed for the reader wanting to know more about their mechanisms of action outside the scope of this review. Substances able to generate reactive oxygen species (ROS) are often used as anti-cancer compounds, producing DNA damage in cancer cells leading to cell apoptosis. Healthy cells may also be affected and cancer cells targeting is required to avoid side effects. Indocyanin green (ICG, Fig. 5), a near infra-red (NIR) fluorophore with fluorescence outside the tissue window is used for bioimaging and is also able to produce oxygen singlet. ICG is rapidly photodegraded in aqueous media and is lacking anchor point for grafting to DDS. Doxorubicin (DOX, Fig. 5) is a standard in oncology with several side effects and has long been a good candidate for DDS functionalization with the clinically used PEG-liposomal version Doxil®. DDS linkers bearing amines like hydrazine react with the ketone group on the C2 side chain to form acidic sensitive systems for release of DOX via the endocytosis-mediated internalization. The conversion of the amine group of the sugar moiety of DOX as carbamate has also been investigated for GNPs applications. ()-Epigalocatechin-3-galate (EGCG, Fig. 5) is a natural


97

Fig. 4. Schematic representation of “zipping” the formation of the bilayer CnTAþ on the surface of gold nanoparticles can promote unidirectional growth or multidirection growth and TEM images of the corresponding synthesized GNPs.

polyphenol extracted from green tea known to inhibit formation and development of tumours but with excessive uptake causing cytotoxicity to normal cells. EGCG has been described as a DNA methyltransferase (DNMT) inhibitor in epigenetics. Paclitaxel (PTX, trade name Taxol®, Fig. 5) is a natural complex compound belonging to taxane family and isolated from the pacific yew tree (taxus brevifolia). PTX is a microtubule stabilizer, blocking cell division during mitosis. It is an insoluble compound formulated in Cremophor EL® (Taxol) consisting of esterified castor oil. More recently an albumin conjugate has been formulated (Abraxane). PTX is thus also a good candidate for alternative DDS solutions. Methotrexate (MTX, Fig. 5), a derivative of folic acid, is also used against cancer and has been subject to various delivery strategies. The platinum based chemotherapies such as cisplatin (cisPt, Fig. 5), carboplatin, oxaliplatin are effective anticancer agents which cause a decrease of proliferation, improve palliative treatments and increase the survival rate, but also present a significant systemic toxicity and many side effects due to their lack of selectivity and specificity towards the tumour cells. It is therefore necessary to improve these metal-based anti-cancer agents. Curcumin (CUR, Fig. 5) has several biological activities including anticancer and has been incorporated into several types of DDS. CUR is a natural compound found in spices active on several anticancer targets (NFkb, protein kinase C, EGFR for tyrosine kinase and HER-2) inducing in turn apoptosis. Additionally, CUR is also known to down regulate the intracellular levels of three major ABC drug transporters, Pgp, MRP-1 and ABCG2, important in multidrug resistance (MDR). CUR suffers poor solubility and bioavailability, reducing its potential anticancer application, and was a good candidate for GNP-based DDS strategy [23]. Radiotherapy is another way to treat cancer tissues by local irradiation using 60Co or proton therapy beams. To avoid energy diffusion to healthy tissues, the maximum of energy has to reach only the cells to treat. When internalized in cancer cells, GNPs showed interesting low energy x-ray emission enhancement, with possible higher absorption within the cells. DU145 human prostate

carcinoma cells were treated with GNPs (diameter ¼ 44 ± 8 nm) at 1 ng particles per cell [24]. Comparison of irradiation between untreated cells, treated Au-free cells and treated cells after GNPs 15e20% increase in the loading showed that GNPs loading gave a therapeutic effect, suggesting that targeting GNPs to cancer cells can also be used to improve radiotherapy. Photodynamic therapy is a non-invasive cancer treatment where a photosensitive agent generates ROS such as H2O2, 1O2, HO., O 2 after irradiation at a specific wavelength. These ROS are responsible for oxidative damage leading to destruction of tumor vasculature, hypoxia, nutrient depletion and finally cell death. Unfortunately, these photosensitive agents deteriorate quickly and do not exhibit specificity or selectivity for cancer cells. 4. Targeting the tumour environment and cancer cells with GNPs Targeting cancer cells and the tumour environment are two separate problems to solve. Functional nanoparticles for anticancer applications requires a safe delivery in the tumour tissue, where the cancer cells can then be reached, recognized and killed by some anti-proliferative effects of the transported anticancer agents. The safe delivery of GNPs in tumors has the same requirement as for other delivery systems: free circulation in the blood stream until the tumour zone is reached and avoiding trapping and elimination by the reticulo-endothelial system (RES). This is usually accomplished by adding stealth properties, e.g. decorating the objects to make them “invisible” to the RES, by covering the particles with polyethylene oxide (PEO) of sufficient length to reduce opsonization, in the so-called pegylation process (PEO are also called polyethylene glycol (PEG)). When dealing with GNPs, these PEG chains are generally modified at one end to introduce a thiol group, used to attach these chains onto the gold surface. The selective passive targeting of tumour with nanoparticles of convenient sizes is achieved by the now well-known enhanced permeability and retention (EPR) effect first described by Maeda.

98


Fig. 5. Examples of (A) anticancer compounds and (B) fluorescent reporters described in this review. Coloured functions indicate groups used for covalent links to GNP.

This physiological particularity of tumours is due to their leaky vasculature, where the endothelial walls are more porous than in the normal blood vessels. Thus objects of convenient sizes can accumulate in tumours while not passing through in healthy tissues. Targeting the angiogenic systems with nanoparticles based on VEGF markers in the context of stem cells has been reviewed [25]. The second point is the recognition of tumour cells and the final cell entry of the nanoobjects. Entry in the cancer cells to release the active molecules has emerged in the recent decades to avoid diffusion of active compounds anywhere outside cells inducing side effects, to improve efficacy as targets are usually intracellular, and to reduce acquired resistance due to the socalled efflux pumps belonging to the ABC membrane transporters family, when multidrug resistance (MDR) genes are stimulated. One advantage of DDS is that when internalized by cells they usually by-pass these resistance mechanisms. The second advantage of DDS is that they can be designed to enter cells by endocytosis-mediated mechanisms, either by active mechanisms, with the recognition of membrane receptors, or by passive mechanisms, involving lipid raft processes. Depending on the particles sizes used to build DDS, different endocytosis mechanisms are also involved (micro/macropynocytosis). Thus the natural cellular endocytosis process is exploited to have cells doing the job, providing that the nanoobject has surface properties favouring the recognition of the targeted cells and/or the internalisation process. Cancer cell targeting depends on over expressed membrane receptors and the receptor substrate must be attached to the DDS surface. The 3-galactosyl-N-acetylgalactosamine disaccharide

Galb1-3GalNAc is overexpressed in 85% of human carcinoma, including breast, colon and prostate carcinoma. It is responsible for aberrant glycosylation leading to glycosylated epitopes constituting tumor-associated antigens. Jacalin is a lectin protein (Fig. 6), which specifically recognizes the disaccharide unit Galb1-3GalNAc. Functionalization of gold nanoparticles with jacalin is used for active targeting of tumour cells overexpressing Galb1-3GalNAc pattern. The effectiveness of this targeting is objectified by fluorescence microscopy of leukemic cells K562 and healthy mononuclear cells treated with GNPs functionalized with jacalin tagged with a fluorescent dye such as fluorescein isothiocyanate (FITC, Fig. 5B). Cell adhesion is observed only on the K562 cell line, healthy cells being not affected by the GNP-jacalin system [26]. Over expression of a5b3 integrins participate in the angiogenesis process, used by tumour cells for their growth. Blocking the integrin binding can induces endothelial cell apoptosis, preventing angiogenesis. The well-known cyclic RGD peptide ligand (Fig. 6) can be used to target the angiogenesis of tumours and selectively deliver active molecules in the tumour environment. Epidermal growth factor receptors (EGFR) are another class of targetable over expressed proteins in angiogenic processes. EGFR2 (HER2) is over expressed in 30% of breast cancer cases associated with poor clinical outcome [27]. Folic acid (FA, vitamin B9, Fig. 6) receptors are over expressed in various cancer cells and in particular in ovarian cancer cells [28]. Folic acid receptors, like several other receptors, can mediate the cellular internalization. When attached to nanoparticles, this internalization creates a diffusion coefficient favouring particles accumulation in the tumour environment and in turn in cancer cells.


99

Fig. 6. Substrates used for cancer cells targeting.

The monoclonal anti-EGFR antibody, Cetuximab (C225), is a unique targeting agent to target EGFR-positive cancer cells. C225 was FDA-approved for the treatment of patients with EGFR positive colorectal cancer (CRC), for NSCLC (non-small cell lung carcinoma), SCCHN (squamous cell carcinoma of the head and neck) and pancreatic cancer. C225 binding to EGFR leads to receptor internalization and degradation, thus inhibiting EGFR-associated pathways. It has been reported that conjugation of cyanin tagged C225 to GNP affects its endocytosis-mediated internalization mechanisms [29]. Nanoconjugation promoted faster uptake and perinuclear localization was found after 30 min compared to the free Cy-C225, still at the membrane. This faster endocytosis for C225GNP resulted in faster co-localisation in Golgi and the transferrin recycling compartments. The involvement of dynamin-2 mediated endocytosis has been evaluated. The results indicated that a shift from dynamin-dependent to dynamin-independent endocytosis can be obtained depending on the cancer cell types. 5. Biodistribution of gold nanoparticles Whatever the drug to deliver and the targeting moieties added on their surfaces, the size and shapes of gold nanoparticles are extremely important for efficient delivery to cancer cells and for their biodistribution as well. 50 nm spherical and 10 45 nm rodshaped gold nanoparticles coated with PEG5000 (surface plasmon resonance (SPR) 540 nm and 830 nm, respectively) were obtained

from commercial source [30]. These PEG-GNPs and GNRs were injected intravenously in mice bearing ovarian tumours xenografts. GNRs showed higher accumulation in tumours (10 fold with a peak at 6 h), lower intake by liver (3 fold less with a peak at 24 h), and long circulation time in blood vessels (4 fold). These PEG-GNRs showed also 4 fold lower uptake by macrophages than the corresponding spherical NPs. In a study with 40 male rats, GNP of spherical shapes with 10 and 20 nm sizes and 50 nm hexagonal GNPs were administered intra-peritoneally with 3 or 7 days schedules. Rat's behaviours were unchanged during the treatment and no mortality occurred. Rats were sacrificed and organs dissected. Histological alterations were observed in heart tissues after 7 days, essentially for 10 and 20 nm GNPs, mainly extravasations of red blood cells with a few scattered lymphocytic infiltrations. Whatever the sizes of GNPs used in this study, oxidative stress in blood and all tissues was detected. The observed higher toxicity for smaller NPs should be the result of protein binding, enhancing the toxic effect by more biological interactions in vivo. This result highlights the necessary stealth coating of such NPs. The importance of the surface charges and their chemical nature has been investigated to determine pharmacokinetics, tumour uptake and biodistribution [31]. Several undecanethiols were used to coat GNPs (Fig. 7), bearing at the other end various chemical groups, neutral (OH) or charged zwitterion (NþMe2-(CH2)3eSO 3 ), cation (NþMe3) or anion (COO). The core particles were 2 nm

Fig. 7. Neutral and charges mercaptoalkyl-PEGs as gold nanoparticles ligands.

100


diameter with final hydrodynamic volume of 9e10 nm. The thiol ligands induced a stabilized hydrophobic interior while the surface was hydrophilic and biocompatible due to the short PEG chain. The plasma dispositions of these GNPs were characterized in male CD1 mice (IV and IP administrations). High peak concentrations in plasma were observed except for the positively charged ammonium GNPs. In contrast fast clearance is observed for positively and negatively charged particles, while the neutral and zwitterionic ones remained detectable after 24 h above 1 mg/ml. This was correlated with higher tumour uptake determined from tumour gold content, with approximately 20 and 30 mg/g at 24 h post injection for neutral and zwitterionic particles respectively. Intra peritoneal injections showed higher pancreas uptake for all the particles types, followed by lower levels in liver and spleen, minor levels in kidneys and lung, and minimal in brain, while in contrast intra venous injections showed higher particles amounts in liver, except for the ammonium version, followed by kidneys and minimal in brain, lung, pancreas and kidneys. Neutral and zwitterionic particles appeared to have the best profiles when taking into account the longer circulation time, lower clearance and distribution after intravenous injection. Thiol-containing PEG can stabilize nanoparticles in various environments. Different PEGGNPs were designed with different PEG molecular weights (MW) and it was showed that high MW-PEG stabilized the GNPs and screened the surface charge better than low-MW PEG [32]. The influence of the ligands used to coat GNPs has been investigated to determine the best cellular uptake on human colorectal cancer cell lines (HCT-116, HT 29, LS154T and SW640) [33]. Three ligands were used: PEG-NH2, b-(2-mercaptoethoxy)-glucose (GLU) and glutathione (GSH) (Table 2). GNPs of 1.6 nm size gave coated particles of 5 nm hydrodynamic size. In Dulbecco's minimum essential GlutaMax medium (DMEM) GLU-GNP were internalized in a dose dependant way with a saturation level of 46.6 cm2/mL for an LC50 of 23.15 cm2/mL corresponding to 1.6 1012 particles per cell at 22 h. GSH-GNPs showed an uptake also dependant on cell density not observed for GLU-GNP. Several combinations of ligands were investigated (Table 2) and the internalization of GSH-GNP in the various cancer cell lines (Table 3). GSH coated GNP appeared to have the highest uptake and combination with a few percentage of GLU rapidly decrease the uptake. The authors suggest that these particles are internalized by creating or using preformed pore at the cell membrane with an energy independent mechanism, as the internalization was not correlated with endocytosis. These results also suggest that the self-organization of one ligand at the particle

Table 3 Cell type-dependent internalization. Rate 1010 particles/cell/22 h

Cell line

HCT-116 HT-29 LS-174T SW620

DMEM

Glucose, þ insulin medium

130.4 87.6 32 89.2

200.4 140.9 68.4 133.2

surface is important for stabilization and that combination of ligands should be carefully analysed to avoid destabilizing effects. The surface charge can be also modulated by loading with charged species (Table 4). GNPs size effect (5, 10, 20, 30, 40, 50 nm) was investigated on pancreas cancer cell lines (PK-1, PK-45, Panc-1) with Atomic Absorption Spectrometry (AAS) [34]. When treated with 11.8 mM GNPs for 24 h, the highest uptake was obtained for 20 nm GNP size, with selective results for 30, 40 and 50 nm sizes with the PK-45 cell line. Uptake was time dependent in all cells. PK-1 cell line was the most sensitive. The uptake appeared dose-dependent without plateau effect in the concentration range used (11.8, 23.6, 47.2 and 94.4 mM of 20 nm GNP for 24 h). When incubated for 24 h with 11.8 mM of 20 nm GNPs at 37 C, 39 C and 41 C no differences in uptake were observed. 6. Functional alkyls or oxa and aza alkyls as reducing, stabilizing or functionalizing agents Thioalkyls are often used to coat GNPs, as well as thio-oxaalkyls, e.g. PEG, bearing a terminal thiol group, and azaalkyls, e.g. polyethyleneimine (PEI), the latter one being branched due to the trivalent nitrogen atoms. Doxorubicine was conjugated to GNP via a linker bearing the lipoic acid at one end for gold coating and a hydrazine group at the other end for linking DOX by the C2 ketone in the hydrazone form [35] (Fig. 8A). The PEG part has molecular weight 3400. Alternatively, in the same work, the amine group on the glycone part of DOX was used to prepare a carbamate version. The intrinsic fluorescence of DOX was quenched upon grafting due to the GNP proximity. The hydrazone version was found more effective for DOX release, with restoration of DOX fluorescence upon endocytosis-mediated release, after excitation at 465 nm and florescence emission at 565 nm, demonstrating the internalization

Table 2 Ligands tested for cellular uptake.

GLU-SH %

GSH %

PEG-SH %

Size (nm)

Uptake rate, 1010particles/cell/22 h

100

5.8e6.4 3.7e3.8 3.9e4.0 5.5e6.4 3.8e3.9

4.4 1.1 134.3 79.3

100 100 50 20 40 60 80

50 80 60 40 20

2.0


101

Table 4 Zeta potential of GNPs before and after ssDN loading. Size of gold nanoparticles (nm)

Zeta potential (mV) Before ssDNA conjugated

After ssDNA conjugated

13 ± 2.6 45 ± 3.1 70 ± 4.9 110 ± 5.1

13.99 17.83 19.14 10.25

27.27 28.69 24.66 19.48

± ± ± ±

1.75 1.31 1.48 0.80

of the hydrazone construct in multidrug resistant MCF-7/ADR cancer cells. The carbamate version was not expected to be cleaved during endocytosis but evident DOX release suggested that

± ± ± ±

1.03 1.07 1.88 0.97

other mechanisms can participate. The reported IC50 were 2e5 mM, close to the free DOX. As many other nanoparticles based delivery systems, the construct was stopped at the nucleus periphery.

Fig. 8. Thioalkyls, oxaalkyls and azaalkyls used to coat and functionalize GNPs. (A) Functional lipoic acid used for double linking to gold. (B) Desymmetrisation of diamino-PEG to introduce thiol for link to gold and anticancer compound in a covalent cleavable way. (C) Plunbagin imine functional GNPs. (D) Functional PEI as coating and reducing agent. (E) Multilayer coverage of GNPs for ONT loading and release upon pH stimuli. (F) Ingredients for the preparation of lipid-embedded GNPs.

102


DOX could be attached to GNPs by converting the glucosamine to thioacetylglucosamine and subsequent disulphide linkage [36] (Fig. 8B). This has been achieved from H2N-PEG2000-NH2, converted to monothioacetamide, used to coat GNPs to give GNP-SPEG-NH2. The terminal amine groups at the surface serve for stabilization, stealth properties and further introduction of functional groups by amide links. These amines groups were partially converted to a reactive pyridinodisulphide moiety GNP-S-PEG-NH-SSPyr. Exchange with the thioacetamido-DOX gave the final construct GNP-S-PEG-NH-SS-DOX. TEM images gave a 5.2 nm diameter for GNP, increased to 16.2 nm after PEG coating and finally 28.2 nm after DOX loading. Elimination of unreacted thioacetamido-DOX was performed by dialysis tubing and 1H NMR was used for validation of the construction. GNP-PEG-SS-Pyr showed no cytotoxicity while GNP-PEG-SS-DOX nanoconjugates demonstrated higher dose-dependent cytotoxicity to drug-resistant HepG2-R cells at lower concentration (IC50 at 8 mM) than free DOX. DOX is a wellknown substrate for efflux pumps, a cancer cell mechanism responsible for resistance where the therapeutic agents are removed from cells. By using these GNPs loaded with DOX, the efflux pump resistance was by-passed, resulting in a lower dose required for the same therapeutic effect. The intracellular amount of GNP-PEG-SS-DOX was determined by ICP-MS and the endocytosis-mediated internalization through lysosome confirmed by Lysotracker staining. The rational for DOX release in this work is based on disulphide reducing enzymes found in cells, possibly with acidic pH assistance. Plumbagin (PB), a generator of ROS of the naphtoquinone family, was studied in this context and GNP delivery (Fig. 8C). The GNP coating was made with NH2-PEG2000-NH2 with subsequent reaction with PB. The author proposed an amide bond for the conjugation, but this may not be the case when regarding the structure of PB and the GNPs shell and the reaction conditions described. An imine formation between the PEG-NH2 and the ketone group can be involved. Internalization in lysosomal compartments was assessed and the ROS properties of PB appeared to be quenched by its conjugation to GNPs. Catechol derivatives are well known antioxidant of the polyphenol family. In turn they can be used as reducing agent for GNP preparation. When linked to PEI chains they can also be used as stabilizers. Ferulic acid is a natural cinnamic acid derivative with a catechol moiety. Coupled to a branched PEI25000 the resulting catecholamide was used to prepare GNPs with subsequent thiopegylation and final RNA loading [37] (Fig. 8D). The GNP sizes were dependant on the substitution degree of the PEI-cathecol: 50.6 ± 5.5, 28.4 ± 6.4 and 15.3 ± 8.7 nm were obtained for 28.4% (PEI-C-1), 15.4% (PEI-C-3), and 6.5% (PEI-C-5), with SPB in the range 520e542 nm corresponding to the increase in size. The diameters of PEI-Catechol micelles were also measured with DLS: 54.6 ± 6.7 nm (PEI-C-1), 30.8 ± 5.2 nm (PEI-C-3), and 13.3 ± 0.9 nm (PEI-C-5) and zeta potential were: 51.3 mV (PEI-C-AuNP-1), 29.5 mV (PEI-C-AuNP-3), and 4.9 mV (PEI-C-AuNP-5), correlated with the diminution of free amine groups. The authors suggested that the oxidation of the catechol part into orthoquinone leads to reaction with the free amine groups in PEI to produce a complex imine network around the GNP. These GNPs were loaded with GFP siRNA and injected into GFP expressing MDA-MB-435 breast cancer cell line. As expected, reduction in GFP expression was determined by decrease in GFP fluorescence indicating also the internalization of GNPs in cells. The PEI-C-AuNP-5-siRNA complexes with an average diameter of 15.3 nm and a surface zeta-potential value of þ5 mV showed significantly higher GFP suppression than PEICAuNP-1 and PEI-C-AuNP-3 (73.8 ± 5.6% and 62.3 ± 2.4%, respectively). A Cy5.5-siRNA version and LysoTracker Red were also used for the determination of the GNP cellular trafficking. Upon

complexation to GNP the Cy5.5 fluorescence was quenched by FRET effect and release restored the fluorescence. PEI-C-AuNP-5 was mostly in the cytosol whereas PEI-C-AuNP-1 was found in the endosomal compartments or bound on the cell membrane. The GNP-siRNA particles were found non-toxic while PEI-SiRNA were toxic, showing the potential of the particles for safe gene delivery. Alternatively to classical cationic GNPs, anionic GNPs were proposed as reversed charged GNP for oligonucleotides (ONT) loading. In this strategy, the GNPs are coated with components able to invert their surface charges upon pH modifications, with the particular objective to destabilize endosomes by a proton pumping effect to favour escape. GNPs were first prepared with mercapto undecanoic acid (MUA) coating. A layer by layer strategy was implemented to prepare the reversible charge system, with first PEI, then PAH-Cit (PAH-Cit: polyallylamine citrate amide) and finally PEI [38] (Fig. 8E). The transfection properties of the construct were demonstrated by loading DNA coding for GFP. A maximum of 7.5 ONT/particle was found the most efficient, higher and lower loading reducing the intake. Involvement of endocytosis and endosome escape was determined by using a siRNA bearing the cyanine-5 fluorophore (ICG family). The results indicated that more siRNA can be delivered with such particles, but may form complexes with PEI in the cytoplasm. Liposomes are organic micelle constructions allowing transport of encapsulated free drugs, like DOX, able to target the tumour environment by EPR. Their important sizes limit the accumulation of liposomes and the drug release is known to be slow while leaks are responsible for early release during blood circulation. To improve this strategy, photothermal assisted release has been studied based on co-administration of GNRs (MeO-PEG5000-SH coated), able to transform near infrared irradiation into heat, with a liposome formulation adjusted to be unstable on heating (DPPC/ Chol/DMPC(1,2-dimyristoyl-sn-glycero-3-phosphocholine)/ PEG2000, 54:30:3:3), with DOX release in 10 min at 43 C [39]. Tumour reduction was more than 3 fold enhanced by combining liposomes and GNRs triggered release, and cancer cells apoptosis was demonstrated. 7. Glycosides as reducing, stabilizing or functionalizing agents In recent years, natural compounds considered as green and renewable resources were studied as alternative to PEGs for GNPs coating. Polysaccharides in particular receive high attention to prepared functional GNPs to deliver anticancer agents. The replacement of platinum derivatives in chemotherapy by gold has been proposed on the basis that gold (I) salts have long been known for their medicinal properties in chrysotherapy for the treatment of rheumatoid polyarthritis and psoriasis. In particular phosphingold(I) complexes have an intrinsic anticancer activity. Combining phosphingold(I) complex and gold nanoparticles and glycopolymers to improve bio-recognition, biocompatibility and targeting of tumor cells was thus proposed as an alternative anticancer treatment. The Reversible Addition Fragmentation chain Transfer (RAFT) polymerization was used to prepare such glycopolymers (Fig. 9A) [40], designed for facilitated attachment after photo irradiation onto gold nanoparticles by introduction of thiolated groups. These thiolated glycopolymeric systems are called glycopolymers dithiocarbamate (DTC). The therapeutic activity was doubled by the association with triphenylphosphine-gold metal complex I (Auþ) by electrostatic interactions between Auþ and DTC (Fig. 9B). Cytotoxicity assays on hepatocellular carcinoma HepG2 cells showed an induction of apoptosis and an inhibition of cellular proliferation higher than the results obtained with cisplatin or cytarabine. This improved efficiency is due to an increased


103

Fig. 9. Glycopolymeric coated gold nanoparticles. (A) Synthesis of glycopolymer by RAFT polimerization method. (B) Glycopolymeric dithiocarbamate coated cold nanoparticles associated with phosphingold (I). (C) Functionalization of GNPs with HA bearing cresyl violet and porphyrin. (D) Curcumin-modified hyaluronic acid as reducing and stabilizing agents in the synthesis of folic acid-targeting GNPs. (E) Synthesis of GNPs with porphyrans as reducing and coating agent for electrostatic DOX loading. (E) Cyclodextrins. (F) Modified thio-cyclodextrins used as coating agents for PTX loading. (G) Chitosans and Pluronics for GNP coating.

cellular uptake caused by galactose units in the glycopolymer that interact with ASGPR triggering endocytosis of the complex. Galactose pattern targets the asialoglycoprotein receptor (ASGPR) that is overexpressed on the surface of cancerous liver cells. This specificity of galactose-ASGPR is correlated with the lack of activity of this assembly on the HeLa and MCF-7 cells, which do not overexpress ASGPR.

Hyaluronic acid (HA) has a strong affinity for CD44 transmembrane receptor overexpressed on the surface of cancer cells and can be used for GNPs active targeting of tumour cells. The recognition and binding of HA to CD44 receptor induces cellular uptake by endocytosis. The GNPs find themselves in the cytosolic compartment where hyaluronidases specifically cleave HA. This idea was exploited by Song and coworkers (Fig. 9C). HA patterns are

104


thiolated to allow attachment onto the GNPs wheras cresyl violet and a porphyrin derivative are grafted onto the chain. The teta(4aminophenyl)porphyrin ligand is sensitive to UV irradiation and, after excitation by photons at 365 nm, generates ROS. Cresyl violet is also photosensitive and emits fluorescence after UV irradiation. Due to the proximity between the electronic surface of GNPs and both biomolecules chromophores, a fluorescence quenching is observed. Once this complex is in the cell compartment, HA is cleaved by hyaluronidases and cresyl violet and porphyrin derivative are released (Fig. 9D). Then there is a ROS emission responsible for the cytotoxic activity and fluorescence emission used for imaging. Spectrofluorimetry data shows that fluorescence is correlated with the concentration and cleavage of HA because in the presence of hyaluronidase inhibitors the fluorescence decreases. As hyaluronidases specifically cleave hyaluronic acid, replacement by other substrates gave no fluorescence. The generation of ROS can be quantified using a commercial probe 20 ,70 -dichlorofluorescin diacetate. The fluorescent measurement showed a correlation between ROS and HA concentration [41]. HA was also coupled with CUR to produce a water-soluble conjugate CUR-HA (CUR 1.3 ± 0.3 mg/100 mg conjugate) possessing HA stabilizing properties and CUR reducing properties used for direct reduction of HAuCl4 to prepare the corresponding CUR-HA-GNPs (63.4 ± 0.2 nm, zeta potential 45.3 ± 1.2 mV). In parallel, FA was activated via NHS method and the intermediate compound reacted with a diamino PEG linker (MW 1500) to produce a NH2-PEG-amidoFA. The remaining amine group was used for amide coupling to the remaining carboxyl groups of the HA coated GNPs under EDC/NHS conditions at basic pH to give CUR-HA-GNPs-PEG-FA (120.6 ± 2.2 nm, z potential 32.3 ± 2.1 mV, SPB 533 nm). The negative potential ensures long storage without aggregation. These particles were found compatible for intravenous injections as no platelets aggregation was observed. The toxicity was evaluated on Human cervical carcinoma cells (HeLa), C6 glioma cells (folate receptor positive) and human epithelial colorectal adenocarcinoma cells (Caco-2). Cell viability was reduced in all cell lines in a dose dependent manner with CUR and CUR-HA-GNP-PEG-FA, the latter having up to 3 fold higher toxicity compared to CUR. Cellular uptake in glioma cells was 55.9% for CUR-HA-GNPs and 95.4% for FA-PEG-GNP-HA-CUR, illustrating the involvement of folate receptors in C6 glioma cells. Uptake was also observed in Caco-2 cells. FITC modification was used to evaluate the cellular trafficking by fluorescence and showed accumulation of the GNPs around and in the nucleus of Hela cells. Porphyrans are complex polygalactosides partly sulphated on the carbon C6 of the galactose units that can be used as reducing agents for GNP synthesis whereas the sulphate group can be used for electrostatic load of positively charged compounds. HAuCl4 was mixed with porphyran at pH 11 to obtain the reduction and production of 13 nm sized porphyran-coated GNPs (hydrodynamic volume 101 ± 4 nm, zeta potential: 31.05 mV) [42] (Fig. 9E). Subsequent DOX in hydrochloride salt form loading was obtained after 24 h incubation with DOX.HCl (14 ± 3 nm, hydrodynamic volume 105 ± 2 nm and zeta potential 19 mV). The loading is assumed to be due to electrostatic interactions between DOX.HCl (glucosamine is in the form NHþ 3 Cl ) and sulphate groups. Buffered conditions showed efficient release of DOX. Endocytosis-mediated internalization is expected to release DOX in cells, where acidity in endosomes/lysosomes will disrupt the electrostatic interactions due to higher amount of Hþ in the vesicles that will replace DOX. The dose dependent toxicities toward cancer cells of GNPs, DOX and GNPs-DOX were determined, GNP-DOX showing the best results, while DOX was 25e50% less efficient. b-cyclodextrin inclusion complexes with PTX were described with GNPs as a support [43] (Fig. 9F). The 7 primary hydroxyl groups in b-cyclodextrin were converted to thiol groups for grafting

to gold particles and partial esterification with rhodamine B allowed for analysis of cellular trafficking. Biotin was coupled to a HS-PEG3400-NH2 as an amide for cell targeting. All the particles prepared were in the 20e40 nm sizes. HeLa, A549 and MG63 cancer cells and NIH3T3 fibroblast as normal cells were used for validations. Cellular GSH activity was found critical for PTX release, with ligand exchange taking place prior to PTX release from thiocyclodextrin. All cancer cells internalized the GNPs, Hela cells having the highest affinity. The toxicity was more than 5 fold higher for cancer cells and correlated with PTX release, inducing apoptotic behaviour. GNRs coated with Pluronics® and chitosan were prepared to improved GNRs-based photothermal therapy [44]. Pluronics are another PEG derivative with insertion of alternative isopropyl motifs. Chitosans are high molecular weight polyglucosamine, partly N-acetylated, also used sometimes in DDS constructs. Pluronics and chitosan-pluronics were incubated with GNRs and gold coating was obtained, several GNRs being found in one pluronic particle (Fig. 9G). The zeta potential of the GNRs loaded particles was identical to the carriers alone, indicating an efficient shielding effect of the pluronics. This strategy offers the advantage of maintaining the gold rods away from proteins able to fix them. Evaluation of these GNPs loaded particles on squamous carcinoma (SCC7) tumor cells and NIH/3T3 fibroblast cells showed no toxicity compared to free GNRs whereas the uptake was superior for GNRs loaded chitosan particles than free GNRs. After irradiation to produce the thermal effect, the GNR-loaded pluronics had a much better photothermal effect on cancer cells than on normal ones and the chitosan-conjugated nanocarriers showed much stronger photothermolysis than the bare nanocarriers and better tumor targeting. In mice the laser irradiation produced a dramatic decrease in tumor size over one week. An improvement of GNPs targeting to cancer cells for radiotherapy has been proposed based on dual folate-glucose receptor [45]. The FA-GNP-Glu nanoparticles were found more effective in selective targeting of KB cells when compared to FA-GNP or GluGNP. KB cells are part of the cells over expressing folate receptors and when compared to the low expressing A549 cells, FA-GNP-Glu internalization in KB cells was approximately 20 fold higher. Submitted to X-ray irradiation at 10 Gy, KB cells treated with FA-GNPGlu showed the highest decrease in viability, indicating a high selectivity and improvement. The isotope 198Au is currently under investigations for radiotherapy and its preparation and applications to prostate cancer has been reviewed [46]. This isotope has a half-life of 2.7 days and produces b emission. 198Au has a better radiation dose rate compared to other commonly used radioisotopes. 198GNP were coated with Gum Arabic (GA), a complex polysaccharide, and injected in a mouse model for biodistribution studies. At 24 h 75% of the GA-GNPs were in the prostate tumour, 11% in the kidney and 17% in urine, while negligible amounts were found in other organs. These particles demonstrated high intrinsic selective prostate tumour targeting. 8. Peptides as reducing, stabilizing or functionalizing agents Peptides anchoring has been another way to directly functionnalize GNPs during synthesis or after their synthesis. Due to their higher toxicities GNPs with sizes below 10 nm are generally not used for therapeutic applications. Studies with 2 nm sized GNPs (SPB 506 nm, zeta potential 41.2 mV) were conducted in order to prepare targeting GNPs based on the RDG variant CRGDK, a ligand of the neuropilin-1 (Nrp-1) receptors over expressed at MDA-MB321 membrane cancer cells and regulating membrane receptormediated processes [47]. These GNPs were also functionalized


with the therapeutic peptide P12 (TSFAEYWNLLSP), known to stimulate the expression of the tumour suppressor gene p53 via binding to MDM2. The small size was obtained by preparing GNPs in the presence of tiopronin (Fig. 10A). This method allows standardized anchoring of the various peptides used in this study via an amide bond under EDC/NHS classical coupling conditions. Dual coupling was also achieved with GNP-P12 followed by CRGDK coupling or in one reaction by mixing P12 and CGRDK. GNP-CRGDK and GNP-P12 conjugations were 90 and 65% respectively, measured by optical density at 570 nm. The CRGDK functionalization increased the intracellular uptake of GNPs as quantified by ICP-MS, with maximal interaction between the CRGDK ligand and the

105

targeted Nrp-1 receptor, improving in turn the delivery of P12 peptide responsible for re-induction of p53 expression, a tumour suppressor gene silenced in this cell line. RGD peptides, singlechain variable fragment (ScFv) peptide recognizing EGFR and an amino terminal fragment (ATF) peptide that recognizes the urokinase plasminogen activator receptor (uPAR) were used as tumour targeting ligand for GNPs and GNRs preparation. Classical preparation of stabilized GNPs with MeO-PEG5000-SH was followed by displacement with HOOC-PEG5000-SH allowing grafting of peptides by the amide coupling under EDC/SulfoNHS conditions. The RGD ligand showed higher accumulation in liver and spleen compared to the other peptides, while tumour targeting was higher with the

Fig. 10. Petide functionalization of GNP. (A) tiopronin synthesis. (B) Preparation of GSH functional GNP as support for multiconjugation. (C) Antibody functionalization of GNP. (D) Antibody functional GNP labelling with iodogen. (E) Technetium incorporation onto GNP with thio-peptide EDDA ligands. (F) Levulinic acid used in GNP-based phototherapy. (G) Dual responsive functional GNPs using a MMP2 cleavable substrate and displacement by GSH.

106


other versions, including GNPs without peptides. These results indicated that the selected peptide-mediated targeting does not really improve the normal PEG-GNP targeting [48]. The GNRs versions with ScFv and EGFR ligands were internalized by cells in a more efficient way, while RGD-GNR remained in the endothelial cells but not in tumor cells. The tiopronin strategy has also been used for DOX loading [49] onto ultra-small (2.7 nm) GNPs. The particles are 20-fold more cytotoxic to B16 melanoma cells than the equivalent concentration of doxorubicin alone, and activity is six times faster. These DOXGNPs are internalized by endocytosis and are observed in the cytoplasm and nuclei, in contrast to previous reports with larger particles, unable to enter the nucleus. These GNP-DOX were also active on DOX resistant cells and showed reduce toxicity toward normal cells. Glutathione (GSH) is the natural tripeptide Glu-Cys-Gly found in cells and responsible for disulphide bioreduction processes. It could be used safely to prepare functional GNP with free carboxylic acids at the surface for advanced functionalization. These carboxyl groups were used to attach FA to gold nanoparticles [50] (Fig. 10B). Synthesis of GNP-GSH-FA nanoparticles was described with a 36.4 ± 4.2 nm hydrodynamic volume, a zeta potential of 15.7 ± 1.8 mV due to the presence of carboxyl group in FA, and a small SPB red-shift (520 nm for GNP to 528 nm for GNP-GSH-FA). The construct showed little toxicity towards HeLa and mouse fibroblast (FB) cells indicating a good biocompatibility. The GNPGSH-FA nanoparticles were modified with FITC, probably on the free GSH NH2 group (not documented in the article) to produce a fluorescent particle for cellular trafficking analysis. Binding to Hela cells due to over expressed folate receptors was followed by internalization, demonstrated by fluorescence in cells, while A549 cancer cells, lacking folate expression did not internalize the particles. Gold nanoparticles were functionalized with anti-HER2 antibodies to target breast cancer cells [51] (Fig. 10C) and interactions with cells were monitored by two photons induced luminescence. Anti IgG and MeOPEG coated particles do not attached to cancer cells. GNP-cetuximab assembly was prepared [52] with a minimum 3:1 ratio of cetuximab:GNP necessary to achieve a direct efficient binding of cetuximab to GNP based on the intrinsic content of binding proteins in cetuximab. A significant internalization was obtained via EGFR receptors. The pro-apoptotic peptide (PAP) targets subcellular organites and more specifically mitochondria when its secondary structure is alpha-helical. Mitochondria are fundamental organelles to the survival of the cells involved in the regulation of energetic metabolism, electrons transfer and transport, regulation of redox equilibria and calcium regulation. The damaging of mitochondria is a signal for apoptosis initiation by caspase activation, membrane depolarization, interaction with Bcl-2 protein that lead to cell death. This PAP is functionalized at one end with a thiol group allowing its anchorage on the GNPs synthesized by Turkevich and Frens method. The effectiveness of GNPs-PAP complex is assessed by cytotoxicity assays on HeLa cells. MTT test showed that the complex is more effective than single PAP with an IC50 of 27.3 mg/L for GNP-PAP against an IC50 of 65 mg/L for PAP used alone. HeLa cells were incubated in the presence of a red fluorescent probe and mitochondrial activity is assessed by monitoring the decay of the fluorescence over time. No more fluorescence was observed after 72 h in cells treated with GNP-PAP, whereas after the same time, the cells treated by PAP remain fluorescent [53]. Methotrexate (MTX) has been conjugated to GNPs in order to overcome its toxicity and drug resistance [54]. The nature of the interactions between MTX and GNPs was confirmed by comparing their FTIR spectra. The NeH stretching peak of the MTX-GNPs shifts

toward a higher wavenumber (3440 cm1) in comparison to that of the free MTX (3394 cm1) suggested the possible binding of the NH2 group of free MTX with the Au surface via Au:N. Various MTXGNPs were designed: 3 nm and 20 nm. The anti-cancer effect of MTX-GNPs was evaluated with lactate dehydrogenase (LDH) in order to observe the death rate of human choriocarcinoma (JAR) cell lines and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) for the survival rate. For JAR cell lines incubated with MTX-GNPs (S), the survival rate (MTT assay) decreased significantly to 47% and the death rate (LDH assay) reached 68.9%. Furthermore, the incubated JAR cell death percentage for MTX-GNPs (L) is only 36.2%. MTX-GNPs (S) have an important cytotoxic effect on the JAR cell lines comparing with MTX-GNPs (L). Small particle size can lead to an easier internalization of the MTX-GNPs (S) in the cellular environment; moreover, the high surface area of these GNPs allows the conjugation of an important quantity of MTX. Peptides are often the ligand of choice for radioelements as well as for radioimmunotherapy, where radioelements as 131I are bound to a monoclonal antibody (Fig. 10D). The radiation is delivered not only to the antibody-bound tumour cell, but also to the neighbouring non-antibody-bound tumour cells through the ‘cross-fire’ effect. 131I (t1/2 ¼ 8.01 days) was used to label C225, a monoclonal antibody that binds specifically to the extracellular domain of the human EGFR (See part 4), in order to synthetize a theranostic agent for radio-immunotherapy, 131I-C225-GNPs-PEG [55]. The labelling reaction occurs at tyrosine and histidine residues. The cytotoxicity of 131I-C225-GNPs-PEG in A549 cells was assessed by MTT assay. Four formulations of gold nanoparticle conjugates (GNPs-PEG, C225-GNPs-PEG, 131I-C225- GNPs-PEG, 131I-C225-GNPs-PEG plus a 200-fold excess of C225, and 131I as the control) at various concentrations (ranged from 0 to 0.625 lg/mL of AuNPs) were added to the wells incubated for 2 h at 37 C, then replaced with fresh medium and incubated for another 24 h. Non-radioactive gold nanoparticles, GNPs-PEG and C225-GNPs-PEG, exhibited good biocompatibility at concentrations up to 0.625 lg/mL, while 131IC225-GNPs-PEG (ranged from 0 to 0.625 lg/mL of AuNPs, 0e185 kBq/mL of 131I) exhibited appreciable and dose-dependent cytotoxicity compared with that of its non-radioactive counterpart. At the maximal dosage (0.625 lg/mL of GNPs, 185 kBq/mL of 131 I), the viability of 131I-C225-GNPs-PEG treated A549 cells dropped to approximately 37%, while that of the blocking group (pretreated with a 200-fold excess of C225) and the control group (treated with 131I) was both higher than 82%. These findings suggested that 131I-C225-GNPs-PEG is a promising radioimmuno theranostic agent for the treatment of high EGFR-expressing tumor cells. The cyclic c[RGDfK(C)] peptide has also been used for cell targeting of radioactive 99technetium delivery with GNPs [56] (Fig. 10E). 99Technetium is used in complex with ethylene diamine diacetic acid (EDDA) and can be further reacted with a hydrazine derivative (HYNIC-GGC) to give the corresponding hepta-coordinated ligand. The cysteine in the cyclic peptide and the technetium complex will serve for gold binding. A general difficulty with radio labelling is to take into account the half-life of the isotope used (99mTc t1/2 ¼ 6 h), explaining the fast reactions for the complex preparation and gold grafting, here 35 min, with 5h30 for biological use. HYNIC GNP functionalization was calculated from the remaining peptides in solution with absorbance at 280 nm and estimated to be 1700/particle (20 nm size). For RGD functionalization, a reversed C18 HPLC method was used for quantification of the remaining peptide and found to be 270 RGD/particle. In this work 99mTc-EDDA/HYNIC-GGC-GNP-c[RGDfK(C)] and 99mTcEDDA/HYNIC-GGC-GNP were prepared to evaluate the impact of RGD targeting. The specific activity was 80e192 GBq/nmol and


introduction of the Tc complex resulted in lower amount of peptides on the surface. The Tc constructs were stable in human plasma after 24 h. The RGD variant showed 2.3 fold cellular uptake than the non RGD variant, indicating a passive uptake. In vivo IP and IV injections showed high cellular uptake in mice. IP injection resulted in higher amount of relative uptake due to the lack of stealth properties towards macrophages for these GNPs when administered IV. The photodynamic therapy of the 5-aminolevulinic acid (ALA, Fig. 10F) leads to the synthesis of protoporphyrin IX (PpIX) responsible for the production of ROS [57]. The irradiation of ALA combined to GNPs (ALA-GNPs) with laser pulses at suitable wavelength converts absorbed light into heat leading to cell damages. Two different-sized GNPs (14 nm: GNPs1 and 136 nm: GNPs2) and free ALA were studied on human cervical cell lines. After photoactivation, ROS production was increased for ALA-GNPs mixtures and especially for ALA-GNPs2. The caspase-3 activity was also evaluated for free ALA, ALA-GNPs1 and ALA-GPNs2. Enhanced caspase-3 activity induces apoptosis and cancer cell damages. After photodynamic therapy of the cell lysates, the caspase-3 activity increased for the ALA-GNPs2 compared with the groups treated with ALA-GNPs1 and free ALA. The high cytotoxic activity of ALAGNPs2 can be explained by the higher maximum absorption of ALA-GNPs2 (650 nm) compared to ALA-GNPs1 (518 nm). After light irradiation, the energy is transferred from GNPs to PpIX molecules which are responsible of ROS formation in the presence of oxygen. Matrix metalloproteinases (MMPs) enzymes are involved in remodelling of the extracellular matrix and their overexpression plays a significant role in the formation of metastases and in the process of tumor angiogenesis. MMP-2 is overexpressed in many cancerous cells such as squamous cell carcinoma SCC-7 and human colon cancer cell HT-29. Grafting of DOX on gold nanoparticles via a peptide substrate of MMP-2 will allow release of the active principle subsequent to cleavage of the substrate by MMP-2 protease [58]. GNPs were synthesized using the conventional technique of Turkevich and Frens and a thio-peptide MMP-2 substrate allowed the grafting onto GNP. The coupling between DOX and the peptide was produced by Solid Phase Peptide Synthesis technical (SPPS). Thanks to the EPR effect, the tumor cells are passively targeted by the functionalized GNPs which are internalized by endocytosis. In the cell compartment, the DOX will be release by two mechanisms: the specific cleavage of the substrate peptide by MMP-2 but also by the phenomenon of exchange of thiolated ligands. Indeed, in the intracellular compartment, high concentrations level are observed in glutathione (GSH) which leads to the exchange between the thiolated peptide and glutathione on GNPs (Fig. 10G). It is also interesting to note that with this organization DOX-Peptide-GNP, the chromophore of DOX is near to the electronic surface of the nanoparticle. This results in a fluorescence quenching, which will be abolished upon release of DOX within the tumor cell. This system combines therapeutic action and imaging by fluorescence emission property. The cytotoxic activity of these functionalized GNPs was assessed by MTT assay on cell lines HT-29, CSC-7 as well as healthy cells. Cell survival rates were 28% and 50% respectively. Healthy cells seem to be not affected by this therapeutic system. 9. Oligonucleotides Synthetic modified single stranded oligonucleotides (ONT, e.g. RNA or DNA) are commonly used to directly functionalize GNPs with the requirement of initial surface functionalization (see PEI multilayer, Fig. 8). On the other hand, ONT can be modified at either 30 or 50 ends depending on the needs by attaching alkylthiols for direct GNPs linkage. In general double stranded ONT are then prepared by reacting the ONT-functional GNPs with the

107

complementary ONT strand. ONT can be used as aptamers to recognize cells, molecules or biological targets or as simple cargos to deliver. Plasmonic scattering imaging has been used to determine the internalization mechanisms of GNPs without the requirement of dye staining [59]. 3D images were reconstructed from 8 layers using a dark-field optical sectioning microscope, and applied to nonsmall lung cancer cells (CL1-0) and HeLa cells with lateral dimensions of about 20 mm and heights about 8 mm. The method allowed time-resolved monitoring with movie production. GNPs were prepared classically with several sizes (Table 3) and coated with single stranded DNA with HS-(CH2)10-GCAGTTGATCCTTTGGATACCCTGG for internalization. This DNA recognizes mucin glycoprotein (MUC1) overexpressed in the extracellular matrix of cancer cells. The amount of particles interacting with cells was 5 fold higher for 45 nm GNP than 70 nm sized ones, and 100 nm particles were almost not interacting. 58% of the 45 nm GNP were internalized, reduced to 43% for 70 nm. DOX intercalates into DNA with a preference for region rich in CG repeats. This property has been used to prepare ONTfunctionalized GNP with increased CG repeats to load several DOX per ONT. The ONT used were also selected to correspond to the over expressed prostate-specific membrane antigen (PSMA). PC3 and LnCap are two prostate cancer cell lines, the latter expressing the PSMA, while the former don't. This difference has been exploited to design a modified ONT duplex with 7 additional extended CGA base repeats in order to load 7 DOX by intercalation [60] instead of only one in the normal PSMA duplex. In turn this aptamer, loaded onto gold nanoparticles, was used both as targeting ligand to LnCap and delivery of DOX and for computer tomography imaging. The synthetic method consisted in preparing a thiol terminated ONT sequence to be attached onto the gold particles. This material was then used to self-assemble as a duplex with the complementary ONT bearing the extra CGA units. The final duplex, rich in CG repeats was able to load an average 7.5 DOX per arm for a final 615 DOX per GNP. Finally the construct showed selective activity toward LnCap cells, compared to PC3. Inclusion of DOX in the duplex resulted in fluorescence quenching and selective targeting of the LnCap cells was assessed by restoration of the fluorescence upon release in cells of DOX from the GNP. This strategy has been adapted to GNP-based siRNA or miRNA delivery. Small interfering RNAs (siRNA) are developed to block disease-associated gene expression by interacting with messenger RNA (mRNA) while microRNAs (miRNA) primarily cleave mRNA. GNPs loaded siRNA [61] were prepared by conventional methods with dodecanethiol in a 5.1 ± 0.5 nm size. These small GNPs were then assembled in a lipid stabilized formulation L-GNP with cholesterol, DC-cholesterol and diolelyphosphatidylethanolamine (DOPE) (Fig. 8F). DOPE is used as lipid stabilizer and to help internalization and endosome escape. Cholesterol also maintains the membrane stability of the construct and also favours endocytosis. The formation of L-GNP resulted in a red shift (517e529 nm) of the SPB due to the closer interaction of the core small GNP. The L-GNP surface has a final positive charge used for siRNA interactions due to the dimethylamino group of the DC-cholesterol. These coated LGNPs showed a spherical granular shape with an average 68.4 ± 2.8 nm diameter, demonstrated by atomic force microscopy (AFM), with zeta potential at 59.4 ± 4.5 mV. Upon siRNA loading the zeta potential decreased to 32.2 ± 3.3 mV. Several siRNA were used to block the expression of various genes: GFP, VEGF, UBB and Hepatithis B Virus Surface Antigen (HBsAg). GFP overexpressing MDA-MB-435 and A549 cancer cells showed reduced GFP-base fluorescence when treated with the GNP-siGFP particles. For VEGF, PC-3 cancer cells and GNP-siVEGF were used. A549 cells were used to validate the interference in UBB gene expression with GNP-

108


siUBB. For hepathitis B, HepG2.2.15 cells were used with GNPsiHBsAg. Results indicated that these L-GNP are good candidate for DDS-based RNA delivery for applications in anti-cancer or vaccines strategies, with reduced cytotoxity when compared to classical PEI constructs and possessed good endosomal escaping properties. Two complementary single miRNA were prepared, one modified at the 30 end with isp18 linkers and a thiol group and one labelled with cyanins (Cy3 and Cy5) at the 50 end (Fig. 11A). They were heated at 60 C to form the corresponding double stranded miRNA, selected to knock down luciferase expression as a final validation test in cells. The miRNA was conjugated to citrate-capped GNPs of 13 nm diameter, with a final 10-25 miRNA per particle [62]. The remaining free GNP surfaces were coated with a short thioalkylPEG. The effective RNA loading was determined by UVeVis with a small red shift. Raman spectroscopy (SERS) showed two specific bands at 1360 and 1590 cm1. Agarose gel electrophoresis was used to confirm the difference between the naked GNPs, the miRNAGNPs and GNPs treated with bis(p-sulfonatophenyl) phenylphosphine (BP), used to increase the negative overall charge of the GNPs. The miRNA-GNPs were found stable to BP treatment using this electrophoresis method. The multiple myeloma cell line MM.1S was used for transfection studies. Internalization was confirmed by fluorescence due to the cyanin dyes. 2 nM Cy5miRNAGNPs led to an observable reduction in luciferase gene expression. miRNA-GNPs appeared more efficient than previously described siRNA-GNPs. GNPs can be used to protect photoligand in phototherapy. Moreover, passive targeting can be improved with ligand that specifically recognizes oncomarkers. Shiao and colleagues have synthesized gold nanoparticles functionalized with DOX and derivative of the porphyrin as photosensitive agent, the TMPyP4 (5,10,15,20-Tetrakis(1-methylpyridinium-4-yl)porphyrin, using an aptamer as linker. This aptamer was obtained by SELEX and shows an affinity for both DOX and TMPyP4. It was functionalized with a thiol to its 50 end for covalent bonding on GNPs. The aptamer affinity for TMPyP4 is related to its recognition sequence rich in guanine residues that form a G-quadruplex structure that fits the TMPyP4. Moreover, its structure has an additional sequence rich in CG pattern which allows the loading of DOX. After irradiation of TMPyP4, ROS are released and interact with

guanine residues of the aptamer that cause DNA brain rupture and release of DOX. It is interesting to note that the graft TMPyP4 onto GNPs enhances the ROS formation of 1.5 fold. Cytotoxicity of this multivalent complex is evaluated by MTT assays on HeLa cells. Cell viabilities in presence of TMPyP4-GNP, GNP, DOX-GNP and TMPyP4/DOX-GNP are 65%, 100%, 83% and 21% respectively. A synergy is observed with improved cytotoxic effect on cancer cells compared to the effect obtained with each biomolecule considered individually [63]. 10. Miscellaneous GNPs functionalizations Combination of EGCG and commercial GNPs has been investigated on MBT-2 murine bladder tumour cells and compared to Vero cells (African green monkey kidney cells) as normal cells [64]. The complex was analysed by SEM. A 50m:1.5 ppm EGCG:GNP ratio gave particles with broad diameter range (20e1200 nm), with zeta potential of 21 mV. The chemical of physical association of EGCG with GNP is not clearly defined. Taken separately EGCG and GNPs gave IC50 at 28.4 mM and 4.3 ppm respectively in viability tests toward MBT-2 lines whereas Vero cells were unchanged. When combined at 12.5 mM and 2 ppm, EGCG and GNPs gave a 2 fold reduction in cells number, indicating a synergistic effect. This effect was correlated with 2 fold increase of apoptosis mediated through changes in Bcl-XL, Bad, and Bax expressions and a 2 fold increase in caspase-3 and 7 expressions. In mice, the combination was administered orally, intraperitoneally (IP) and intratumour (IT). IP and IT showed good response correlated with suppression of VEGF expression. Cell penetration without membrane receptor in transfection studies involves various methods, including viral carriers, electroporation or liposomes, but with always some limitations in efficacxy. A femto second laser was used to stimulate this process with GNP and to avoid the high temperature increase observed with nano second lasers, inducing toxic side effects [65]. In this work commercial GNP were used (8 mg/ml, diameter 100 nm). The 8 min. laser treatment consisted in a central wavelength of 800 nm, a repetition rate of 1 kHz, and maximum output pulse energy of 5 mJ and bottom irradiation. Internalization was observed only for cells surrounded by GNP, showing great promises for this therapeutic strategy in skin cancers. Spherical and rod-shape particles (GNPs and GNRs respectively)

Fig. 11. (A) Principle for ONT functionalization of GNPs. (B) Principle of Pi-stacking to associate aromatic fluorescent reporters to polystyrene-coated GNP.


were prepared and functionalized with ICG [66]. GNRs were first coated with PSMA (poly(styrene-maleic acid), Fig. 11B) via carboxyl groups interactions with Au (broad SPB 950 nm). A basic treatment exposed the carboxylate groups to the surface with a final GNRs zeta potential of 10.7 mV. This treatment was also claimed to reduce the toxicity of the remaining CTAB traces. Subsequent ICG mixing resulted in ICG inclusion through pi-stacking between the phenyl groups of PSMA and the indole groups of ICG, revealed two SPB bands (720e770 nm and 830e900 nm). The number of ICG per GNR was estimated to be 28,100 based on UV analysis. Submitted to laser irradiation at 808 nm (22.5 W/cm2), the nanoparticles temperature raised to 69 C after 8 min irradiation revealing a synergistic effect for GNR-PSMA-ICG construct with almost two fold increase in singlet oxygen formation. The two photon luminescence of this material was also investigated and showed also a synergistic effect compared to ICG and GNP alone. In order to apply these particles on A549 cancer cells, an antibody directed toward EGFR was conjugated to ICG and then included in a new GNR particle in the form GNR-PSMA-ICG-AbEGFR to obtain cell recognition and internalization. Cell viability decreased to 70% after 1 day incubation and down to 45% after 4 days. GNP-PEI-ICG-AbEGFR particles were also prepared in various sizes and exhibited size-dependant A549 cells killing. The viability of A549 cells treated with ICG alone, 13 nm GNP-PEI-ICG NPs-, 50 nm GNP-PEI-ICG NPs- and 100 nm GNP-PEI-ICG NPs after 2 min irradiation (20 W/cm2) fell to approximately 83%, 34%, 20% and 8% respectively, showing a sizedependant improvement on PDT and PTT treatment compared with ICG alone. Overall these results indicated that enhancement of ICG properties can be achieved, but the inclusion strategy can still deliver free ICG sensitive to photodegradation. 11. Detection High throughput screening (HTS) is a key methodology to analyse rapidly many samples. In the context of anticancer patientoriented therapies, practical and sensitive methods must be elaborated to identify oncomarkers in patient samples. Oncomarkers such as a-fetoprotein (AFP), human chorionic gonadotrophin b (bhCG), carcinoma antigen 125 (CA125) or carcinoembryonic antigen (CEA) are present in low concentrations at the early stages of the disease and the presence of one of these markers is not specific. To address this problem, new multivalent and ultrasensitive detection systems must be developed. This can be accomplished by the use of antibodies directed towards these oncomarkers [67]. These antibodies are deposited on cell plates, followed by samples injections. The oncomarkers present in the samples bind to their respective antibodies. In a second pass, GNPs coated with the same antibodies are used to be fixed onto vials where the corresponding oncomarkers are bound (Fig. 12A). The method demonstrated high throughput stability, reproducibility and lower detection limits compared to other immunoassays, contributing to the increasing demand for patient-oriented diagnostics. Immunoassays involving capillary electrophoresis (CE) and chemiluminescence (CL) were designed to detect oncomarkers. CE allows microseparation and presents many advantages as short analysis time, high resolution, small sample consumption and simplicity of use. CL is used as detection technique due to its wide range of linearity, low background and simple apparatus. Unfortunately, coupling CE and CL displays a lack of sensitivity because of the very small amount of analyte used. Consequently, GNPs functionalized with DNAzymes and antibodies are very interesting as label for CL signal amplification. The important surface area/volume ratio allowed increasing the number of catalytic unit and thus amplifying the signal. An assay was proposed using antibodies against carbohydrate antigen 19-9 (CA19-9), one of the most

109

widespread carbohydrate oncomarker and DNAzymes with a richguanine DNA sequence (50 HS-AAAAAAGGGTTGGGCGGGATGGGT30 ) forming a G-quadruplex structure able to bind hemin molecule [68]. The G-quadruplex-hemin association shows a peroxidase-like activity and catalyses the chemiluminescent reaction between H2O2 and luminol. The DNA sequence is functionalized with a thiol at the 50 in order to establish a strong covalent binding with GNP. The use of GNP as a platform for CA19-9 antibodies and DNAzymes allowed obtaining a bigger contact surface, a decrease of background signal and consequently an improvement of the chemiluminescent signal (Fig. 12B). The performance of this CE-CL ImmunoAssay (CE-CLIA) showed a good correlation compared to conventional ELISA method and displayed a wide linearity range and a very low detection limit. Combining CL, poly-DNAzymes and multiples row detection, Zong et al. have developed an advanced sandwich assay [69]. Specific capture antibody for oncomarkers were grafted onto an epoxy-functionalized support and incubated with markers. Then, biotinylated detection antibody were added allowing the formation of an antibody/antigen/biotinylated antibody sandwich complex. After washing, GNP with poly-DNAzymes partially biotinylated complex and streptavidin were added. Biotin and streptavidin form a stable complex between the detection antibodies and nanoparticles. Finally, luminol and H2O2 yielded the generation of a chemiluminescent signal amplified by the multilayer structure of DNAzymes (Fig. 12C). A strategy was proposed to detect circulating tumour cells (CTC) based on oncomarker recognition by nanowires (NW), where the signal can be amplified with GNP bearing RNA [70] (Fig. 12D). In this work prostate cancer was used to validate the concept, and several antisense oligonucleotides (ASO) were used to found the best responding system. PCA3 gene was selected for this study, an over expressed specific prostate cancer marker found in peripheral blood and urine. The nanowires were synthetized from rhodium by templated electrodeposition in commercial alumina membranes of approximately 320 nM in cross-sectional diameter and 4e6 mM in length and coated with ASO. ASO coated GNPs were also prepared, both targeting the PC3 marker. A dual binding of the ASO-GNP and ASO-NW to PC3 gave a frequency signal modification that could be detected. Deng et al. have proposed a theranostic approach with GNPs for in-vitro detection of oncomarkers and in-vivo imaging tools [71]. The strategy exploits the quenching of fluorescent dyes due to the electronic surface effect of GNPs. A first fluorophore was used for in vitro assay (FITC, fluoresces in the visible at 520 nm after excitation at 450 nm) and a second one was used for the in vivo application with emission in the near infra-red (NIR) at 810 nm when excited at 450 nm, a strategy that overcomes the strong absorption of visible light by tissue in classical fluorescence setting. The two dyes were grafted onto the nanomaterial via a peptide substrate of matriptase, a transmembrane type II serine protease, overexpressed on the surface of many cancerous cells. Matriptase is involved in proteolysis that regulate the cellular microenvironment, the adaptation and cell survival by the cleavage of proenzymes which results in the formation of pro-oncogenic enzymes. It is an attractive target for diagnostic, prognostic and therapy because its overexpression is correlated with the metastatic spread of tumours. In the detection mode, the presence of matriptase promoted the peptide substrate linker cleavage and, on release, FITC fluorescence was restored. The second NIR probe emits NIR fluorescence when released in vivo. MIL (multiphoton induced luminescence) and CARS (anti-Stokes Raman scattering) microscopy have been combined to produce 3D imaging modalities with 300 nm lateral and 2 mm axial resolutions for the determination of GNP uptake in cells [72]. GNPs were coated with PEG5000-SH by sonication of the mixture reaction consisting

110


Fig. 12. Bioassays involving GNPs. (A) Typical “sandwich” assay with detecting entity on both GNP and the support. (B) Combination of antibodies and DNAzymes in CE-CL strategy. (C) Amplification of method (B) with streptavidin-biotin affinity. (D) Signal amplification with the combined use of ONT-coated nanowires and GNPs.

by GNPs/PEG5000-SH (1/1000, v/v). Once the functionalization step was completed, the coated GNPs were purified and concentrated by centrifugation. Computed tomography (CT) is used to diagnose cancer due to its important spatial and density resolution but still needs suitable contrast agents. Highly branched, monodispersed synthetic nanoparticles like dendrimers with entrapped gold nanoparticles (DEGNP) are good candidates for CT and can be pegylated to extend their blood circulation time. Commercially available poly(amidoamine) [PAMAM] 5th generation [G5] dendrimers allows entrapping of GNPs due to internal free spaces, making them a good nanoplatform for the synthesis of multifunctional DEGNP. After the administration of the PEGylated DEGNP [G5] into the tumour mice [73], an obvious enhancement with a significantly higher CT value was obtained that remains after 6 h post-injection when compared with CT before GNP injection.

12. Clinical trials A phase I dose escalation study was reported with GNPs coated with the recombinant tumour necrosis factor alpha (TNFa). TNFa was discovered 30 years ago but suffers from endotoxic shock and sepsis side effects with dose-limiting toxicities (DLT) being hypotension, hepatotoxicity, malaise and fatigue when administered IV. Recent studies indicated that targeting TNFa to tumour sites may greatly improve its efficiency with less toxic effects. GNPs were coated with PEG-thiol to avoid RES and TNFa was conjugated through the thioproteins found in the TNFa, the construct being called CYT-6091. CYT-6091 gave dramatic tumour reduction in TNFsensitive models. The study was conducted with 30 patients with advanced and/or metastatic, histologically documented solid organ cancers, and for which conventional treatments were no longer responding. The dose escalation and schedules were: 50, 100, 150, 250, 300, 400, 500 and 600 mg/m2, in two doses (day 1 and 15)


constituting one course and patient monitoring being made after 48 h treatment. The treatment was well tolerated and there were no DLTs. The pharmacokinetic analysis indicated that the half-life of CYT-6091 was 5 fold longer with GNP (~130 min vs. ~28 min). Electron micrographs of the patient biopsies 24-h treatment showed GNP trafficking in tumours, with fenestrations of 200e400 nm in size. The 27 nm sized CYT-6091 allowed thus tumour targeting via the EPR effect. Importantly, naked GNP bound after 24 h while CYT-6091 was found bounded after 30 min, indicating the critical impact of CYT in GNP biodistribution.

[21]

[22] [23] [24] [25] [26]

References

[27]

[1] a) S. Jain, D.G. Hirst, J.M. O'Sullivan, Br. J. Radiol. 85 (2012) 101; b) E.C. Dreaden, A.M. Alkilany, X.H. Huang, C.J. Murphy, M.A. El-Sayed, Chem. Soc. Rev. 41 (2012) 2740; c) A.M. Alkilany, L.B. Thompson, S.P. Boulos, P.N. Sisco, C.J. Murphy, Adv. Drug Deliv. Rev. 64 (2012) 190. [2] a) M.-C. Daniel, D. Astruc, Chem. Rev. 104 (2004) 293; rez-Juste, I. Pastoriza-Santos, L.M. Liz-Marza n, P. Mulvaney, Chem. Rev. b) J. Pe 249 (2005) 1870; c) P. Jain, X. Huang, I. El-Sayed, M. El-Sayed, Plasmonics 2 (2007) 107; d) K.L. Kelly, E. Coronado, L.L. Zhao, G.C. Schatz, J. Phys. Chem. B 107 (2003) 668; e) S. Link, C. Burda, M.B. Mohamed, B. Nikoobakht, M.A. El-Sayed, J. Phys. Chem. A 103 (1999) 1165; f) S. Link, C. Burda, B. Nikoobakht, M.A. El-Sayed, Chem. Phys. Lett. 315 (1999) 12. [3] a) B. Nikoobakht, M.A. El-Sayed, Chem. Mater. 15 (2003) 1957; n, b) N. Malikova, I. Pastoriza-Santos, M. Schierhorn, N.A. Kotov, L.M. Liz-Marza Langmuir 18 (2002) 3694; c) S.S. Shankar, A. Rai, B. Ankamwar, A. Singh, A. Ahmad, M. Sastry, Nat. Mater. 3 (2004) 482; d) Y. Sun, Y. Xia, Science 298 (2002) 2176; e) J. Zhang, M.R. Langille, M.L. Personick, K. Zhang, S. Li, C.A. Mirkin, J. Am. Chem. Soc. 132 (2010) 14012; f) T.K. Sau, C.J. Murphy, J. Am. Chem. Soc. 126 (2004) 8648. [4] a) A.V. Simakin, V.V. Voronov, G.A. Shafeev, R. Brayner, F. Bozon-Verduraz, Chem. Phys. Lett. 348 (2001) 182; b) Y. Ying, S.-S. Chang, C.-L. Lee, C.R.C. Wang, J. Phys. Chem. B 101 (1997) 6661; c) M. Tsuji, N. Miyamae, M. Hashimoto, M. Nishio, S. Hikino, N. Ishigami, I. Tanaka, Colloids Surf. A: Physicochem. Eng. Asp. 302 (2007) 587; d) S. Koeppl, C. Solenthaler, W. Caseri, R. Spolenak, J. Nanomater. 2011 (2011) 1. [5] a) J. Turkevich, P.C. Stevenson, J. Hillier, Discuss. Faraday Soc. 11 (1951) 55; b) G. Frens, Kolloid-Z.u.Z. Polym. 250 (1972) 736. [6] M. Brust, M. Walker, D. Bethell, D.J. Schiffrin, R. Whyman, J. Chem. Soc. Chem. Comm. (1994) 801. [7] M.N. Martin, J.I. Basham, P. Chando, S.-K. Eah, Langmuir 26 (2010) 7410. [8] a) J.W. Slot, H.J. Geuze, Eur. J. Cell. Biol. 38 (1985) 87; , T. Napporn, J. Rousseau, K. Servat, K.B. Kokoh, Elecb) A. Habrioux, S. Hebie trocatal 2 (2011) 279. [9] N.R. Jana, L. Gearheart, C.J. Murphy, Chem. Comm. 7 (2001) 617. [10] V.K. LaMer, R.H. Dinegar, Theory, J. Am. Chem. Soc. 72 (1950) 4847. [11] a) Y. Xia, Y. Xiong, B. Lim, S.E. Skrabalak, Angew. Chem. Int. Ed. 48 (2009) 1; b) A.R. Tao, S. Habas, P.D. Yang, Small 4 (2008) 310. [12] N. Semagina, L. Kiwi-Minsker, Catal. Rev. 51 (2009) 147. [13] a) T.K. Sau, C.J. Murphy, Langmuir 20 (2004) 6414; , K.B. Kokoh, K. Servat, T. Napporn, Gold Bull. 46 (2013) 311. b) S. Hebie [14] a) Y.Y. Yu, S.S. Chang, C.L. Lee, C.R.C. Wang, J. Phys. Chem. B 101 (1997) 6661; b) S.S. Chang, C.L. Lee, W.C. Lai, C.R.C. Wang, Abstr. Pap. Am. Chem. Soc. 215 (1998) U441; c) S.X. Huang, H.Y. Ma, X.K. Zhang, F.F. Yong, X.L. Feng, W. Pan, X.N. Wang, Y. Wang, S.H. Chen, J. Phys. Chem. B 109 (2005) 19823. [15] a) C. Tollan, J. Echeberria, R. Marcilla, J. Pomposo, D. Mecerreyes, J. Nanoparticle Res. 11 (2009) 1241; , L. Cornu, T.W. Napporn, J. Rousseau, B.K. Kokoh, J. Phys. Chem. C b) S. Hebie 117 (2013) 9872. [16] J. Sun, M. Guan, T. Shang, C. Gao, Z. Xu, J. Zhu, Cryst. Growth Des. 8 (2008) 906. [17] H.-L. Wu, C.-H. Kuo, M.H. Huang, Langmuir 26 (2010) 12307. [18] a) C.-J. Huang, Y.-H. Wang, P.-H. Chiu, M.-C. Shih, T.-H. Meen, Mater. Lett. 60 (2006) 1896; b) H.Y. Ma, B.S. Yin, S.Y. Wang, Y.L. Jiao, W. Pan, S.X. Huang, S.H. Chen, F.J. Meng, Chemphyschem 5 (2004) 68; c) L.M. Shen, J.L. Yao, R.A. Gu, Spectrosc. Spectr. Anal. 25 (2005) 1998. [19] G. Han, C.C. You, B.J. Kim, R.S. Turingan, N.S. Forbes, C.T. Martin, V.M. Rotello, Angew. Chem. Int. Ed. 45 (2006) 3165. [20] a) S. Dadras, P. Jafarkhani, M.J. Torkamany, J. Sabbaghzadeh, J. Phys. D. Appl. Phys. 42 (2009) 025405; b) A.V. Kabashin, M. Meunier, J. Appl. Phys. 94 (2003) 7941; c) V. Amendola, G.A. Rizzi, S. Polizzi, M. Meneghetti, J. Phys. Chem. B 109 (2005) 23125; d) A.V. Kabashin, M. Meunier, C. Kingston, J.H.T. Luong, J. Phys. Chem. B 107 (2003) 4527;

[28] [29] [30] [31]

[32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52]

[53] [54] [55] [56]

[57] [58] [59] [60] [61] [62] [63] [64]

111

e) V. Amendola, S. Polizzi, M. Meneghetti, J. Phys. Chem. B 110 (2006) 7232; f) F. Mafune, J.-Y. Kohno, Y. Takeda, T. Kondow, H. Sawabe, J. Phys. Chem. B 105 (2001) 5114. a) J.L. Marignier, J. Belloni, M.O. Delcourt, J.P. Chevalier, Nature 317 (1985) 344; b) J. Attia, S. Remita, S. Jonic, E. Lacaze, M.C. Faure, E. Larquet, A. Goldmann, Langmuir 23 (2007) 9523; c) W. Abidi, H. Remita, Recent Pat. Eng. 4 (2010) 170. R. Bhat, V.G. Sharanabasava, R. Deshpande, U. Shetti, G. Sanjeev, A. Venkataraman, J. Photochem. Photobiol. B: Biol. 125 (2013) 63. S. Manju, K. Sreenivasan, J. Colloid. Interface Sci. 36 (2012) 8144. J.C. Polf, L.F. Bronk, W.H.P. Driessen, W. Arap, R. Pasqualini, M. Gillin, Appl. Phys. Lett. 98 (2011) 193702. R.K. Ambasta, A. Sharma, P. Kumar, Vasc. Cell. 3 (2011) 26. V.S. Marangoni, I.M. Paino, V. Zucolotto, Colloids Surf. B: Biointerfaces 112 (2013) 380. D.J. Slamon, G.M. Clark, S.G. Wong, W.J. Levin, A. Ullrich, W.L. McGuire, Science 235 (1987) 177. S.D. Weitman, R.H. Lark, L.R. Coney, D.W. Fort, V. Frasca, V.R. Zurawski Jr., B.A. Kamen, Cancer Res. 52 (1992) 3396. S. Bhattacharyya, R. Bhattacharya, S. Curley, M.A. McNiven, P. Mukherjee, Proc. Nat. Acad. Sci. 107 (2010) 14541. M.A.K. Abdelhalim, Lipids Health Dis. 10 (2011) 233. R.R. Arvizo, O.R. Miranda, D.F. Moyano, C. Walden, K.G.R. Bhattacharya, J.D. Robertson, V.M. Rotello, J.M. Reid, P. Mukherjee, PLoS One 6 (2011) e24374. T. Thomas, R.J. Mascarenhas, O.J. D'Souza, P. Martis, J. Dalhalle, B.E.K. Swamy, J. Colloid Interface Sci. 404 (2013) 223. T. Lund, M.F. Callaghan, P. Williams, M. Turmaine, C. Bachmann, T. Rademacher, I.M. Roitt, R. Bayford, Biomaterials 32 (2011) 9776. J.D. Trono, K. Mizuno, N. Yusa, T. Matsukawa, K. Yokoyama, M. Uesaka, J. Radiat. Res. 52 (2011) 103. F. Wang, Y.-C. Wang, S. Dou, M.-H. Xiong, T.-M. Sun, J. Wang, ACS Nano 5 (2011) 3679. Y.-J. Gu, J. Cheng, C.W.-Y. Man, W.-T. Wong, S.H. Cheng, Nanomedicine 8 (2012) 204. Y. Lee, S.H. Lee, J.S. Kim, A. Maruyama, X. Chen, T.G. Park, J. Control. Release 155 (2011) 3. S. Guo, Y. Huang, Q. Jiang, Y. Sun, L. Deng, Z. Liang, Q. Du, J. Xing, Y. Zhao, P.C. Wang, A. Dong, X.-J. Liang, ACS Nano 4 (2010) 5505. A. Agarwal, M.A. Mackey, M.A. El-Sayed, R.V. Bellamkonda, ACS Nano 5 (2011) 4919. C.K. Adokoh, S. Quan, M. Hitt, J. Darkwa, P. Kumar, R. Narain, Biomacromolecules 15 (2014) 3802. Y. Song, Z. Wang, L. Li, W. Shi, X. Li, H. Ma, Chem. Comm. 50 (2014) 15696. V. Venkatpurwar, A. Shiras, V. Pokharkar, Int. J. Pharm. 409 (2011) 314. D.N. Heo, D.H. Yang, H.-J. Moon, J.B. Lee, M.S. Bae, S.C. Lee, W.J. Lee, I.-C. Sun, I.K. Kwon, Biomaterials 33 (2012) 856. W.I. Choi, J.-Y. Kim, C. Kang, C.C. Byeon, Y.H. Kim, G. Tae, ACS Nano 5 (2011) 1995. X. Li, H. Zhou, L. Yang, G. Du, A.S. Pai-Panandiker, X. Huang, B. Yan, Biomaterials 32 (2011) 2540. R. Kannan, A. Zambre, N. Chanda, R. Kulkarni, R. Shukla, K. Katti, A. Upendran, C. Cutler, E. Boote, K.V. Katti, WIREs Nanomed. Nanobiotechnol 4 (2012) 42. A. Kumar, H. Ma, X. Zhang, K. Huang, S. Jin, J. Liu, T. Wei, W. Cao, G. Zou, X.J. Liang, Biomaterials 33 (2012) 1180. X. Huang, X. Peng, Y. Wang, Y. Wang, D.M. Shin, M.A. El-Sayed, S. Nie, ACS Nano 4 (2010) 5887. X. Zhang, H. Chibli, R. Mielke, J. Nadeau, Bioconjugate Chem. 22 (2011) 235. Z. Zhang, J. Jia, Y. Lai, Y. Ma, J. Weng, L. Sun, Bioorg. Med. Chem. 18 (2010) 5528. E.S. Day, L.R. Bickford, J.H. Slater, N.S. Riggall, R.A. Drezek, J.L. West, Int. J. Nanomedicine 5 (2010) 445. J.A. Khan, R.A. Kudgus, A. Szabolcs, S. Dutta, E. Wang, S. Cao, G.L. Curran, V. Shah, S. Curley, D. Mukhopadhyay, J.D. Robertson, R. Bhattacharya, P. Mukherjee, PLoS One 6 (2011) e20347. W.H. Chen, J.X. Chen, H. Cheng, C.S. Chen, J. Yang, X.D. Xu, Y. Wang, R.X. Zhuo, X.Z. Zhang, Chem. Comm. 49 (2014) 6403. N.T.T. Tran, T.-H. Wang, C.-Y. Lin, Y. Tai, Biochem. Eng. J. 78 (2013) 175. H.-W. Kao, Y.-Y. Lin, C.-C. Chen, K.-H. Chi, D.-C. Tien, C.-C. Hsia, M.-H. Lin, H.E. Wang, Bioorg. Med. Chem. Lett. 23 (2013) 3180. E. Morales-Avila, G. Ferro-Flores, B.E. Ocampo-García, L.M. De Leon-Rodríguez, C.L. Santos-Cuevas, R. García-Becerra, L.A. Medina, L. Gomez-Olivan, Bioconjugate Chem. 22 (2011) 913. n, C. Go mez, J. Pharm. Sci. 102 M. Benito, V. martín, M.D. Blanco, J.M. Teijo (2013) 2760. W. Chen, X. Xu, H. Jia, Q. Lei, G. Luo, S. Cheng, R. Zhuo, X. Zhang, Biomaterials 34 (2013) 8798. S.-H. Wang, C.-W. Lee, A. Chiou, P.-K. Wei, J. Nanobiotechnology 8 (2010) 33. D. Kim, Y.Y. Jeong, S. Jon, ACS Nano 4 (2010) 3689. W.H. Kong, K.H. Bae, S.D. Jo, J.S. Kim, T.G. Park, Pharm. Res. 29 (2012) 362. E. Crew, S. Rahman, A. Razzak-Jaffar, D.K. Mott, M. Kamundi, G. Yu, N. Tchah, J. Lee, M. Bellavia, C.-J. Zhong, Anal. Chem. 84 (2012) 26. Y.S. Shiao, H.H. Chiu, P.H. Wu, Y.F. Huang, ACS Appl. Mater. Interfaces 6 (2014) 21832. D.-S. Hsieh, H. Wang, S.-W. Tan, Y.-H. Huang, C.-Y. Tsai, M.-K. Yeh, C.-J. Wu,

112


Biomaterials 32 (2011) 7633. Boulais, R. Lachaine, J.-J. Lebrun, M. Meunier, [65] J. Baumgart, L. Humbert, E. Biomaterials 33 (2012) 2345. [66] W.-S. Kuo, Y.-T. Chang, K.-C. Cho, K.-C. Chiu, C.-H. Lien, C.S. Yeh, S.-J. Chen, Biomaterials 33 (2012) 3270. [67] C. Zong, J. Wu, C. Wang, H. Ju, F. Yan, Anal. Chem. 84 (2012) 2410. [68] M. Shi, S. Zhao, Y. Huang, L. Zhao, Y.M. Liu, Talanta 124 (2014) 14. [69] C. Zong, J. Wu, J. Xu, H. Ju, F. Yan, Biosens. Bioelectron. 43 (2013) 372.

[70] J.A. Sioss, R. Bhiladvala, W. Pan, M. Li, S. Patrick, P. Xin, S.L. Dean, C.D. Keating, T.S. Mayer, G.A. Clawson, Nanomedicine 8 (2012) 1017. [71] D. Deng, D. Zhang, Y. Li, S. Achilefu, Y. Gu, Biosens. Bioelectron. 49 (2013) 216. [72] G. Rago, B. Bauer, F. Svedberg, L. Gunnarsson, M.B. Ericson, M. Bonn, A. Enejder, J. Phys. Chem. B 115 (2011) 5008. [73] C. Peng, L. Zheng, Q. Chen, M. Shen, R. Guo, H. Wang, X. Cao, G. Zhang, X. Shi, Biomaterials 33 (2012) 1107.

Surface chemistry of gold nanoparticles mediates their exocytosis in macrophages.

Surface chemistry of thiomalic acid adsorption on planar gold and gold nanoparticles.

"Click" chemistry mildly stabilizes bifunctional gold nanoparticles for sensing and catalysis.

Gold nanoparticles for photoacoustic imaging.

Selective colorimetric NO(g) detection based on the use of modified gold nanoparticles using click chemistry.

Gold nanoparticles for in vivo cell tracking.

Gold nanoparticles for photothermally controlled drug release.

Gold nanoparticles for nucleic acid delivery.

Gold Nanoparticles for In Vitro Diagnostics.

Radiofrequency heating pathways for gold nanoparticles.

Gold nanoparticles and vaccine development.

Chemistry. Tracking the merry dance of nanoparticles.

Monolayer-capped gold nanoparticles for disease detection from breath.

Colorimetric aptasensor using unmodified gold nanoparticles for homogeneous multiplex detection.

Gold nanoparticles enhanced electroporation for mammalian cell transfection.

Strategies for the biofunctionalization of gold and iron oxide nanoparticles.

Liposomes as nanoreactors for the photochemical synthesis of gold nanoparticles.

Peptide-coated gold nanoparticles for modulation of angiogenesis in vivo.

Highlights from the latest articles in gold nanoparticles for theranostics.

Metallamacrocycle-modified gold nanoparticles: a new pathway for surface functionalization.

Silver and gold nanoparticles for sensor and antibacterial applications.

Triphenylarsonium-functionalised gold nanoparticles: potential nanocarriers for intracellular therapeutics.

Thiol-modified gold nanoparticles for the inhibition of Mycobacterium smegmatis.

Gold nanoparticles: testbeds for engineered protein-particle interactions.