Nanodiamond-mediated drug delivery and imaging: challenges and opportunities.

Review

Nanodiamond-mediated drug delivery and imaging: challenges and opportunities 1.

Introduction

2.

Background on nanodiamond particles

3.

Imaging capabilities of nanodiamonds

Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Selcuk Universitesi on 12/24/14 For personal use only.

4.

Biocompatibility of nanodiamonds

5.

NDs as therapeutic carriers

6.

Demonstrations of ND-mediated drug delivery

7.

Conclusion

8.

Expert opinion

V Vaijayanthimala, Dong Keun Lee, Sue V Kim, Albert Yen, Nathaniel Tsai, Dean Ho, Huan-Cheng Chang & Olga Shenderova† †

International Technology Center, Ada´mas Nanotechnologies, Inc., Raleigh, NC, USA

Introduction: The field of nanoparticle-based therapeutic systems is rapidly expanding encompassing a wide variety of practices ranging from detection to diagnosis to treatment. Recently a great potential of nanodiamond (ND) particles as a multimodal imaging/therapy platform has been demonstrated. Areas covered: This review describes a unique set of properties of ND particles attractive for drug delivery and imaging applications and highlights the most recent ND-based multimodal imaging/therapy approaches and related biocompatibility studies. The spectrum of major advancements includes marked improvements in tumor treatment efficacy and safety based on integration of ND with doxorubicin (DOX). Recent progress of ND-mediated drug delivery in orthopedic, dental and ophthalmic applications is also discussed. Expert opinion: ND particles possess a unique set of properties attractive for drug delivery applications, including exceptional biocompatibility, large carrier capacity and versatile surface chemistry properties, which enhance drug binding and provide sustainable drug release. Other unique attributes of NDs embrace bright stable fluorescence based on crystallographic defects. A roadmap toward a clinical translation comprises identification of NDtherapeutic compounds that display marked improvements over clinical standards with respects to efficacy, safety and cost. Keywords: cancer, drug delivery, imaging, nanodiamond, photoluminescence, targeting Expert Opin. Drug Deliv. [Early Online]

1.

Introduction

Criteria for selection of drug delivery platforms providing high drug efficacy while decreasing side effects include the timing of drug administration, dosages and drug release patterns [1,2]. Advanced drug delivery systems demonstrate improved bioavailability by preventing premature degradation, enhancing uptake, maintaining drug concentrations within the therapeutic window by controlling the drug release rate and reducing side effects by targeted drug delivery [3]. A range of reports has shown that through the use of nanoparticle platforms for drug delivery, existing chemotherapeutic efficacy can be improved while side effects may be decreased [1-3]. Many benefits that are offered by nanotechnology for drug delivery, such as improved circulation half-life, enhanced permeation and retention (EPR), and reduced toxicity, are also advantageous for imaging. Over the last decade, there has been a growing trend toward the expansion of nanotechnology-based contrast agents for biological and medical applications [4]. In general, it is believed that these agents with multiple functionalities will improve medical diagnostics through enhanced contrast specificity and efficiency, as well as improved therapeutic outcomes [5]. More specifically, the high fidelity biocompatible imaging of whole living organisms may be enabled through nanomaterial-mediated

10.1517/17425247.2015.992412 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted

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Physical and chemical properties, biocompatablity and low toxicity of nanodiamonds open up bright perspectives for using them as theranostic agents. The surface structure of NDs allows electrostatically mediated drug uploading as well as conjugation with variety of ligands and antibodies facilitating targeted drug delivery. The use of NDs as delivery agents helps in the interaction with target cells and optimization of the drug complex penetration inside the cells, for controlled drug release and delivery of water-insoluble drugs, among others. Integration of DOX with NDs provides a highly efficient method of treating drug-resistant tumors. NDs not only improve the circulatory lifetime of DOX but also allow lethal doses of the drug DOX to be administered systemically while reducing toxicity. Nanodiamonds containing color centers possess bright fluorescence without photobleaching and blinking, making NDs ideal bioprobes for molecular imaging and cell labeling. ND spectroscopic properties and the methods to adapt them for imaging and sensing applications are still under investigation. New methods of bioimaging based on NDs containing color centers include background free imaging based on modulation of ND fluorescence by magnetic field, multiphoton excited fluorescence, energy transfer as well as fluorescence lifetime analysis and imaging.

This box summarizes key points contained in the article.

platforms [6]. Imaging has helped to advance human health globally, thereby supporting and improving various methods of drug delivery. While the nanotechnology field has made significant strides toward realizing drug delivery and medical imaging, basic research and clinical translation represent two distinct domains with sometimes starkly different requirements for success. As the field of nanotechnology related to medicine comes to fruition, the importance of demonstrating enhanced therapeutic and imaging capabilities that are uniquely mediated by specific nanomaterial platforms over unmodified clinical standards or other nanoparticles in development will become increasingly apparent. The process of achieving scalable production, passing regulation and demonstrating substantial patient benefit toward eventual approval of a new drug, therapeutic or imaging can be prohibitively expensive. As such, identifying nanotechnology-enabled imaging agents that are best suited for specific indications while addressing all the aforementioned attributes will largely define the widespread acceptance of nano-enabled clinical therapy and imaging. In this review, the accession of these issues through a variety of studies with nanodiamonds (NDs) is discussed. Intrinsic stable fluorescence of NDs together with the excellent biocompatibility and versatile surface chemistry for attaching 2

various biomolecules, including imaging contrast agents, makes ND a potential translationally significant candidate for simultaneous imaging and therapeutic applications [1,7-10].

2.

Background on nanodiamond particles

Despite the fact that ND particles were discovered > 50 years ago and experienced mass production in the early 80s [11], for a long time they were in the shadow of their more famous sp2 carbon cousins. Two recent major breakthroughs, the production of individual ND particles 4 -- 5 nm in size [12] and ND particles containing nitrogen-vacancy (NV) color centers exhibiting stable luminescence and unique spin properties [9,13] have brought ND particles to the forefront of materials research and applications [7]. ND particles are produced by detonation of carbon-containing explosives (so-called detonation nanodiamonds [DNDs], Figure 1) or by grinding microdiamond powders manufactured by static high-pressure, high-temperature (HPHT) synthesis in special presses. These two classes of ND particles have different structure and, correspondingly, different niche applications. Major differences between DND and HPHT ND in terms of applications are the size of primary particles (monocrystallites) and the state and content of nitrogen impurities in the core of the ND particles. Advantages of DND include the few nanometers size of primary particles (Figure 1A) and production on an industrial scale in tons quantities annually [14]. As produced DNDs form tight unbreakable by sonication aggregates. Based on recent advances in DND deaggregation [12], colloidal solutions of primary ND particles 4 -- 5 nm in diameter have become available on the market. Monocrystalline ND particles obtained by processing HPHT synthetic diamond currently are commercially available with the smallest average particle size around 20 nm; these particles exhibit blocky shapes. The availability of individual 4 -- 5 nm ND particles produced by detonation of explosives has opened broad prospective applications of NDs in drug delivery. Indeed, the specific surface area of ND particles with 4 and 30 nm in diameter are 428 and 57 m2/g, respectively, making a noticeable 7 difference in the adsorption and load-carrying capacity of the nanoparticles. HPHT NDs contain N as a natural impurity in the form of substitutional nitrogen (Ns) with concentration of up to 300 ppm (for HPHT ND type Ib). ND produced from natural diamond type Ia (Ns concentration up to 3000 ppm) is also available. High-energy particle irradiation of type Ib ND followed by annealing causes formation of NV centers with red emission [15], while ND from diamond Ia demonstrates green luminescence originating from formation of NV-N centers after irradiation and annealing. Up to now, NDs synthesized from explosives were not among the preferred candidates for imaging applications based on NV centers. While DND contains up to 2 -- 3 wt% of N, N conglomerates in DND are optically inactive [16].

Expert Opin. Drug Deliv. (2014) 12(6)

Nanodiamond-mediated drug delivery and imaging: challenges and opportunities

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[011]

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3 nm N subst NV center N conglomerate

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Figure 1. (A) HRTEM of detonation nanodiamond particles demonstrating highly ordered diamond core. The current HRTEM image shows a faceted particle, whereas most DNDs have a rounded shape. The inset is a fast Fourier transform of the micrograph, which confirms that this nanodiamond has a highly ordered diamond core. (B) A schematic model illustrating the structure of a single ~ 5 nm nanodiamond particle after oxidative purification [7].The diamond core is covered by a layer of surface functional groups and by chains and patches of graphitic sp2 carbon (shown in black). The majority of surface atoms are terminated with oxygen-containing groups (shown in red). Some hydrocarbon chains (shown in green) and hydrogen terminations are also present. Figure 1A is reprinted with permission from [90] 2011 American Chemical Society. Figure 1B is courtesy of Mochalin V, Drexel University. DNDs: Detonation nanodiamonds.

These two major breakthroughs, the production of individual ND particles 4 -- 5 nm in size and ND particles containing impurity defects exhibiting stable luminescence and optically modulated spin properties useful for quantum sensors applications [17], are related to ND particles synthesized by different techniques, detonation of explosives and grinding of HPHT diamond, correspondingly. Thus, these two important characteristics are not currently available through a single synthesis route. Interestingly, Si-V luminescent color centers were detected recently in ~ 2 nm meteoritic NDs [18]. Synthesis of NDs a few nanometers in size with specific color centers remain an important goal. Possibly, new methods of ND particle synthesis are needed to address this goal. 3.

Imaging capabilities of nanodiamonds

Fluorescence imaging is a common method for monitoring cellular interactions and dynamics at the single-molecule level. An ideal fluorescent nanoparticle should have perfect photostability and be biocompatible. Organic dye molecules have long been used in biolabeling applications, but they suffer from photobleaching, which precludes tracking over extended times. Quantum dots (QDs), on the other hand, can overcome this problem by providing better photostability and higher brightness. However, blinking, degradation and toxicity of QDs provide the motivation for examining alternative fluorescent probes. ND, a new member of the nanocarbon family [7], can contain high-density ensembles of crystallographic defects as photostable fluorophores and holds great potential to overcome the aforementioned limitations. ND particles serve as a platform for several imaging capabilities. In addition to the most well-known fluorescence properties of NDs, characteristic Raman signals of pristine NDs and

photoacoustic (PA) signals from extensively radiationdamaged NDs can also be utilized for imaging purposes [19]. Moreover, ND particles after proper surface modification can be externally labeled with molecular contrast agents for fluorescence imaging, MRI, and positron emission tomography (PET). Figure 2A summarizes the imaging modalities that have been developed to detect NDs in vitro and in vivo. Imaging based on light scattering by nanodiamonds

3.1

Diamond has the highest refractive index of all dielectric materials, n = 2.42 [20]. The index is about twice as large as that of the intracellular medium and therefore illumination of NDs in cells can yield a higher backscattered light intensity than the corresponding illumination of cellular compartments. A study by Smith et al. [21] showed that the light scattering signal of 55-nm NDs can be up to 300-fold brighter than that of the cell organelles of similar size. Raman scattering is a noninvasive technique and thus can be used for detection and imaging of NDs in biological specimens. For NDs in the size range of ~ 50 nm, they can be readily detected by Raman scattering from the diamond matrix with a sharp and narrow peak at 1332 cm-1, as demonstrated by Perevedentseva et al. [22] in the investigation of lysozymeconjugated NDs interacting with Escherichia coli. Similar studies using ND as a marker revealed the location of growth-hormone receptors in lung cancer cells [23]. Imaging utilizing crystallographic defects in nanodiamonds

3.2

The vacancy-related defects in diamond have been intensively studied over the past few decades [24]. Vacancies can be


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Crystallographic defects Photoluminescence imaging Cathodoluminescence imaging Photoacoustic imaging

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Nano diamond

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ing er ng att eri sc catt ing s pp ht a Lig igh yle n m Ra ama R

E Ma Fluo xter gn res nal Po etic cen lab sitr on reso ce im els em nan ag iss ce ing ion im im agin ag ing g

A.

ND-NV core

Figure 2. (A) Nanodiamond-based bioimaging modalities; (B) structure of an NV center in diamond; (C) fluorescent ND containing NV centers covered with a porous silica shell; (D) merged bright-field and fluorescence image of a live cell (HeLa cell) labeled with fluorescent NDs by endocytosis; (E) in vivo imaging of a worm (C. elegans) fed with fluorescent NDs. ND: Nanodiamond; NV: Nitrogen-vacancy.

produced by irradiating with high-energy particle beams such as electrons, protons, neutrons, helium ions or g-rays. Depending on the type of diamond used, annealing at high temperature (600 C and above) results in the movement of vacancies toward nitrogen atoms and formation of NV, H3 or N3 color centers. Diamonds with any of these color centers are collectively referred to as fluorescent NDs (FNDs) [25] and used for photoluminescence (PL) and cathodoluminescence (CL) imaging [26]. For NDs subjected to extensive irradiation without annealing, which results in amorphization of the diamond lattice and leads to high light absorbance in the infrared, they are denoted as INDs and used for PA imaging [27]. Photoluminescence imaging NDs containing negatively charged NV- color centers have caught on as an attractive alternative with stable in time brightness, which does not photobleach or photoblink [28]. Upon excitation with green--yellow light, the NV-center emits bright red fluorescence at ~ 700 nm with a quantum yield close to 1 in bulk diamond. Moreover, surface modification of NDs and solution pH does not affect its fluorescence properties [29] since the NV centers are shielded by the crystal lattice. The emission from NV- defects peaks in the red and near-infrared (NIR) spectral region, representing a spectral window of low absorption attractive for biological labeling due to greater penetration of light in surrounding tissue. The first report of the use of FNDs for biological labeling appeared in 2005 [25], demonstrating that FNDs containing 3.2.1

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NV centers can be spontaneously internalized by cells and have very low cell toxicity. Subsequent studies revealed the spontaneous internalization and efficient detection of FNDs in cells with confocal microscopy [28-30]. Additionally, improved intracellular contrast can be achieved by time-gated imaging taking advantage of the long fluorescence lifetime (~ 20 ns) of FND, in comparison to the short lifetime (~ 3 ns) of cell and tissue autofluorescence [29]. The perfect photostability of FNDs enabled super-resolution imaging by stimulated emission depletion (STED) [31]. Using this technique, Tzeng et al. [32] were able to distinguish single FND particles and aggregates in cells with subdiffraction resolution of ~ 40 nm, indicating an unprecedented level of insight that can be achieved. To demonstrate the feasibility of multiphoton imaging, Hui et al. [33] used a femtosecond infrared laser to detect the presence of single FND particles (~ 40 nm) in cells. The technique is advantageous in having longer penetration depths of photons in tissues of living organisms. It excites fluorophore in the focal spot reducing cell autofluorescence and providing better image contrast. Moreover, longer excitation wavelength reduces photodamage to cells. A major hurdle associated with in vivo imaging is the unavoidable autofluorescence derived from endogenous molecules. Igarashi et al. [34] improved the image contrast of FNDs in vitro as well as in vivo based on the spin property of the NV-- center. The authors acquired wide-field fluorescence images with and without microwave irradiation in resonance with the crystal-field splitting (2.87 GHz) of the ground-state spin, and then performed subtraction between these two




images pixel by pixel. As the alternative microwave irradiation modulated only the fluorescence intensity of the NV- center, the operation effectively removed background autofluorescence signals and significantly improved the image contrast. Similarly, Hegyi and Yablonovitch [35] applied the optically detected magnetic resonance technique to image FNDs in tissue in the field-free region using an amplitude-modulated microwave source. More recently, Sarkar et al. [36] utlized a modulated external magnetic field to achieve contrast enhancement of FNDs in vivo. The magnetic field mixes the spin levels at the ground state, resulting in modulation of the FND fluorescence. As a demonstration of the potential translational relevance of the work, where unique improvements to imaging efficiency were observed, this technique improved the image contrast by nearly two orders of magnitude, allowing for wide-field imaging of FNDs in sentinel lymph nodes of mice. Cathodoluminescence imaging A new achievement in ND-based bioimaging includes correlative light and electron microscopy (CLEM) based on the multicolor CL from FNDs. The NDs contain spectrally distinct color centers and defects (such as dislocation, NV and H3) that are stable under prolonged electron-beam exposure [37,38]. CL occurs when a high-energy electron strikes a luminescent material and produces photons emitted at a characteristic wavelength. Although CL imaging can provide a spatial resolution better than 5 nm, this method is not commonly used for imaging of biological specimens due to the limitation of low fluorescence intensity and rapid sample degradation. Nawa et al. [39] have recently demonstrated twocolor images of green and red FNDs in living HeLa cells using a CLEM microscope with a spatial resolution of 150 nm, which is essentially limited by the size of the particles. As this new method of imaging can reveal structural details with excellent spatial resolution in living biological systems, the use of NDs for ultra-high resolution CL imaging in combination with transmission electron microscopy (TEM) may significantly advance certain fields such as pathology, where improved abilities to spatially differentiate within tissues or even cells may improve the clinician’s diagnostic sensitivity. 3.2.2

Photoacoustic imaging In PA imaging, the specimen is generally lit up with short and focused laser pulse, so that the subsequent absorption of laser energy results in specimen heating and generation of ultrasonic waves. The resultant emission is detected with a transducer and finally reconstructed to produce a threedimensional image of the test specimen. INDs, prepared with extensive ion irradiation, have recently been shown to be an ideal optical contrast agent for PA imaging in deep biological tissues due to their low toxicity and high optical absorbance. Zhang et al. [40] injected INDs into rodents and were able to clearly image the particles located 3 mm below the skin surface with a PA signal enhancement of > 500% using 3.2.3

an 820-nm laser. High-power laser illumination did not degrade the PA signals from INDs, while the opposite was observed with gold nanorods, demonstrating a potentially unique application of NDs as multimodal imaging contrast agents for biomedical applications.

Imaging based on external labels on nanodiamonds

3.3

While ND core is chemically inert, its chemically reactive surface allows attachment of organic dyes or carbon dots for fluorescence imaging [41]. ND particles conjugated with poly-L-lysine [42] were covalently labeled with organic dyes by carboxyl-to-amine cross-linking and were used for in vivo fluorescence imaging of DND clearance in mice [43]. In another study, aminated ND was directly conjugated with the N-hydroxysuccinimide (NHS) ester of carboxytetramethylrhodamine (TAMRA) and used for fluorescent cellular tracing of NDs in cytotoxicity studies [44,45]. Surprisingly, NDs conjugation with otherwise nonfluorescent moieties, like octadecylamine (ODA), made them fluorescent [46]. Incorporation of ND-ODA into the biodegradable polymer poly-lactic-acid (PLA) significantly improved its mechanical strength and allowed optical monitoring of the replacement of PLLA--ND-ODA composite with growing bone tissue, facilitating the composites use for manufacturing fixation devices for bone fracture surgery [47]. A different strategy was exploited for production of allcarbon PL nanostructures by using specific acid treatment of detonation soot, that produced tiny rounded sp2 carbon species (carbon dots), covalently attached to the surface of ND particles [48]. These carbon-dot-decorated NDs demonstrated stable red/NIR PL and were used in in vitro cell culture imaging. By creating carbon dots on the surface of deagglomerated 5 nm detonation ND, dye-free small NDs with bright red luminescence can be produced. Another external label includes gadolinium (Gd) conjugates useful for MRI imaging [49,50]. Of particular interest is that Gd(III)-conjugated ND complexes show superior performance in MRI with contrast enhancement as high as 10-fold [49], which is among the highest relaxivity values per Gd(III) ion reported to date compared to all other clinical and nanoparticle agents. An order of magnitude decrease in Gd dosing while maintaining requisite contrast levels would represent a major advance. The development of this technique not only enables detection of NDs by MRI but also represents an important advance in improving the efficiency of the MRI contrast agents in the field. Finally NDs with chemically grafted radiotracers such as 18 F [51] and188Re [52] were successfully used for studies of NDs biodistribution in small animals. Girard et al. [53] succeeded in radioactive labeling of detonation NDs with 3H atoms using a tritium microwave plasma. Interestingly, while 93% of 3H atoms were tightly bonded to the surface, up to 7% were embedded into the diamond core, ensuring a highly


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stable radiolabeled ND, with surface available for further functionalization. NDs conjugated with radiotracers open the way to biodistribution and pharmacokinetics studies of NDs by PET.


4.

Biocompatibility of nanodiamonds

Biocompatibility is one of the critical determinants that should be considered before subjecting any material to drug delivery applications. Key factors that decide the biocompatibility include host reactions toward the nanomaterial and degradation of the same nanomaterial in the body and/or its excretion. Knowledge on cellular and tissue responses is essential for design and development of any materials for drug delivery applications. Toward a translational roadmap for ND-based therapeutic and imaging agent implementation in a clinical setting, there are several important production considerations that must be taken into account with regard to maintaining Chemistry, Manufacturing and Controls (CMC), Good Laboratory Practice (GLP), and Good Manufacturing Practice (GMP) compliance, all of which are important components of regulatory evaluation [1]. These are critical elements of the transition of ND platforms from the bench top toward the clinic as they govern batch to batch consistency during synthesis among other properties, which will inevitably play a role in ensuring that the biological response to their administration stays within known limits to maintain the safety of implementation. Effects of NDs on cells Previous studies by a number of groups have shown that the uptake of DNDs is nontoxic to different cell lines [54-56]. Among all the tested nanocarbon materials, the biocompatibility trend observed was in the order of ND > carbon black (CB) > multi-walled carbon nanotubes (MWCNT) > singlewalled carbon nanotubes (SWCNT) [55]. Other methods used to assess the toxicity of NDs including reactive oxygen species (ROS) production and luminescent ATP production further confirmed the lack of toxicity of ND particles. From a comparison of HeLa cells treated with MWCNTs, graphene oxide (GOs) and NDs, the NDs showed the highest biocompatibility [56]. Vaijayanthimala et al. [57] have also evaluated the biocompatibility of FNDs in cancer cells and preadipocytes at both clonal and population levels. The proliferative potential was not affected by FNDs in HeLa cells, neither did the adipogenic and osteogenic differentiation abilities of 3T3-L1 pre-adipocytes and 489--2 osteoprogenitors. Hemocompatibility of nanoparticles is a major concern when they are administered through the bloodstream. In hemocompatibility studies of HPHT-NDs, Li et al. [58] demonstrated the excellent biocompatibility of purified and acidwashed NDs with negligible hemolytic and thrombogenic activities. In contrast, GOs showed considerable hemolytic 4.1

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activity. Cell viability assays with human primary endothelial cells further confirmed the non-cytotoxic behavior of NDs. Efficient application of stem cells to the treatment of various diseases needs safe tracking of stem cells in vivo to study their fate over time that can be done using fluorescent nanoparticles. Xing et al. [59] conducted genotoxicity tests on NDs and observed that embryonic stem cells displayed little increase in the expression of DNA repair proteins. However, the extent of DNA damage caused by NDs was much less compared to MWCNTs. Another study by Blabber et al. [60] showed that labeling with NDs of human adipose-derived mesenchymal stem cells did not alter cell morphology, differentiation and CD marker expression profile of these cells. Despite these facts, ND labeling does increase secretion of intracellular proteins but without inducing stress response or changing cellular function. Effect of NDs on model organisms/animals To test the toxicity of NDs in whole organisms, Mohan et al. [61] introduced FNDs into Caenorhabditis elegans by both feeding and microinjection (Figure 2B). Lifespan, brood size and ROS measurements showed that both the longevity and reproduction capability of C. elegans were not altered when treated with FNDs. Moreover, embryogenesis was not affected, proving that NDs are biologically inert. Chang et al. [62] microinjected FND into the yolk cell of a zebra fish embryo at the one-cell stage. The ND-injected embryo developed into a whole fish without any apparent abnormalities during their embryogenesis. Marcon et al. [63] have assessed the in vivo toxicity of DNDs with different surface modifications (-OH, -NH2 or -COOH) in Xenopus laevis. Microinjection of ND-COOH into early-stage embryos caused significant embryotoxicity and teratogenicity, while NDs with NH2 and OH are only slightly toxic. Using mice as a model animal, Yuan et al. [64] has examined the effect of DNDs on mouse respiratory system after intratracheal instillation.Ultrastuctural and histopathological investigations revealed the absence of adverse effects in lungs. In addition, no lipid peroxidation in mice lung was observed. On the other hand, Zhang et al. [65] have observedan acute toxicity of DNDs in mice after intratracheal instillation due to the high administred dose of the particles (20 mg/kg body weight). Histomorpholoy and elevation of biochemical markers indicated a dose-dependent toxicity of DNDs in lung, liver, kidney and blood. The observed toxicity was much smaller than that caused by CNTs and other carbon nanoparticles. Similarly, Puzyr et al. [66] have studied the long-term toxicity of DND hydrosols on mice and offspring by oral administration of ND hydrosols up to 6 months. DNDs did not cause death or damage to internal organs such as lung, liver, kidney, spleen and heart, and the reproduction ability was not affected by DNDs. More recently, Vaijayanthimala et al. [67] studied the long-term stability and biocompatibility of FNDs in rats after intraperitoneal injection for a period of 5 months. No significant difference was observed in food consumption, water 4.2



consumption and organ index of control and FND-treated rats. No necrosis, inflammation or tissue reaction was observed in histopathological analysis providing further convincing evidence that NDs are nontoxic. The LD50 values of NDs were not reported and are too low to be detected. This is also hard to define as a standard value given the different types of NDs used and additional studies will aim to ascertain an accurate LD50 value for the various ND classes.


5.

NDs as therapeutic carriers

Up to date, adsorption was the basic mechanism most commonly used for loading drugs onto ND particles [1,43,68]. Rational design of ND drug delivery systems include control over loading capacity of drugs of interest on NDs, possibility for a triggered release of the drug as well as control over kinetics of the drug release. In order to be able to control these processes, understanding of the basic mechanisms of the ND--drug interactions as well as precise knowledge of the ND surface structure and composition are necessary. Binding between ND particles and drug, gene or protein molecules are due to interactions between functional groups on the surfaces of the NDs and a molecule of interest [69,70]. As such, ND surfaces can be functionalized with surface groups tailored for optimal adsorption and controlled release of the drug. One of the hurdles in the control of the ND--drug interaction is the absence of a ND ‘standard,’ a material with uniform particle sizes (preferentially 4 -- 5 nm), with precisely identified type(s) and amount of surface groups and a low metallic impurities content at the ppm level. Different vendors use different DND processing techniques resulting in DND with very different surface chemistry [71]. A model of ND with the most common and important features is illustrated in (Figure 1B), accentuating the rather complicated composition of the ND surface. Qualitative and quantitative analysis of these chemistries is still a big challenge [71]. Surface groups are responsible for electrostatic interaction, van der Waals forces and hydrogen bonding with drug molecules. The majority of the functional groups on ND become charged in solution with the degree of dissociation depending on the pKa of the surface group and the pH of the solution. Depending on the method of detonation soot oxidation, as received NDs from different vendors have either positive or negative zeta potentials [71]. Electrostatic attraction between charges associated with NDs surface groups and oppositely charged drug molecules result in relatively strong binding, which can be changed if the degree of dissociation of NDs surface groups is altered by a change in the pH of the colloid. This model was used for explanation of the observed adsorption capacity and binding of drug molecules [69,70] and toxins [72] on NDs surface modified with different functional groups. A complication arises from the fact that quite often ND surfaces are amphoteric with both acidic and basic groups coexisting on their surfaces. At the same time, if drug

molecules contain both positive and negative charges within their structure, amphoteric ND surface can be preferable for multiple binding points, although elaboration of the binding mechanisms would be more difficult. Mochalin et al. performed equilibria studies between aqueous solutions of doxorubicin or polymyxin B with NDs in isothermal conditions for NDs with polyfunctional and aminated surfaces [68]. Although the adsorption of both drugs on all studied NDs followed the Langmuir isotherm, the parameters of adsorption such as the maximal monolayer capacity and binding strength demonstrated strong dependence on the ND surface chemistry. Paci et al. performed a detailed investigation of the types of the surface groups on NDs deagglomerated to primary 4 nm particles produced by the NanoCarbon Research Institute [70]. These NDs have been successfully used as Dox drug carriers in several in vivo studies and the ND--Dox complexes demonstrated very high efficacy in cancer treatments [1,43,68]. The authors used a set of experimental and computational techniques to quantitatively characterize functional groups on the surface of these NDs and concluded that the groups are amphoteric, with high concentrations of phenols, pyrones and sulfonic acid groups. The presence of phenols and pyrones is consistent with the presence of graphitic surfaces formed during bead milling [12]. The sulfonic acid groups originate from the sulfuric acid used in the ND purification process. It was concluded that the positive surface potential of these NDs originate from the presence of pyrones, consistent with a discussion of the origin of positive zeta potential in NDs by Schrand et al. [71]. The comprehensive model presented by Paci et al. explained a number of recent experiments on binding and release of drugs on NDs. For example, an explanation is required for a strong adsorption of Dox molecules, which have a positive charge in a solution with a pH lower than ~ 8, while NanoCarbon NDs have also a positive zeta potential. Paci et al. attributed Dox binding through negatively charged Ph-SO3- or Ph-O- groups, which were detected on the surface (along with positively charged pyrones responsible for the overall-positive surface charge). Addition of salt to the colloid to promote binding [68] makes the electrical double layer more compact, so that the repulsive effects of the overall-positive potential of the surface is reduced and positively charged Doxorubicin (DOX) molecules can approach the surface. It is also important to add that ND--Dox complexes form ~ 50 nm agglomerates, which are ideal in size for EPR-based accumulation in tumors. To further increase the loading capacity of NDs in combination with PL imaging modality, Rosenholm et al. recently adapted the core--shell approach for fabrication of fluorescent NDs containing NV centers covered with a mesoporous silica (MSN) shell (Figure 2C) [73,74]. Porous silica nanoparticles have been studied extensively as drug carriers, owing to their high loading capacity, stemming from the characteristic high specific surface areas (up to ‡1000 m2/g). MSN nanoparticles are especially suitable for a hydrophobic drug cargo that is


7



generally challenging to formulate. The class of drug molecules poorly soluble in water composes about ~ 40% of the drugs on the market and ~ 90% of drugs in the development pipeline [75]. For ND@MSN, loading degrees of ‡ 100 wt% (drug/carrier) have been reported for the red carbocyaninedye DiI, with similar loading degrees obtained for hydrophobic drugs (dexamethasone, furosemide and prednisolone) [73,74]. The core--shell design of the FND@MSN@cop particles possessing bright internal fluorescence facilitated monitoring of the intracellular fate of the particles and release of the model drugs. Selective cellular uptake and retention is one of the key criteria in targeted drug delivery. Vaijayanthimala et al. [57] used an array of metabolic and cytoskeletal inhibitors to examine the uptake mechanism of NDs (140 nm) in cancer cells (HeLa cells) and pre-adipocytes (3T3-L1 cells). The cells were treated with inhibitors for various endocytic pathways before ND treatment. The results substantiate that the ND uptake in both cells was through energy-dependent clathrinmediated endocytosis. Similar uptake mechanism was reported by Orestis et al. [30]. In a comparative study of a cellular uptake and cytotoxicity of carbon nanomaterials such as MWCNTs, GO and NDs in HeLa cells, the highest cellular uptake with least cytotoxicity was observed for NDs (10 nm) [56]. ND size can certainly have an effect on ND uptake. However, recent studies using both detonation and fluorescent NDs show that they can be readily taken up with minimal to no impact on cell health. The toxicity of NDs is too low to allow the systematic study of their size dependence. A recent study showed that size, shape, surface charge and functionality determines the internalization pathway and their destination in cells [76]. Experiments were performed in the presence of different inhibitors to study the specific entry route of poly-L-lysine conjugated FNDs. The results revealed that cellular uptake mechanism of PLL-FNDs is similar to that of FNDs, that is, through energy-dependent, clathrinmediated endocytosis in both HeLa cells and 3T3-L1 cells. In addition, there was a two- to threefold increase of cellular uptake of PLL-FNDs in a serum containing medium compare to pristine FNDs. A similar kind of study was carried out using folate conjugated FNDs and transferrin-coupled FNDs. Both particles enter through receptor-mediated endocytosis with enhanced uptake, which is further confirmed by competitive inhibition assays [77,78]. The above studies demonstrate that the surface charge of NDs influences the amount of FND uptake in cells as well as tailored surface chemistry facilitates targeting thus reducing cytotoxicity [76]. A drastically different approach for intercellular delivery of ND conjugates with biologically active molecules was demonstrated by Grichko et al. using the Bio-Rad PDS-1000/He ballistic bombardment instrumentation, that uses He pressure and vacuum circuits to generate a shock wave to precisely target small areas of a cell culture or tissue with NDs ‘bullets’ [79]. The immobilization of DNA onto NDs permitted plasmid 8

intercellular delivery via ballistic bombardment. The particular advantages of using ND as a carrier for DNA include its ability to stabilize DNA under long-term storage as compared with traditionally used heavy metal nanoparticles. NDs can be simultaneously uploaded with different types of drug/DNA introducing multifunctionality and extending applications of the ballistic methods to vaccine delivery and gene therapeutics.

Demonstrations of ND-mediated drug delivery

6.

Among the several promising applications of NDs in biomedicine, drug delivery via ND has been widely explored due to their versatility as carriers for a broad array of compounds ranging from small molecules to proteins and nucleic acids (Figure 3). In several studies, ND--drug compounds have demonstrated marked improvements over clinical and commercial standards. For example, ND-based doxorubicin administration against drug-resistant cancers resulted in marked tumor regression while drug alone resulted in virtually no efficacy. Furthermore, even lethal doses of drug were rendered even more efficient and well tolerated when delivered via ND compared to drug alone [43]. It should also be noted from Chow et al., that even systemically injected ND--drug complexes do not result in early drug release, demonstrated by the fact that there was no apparent myelosuppression and enhanced drug efficacy in highly resistant tumors. Chow et al. also showed that the ND--Dox half-lives in a preclinical model were ~ 8.4 h [43]. ND-based gene delivery has also resulted in a one order of magnitude improvement in transfection efficacy compared to commercial standards [80]. Importantly, comprehensive evaluation of ND administration both in vitro and in vivo has demonstrated that they are well tolerated and also capable of sequestering drug activity to reduce systemic side effects such as myelosuppression [81]. As such, ND-based therapeutic delivery serves as a promising route toward ND implementation in a translational setting. Nanodiamond-embedded contact lenses Drug-eluting contact lenses allow for convenient, localized drug delivery into the eye. This treatment approach has been explored for various ocular disorders, including glaucoma, a disease that leads to blindness in millions of people worldwide. Typically, a drug is loaded into a lens via drug soaking or molecular imprinting. However, both loading methods culminate with the burst release of drug, necessitating high drug dosages or frequent lens application. The wet storage conditions required for most soft contact lenses also contribute to premature drug elution. To circumvent the shortcomings associated with conventional drug-eluting contact lenses, Kim et al. fabricated a ND-embedded contact lens that only releases timolol maleate (TM), an antioxidative glaucoma drug, upon biological stimulation [82]. ND-loaded polymer matrices have been shown to be capable of ultra-sustained drug elution. With this principle 6.1



ND-PEI800 ND-PEI800/DNA

Cancer drug delivery

DNA/siRNA delivery


1

Shell

2

Microchannel to reservoir

3 Core tip 4

Liquid-air interface

5 6

7

Meniscus 8 Microfluidic ND delivery

MR imaging

Figure 3. Nanodiamonds are capable of markedly enhancing a broad array of applications ranging from imaging to drug delivery. With regard to therapeutic applications, NDs have demonstration major improvements over commercial and clinical standards by improving both the efficacy and safety of administration. NDs: Nanodiamonds.

in mind, polyethyleneimine-functionalized NDs were crosslinked with N-acetylated chitosan, a lysozyme-cleavable polysaccharide, and mixed with TM to form a TM-loaded ND nanogel. The TM-loaded ND nanogels were subsequently integrated into a standard poly-2-hydroxyethyl methacrylate (polyHEMA) lens matrix and casted into a TM-eluting contact lens (Figure 4). Despite the addition of ND nanogel to the polyHEMA matrix, the optical clarity, water content and oxygen permeability of the ND nanogel-embedded lens was comparable to a standard polyHEMA lens. Furthermore, the ND nanogelembedded lenses exhibited higher mechanical strength than polyHEMA lenses. In contrast to drug-soaked and molecularly imprinted lenses, which immediately released TM upon immersion in a saline solution, ND nanogel-embedded lenses sequestered TM for an indefinite period of time. Upon treatment with lysozyme, a chitosan-cleaving enzyme found in lacrimal fluid, TM was slowly released from the ND nanogelembedded lenses over 48 h. Most importantly, the biological activity of the eluted TM was preserved. Eluted TM was equally effective at rescuing the viability of hydrogen peroxide-treated trabecular meshwork cells as standard TM. The results obtained by Kim et al. indicate that ND nanogel-embedded contact lenses can facilitate sustained therapeutic release of drug, eliminating the need for repeated dosages [82]. Since lysozyme is present in lacrimal fluid,

lysozyme-mediated elution can be utilized for any ocular therapeutic purpose. This lysozyme-mediated elution mechanism also prevents inadvertent drug release under aqueous storage conditions. The optical clarity, water content and oxygen permeability of ND nanogel-embedded lenses are also preserved in conjunction with an improvement in mechanical strength, demonstrating the lenses’ clinical and commercial potential. Localized drug delivery for glioblastoma therapy Finding an effective treatment of glioblastoma, the most common and deadliest brain tumors in existence, has become a distinctive challenge in nanomedicine-mediated treatment and an indication where ND drug delivery may enable important therapeutic advances [83,84]. The difficulty in penetrating the blood--brain barrier (BBB) and the poor retention of conventional treatments has made it necessary to explore other options. Xi et al. studied the use of convection-enhanced delivery (CED) to administer the NDs adsorbed with DOX as a potential drug delivery system [84]. In CED, drugs are delivered through catheter(s) placed stereotactically directly within the tumor mass or around the tumor or the resection cavity. DOX has been used for many different cancers but not for malignant brain tumors because of its inability to penetrate the BBB. However, CED is a process that enables drug therapeutics to cross the BBB using a localized approach. Furthermore, CED can alleviate side effects by maintaining a lower 6.2


9


Lysozyme


Figure 4. A nanodiamond-embedded contact lens exhibited improved lens mechanical properties while simultaneously preserving water content and mediating lysozyme-dependent drug release.

level of drug concentration within the rest of the body while sustaining an effective dose within the intracerebral area to effectively eliminate tumor cells. In addition to finding an improved drug delivery technique, the study also examined potential carriers for the therapeutic agents. Integration of DOX with NDs provides a highly efficient method of treating drug-resistant tumors. NDs not only improve the circulatory lifetime of DOX but also allow lethal doses of the drug DOX to be administered systemically while reducing toxicity. By combining two means of improved drug delivery, Xi et al. observed how ND--Dox performed in brain tumor treatment in glioblastoma cells lines and in normal rodent parenchyma. They conducted their study by incubating cells with fluorescein isothiocyanate (FITC), NDs--FITC, DOX and ND--DOX. They found that the drug distribution and retention improved when ND was complexed with DOX in comparison to the other formulas. In vivo, ND--Dox prevented cell growth and induced cell dose to a greater degree than unmodified DOX over time, further supporting the biocompatibility of NDs. When ND--DOX and unmodified DOX were delivered through CED in vitro, the ND--Dox was more localized at the injection site while the DOX was distributed throughout the chamber of the cell slides. Furthermore, toxicity studies indicated extensive damage to the brain tissue when DOX was administered alone while ND--Dox resulted in minimal tissue damage. Lastly, intracranial bioluminescence xenograft rodent model studies were conducted to show that increased dosage of ND--DOX could improve efficacy without compromising the health of the tumor-bearing rats, thereby markedly improving drug tolerance (Figure 5). This study examined the efficacy of CED of ND--Dox and found that this method of treatment allowed the therapeutic agents to cross the BBB while maintaining limited drug distribution and minimal toxicity. Targeted drug delivery for breast tumor treatment

6.3

Due to common limitations of chemotherapy such as poor water solubility, short circulation time, high toxicity and drug resistance, significant efforts have been devoted to the development of new technologies for overcoming those challenges. NDs possess large carrier capacity, the ability to overcome chemoresistance, and relatively favorable biocompatibility 10

properties compared to other nanocarbons and nanomaterials in general. Because of these properties, NDs have previously been used to treat multiple drug-resistant tumor models (breast and liver) where even lethal dosages of the doxorubicin chemotherapeutic were rendered completely tolerable with marked improvements to tumor treatment efficacy [1,43,84]. ND and mitoxantrone have also previously been applied to drugresistant breast cancer cells [85]. To address a particularly challenging form of cancer classified as triple negative breast cancer, recent preclinical work by Moore et al. developed ND--lipid hybrid particles (NDLPs) that were functionalized with antibody to target the EGFR on implanted MDAMB-231 (triple negative breast cancer) cells and tumors in mice while also delivering the epirubicin chemotherapeutic which led to marked improvements in treatment efficacy and improvement of chemotherapeutic tolerance [86]. Furthermore, the scalability, chemical stability and biocompatibility also prove that NDLPs are a promising future chemotherapeutic delivery vehicle. The syntheses of NDLPs involved a rehydration process of lipid thin films made of cholesterol and biotinylated lipids in the presence of concentrated ND solutions. Then, the NDLPs were further functionalized for selective targeting with antiEGFR antibody using streptavidin. In order to confirm NDLP formation and antibody presence, comprehensive analysis including dynamic light scattering (DLS) analysis for size and zeta potential of particles, flow cytometry for quantification of NDLPs among NDs and lipids, cryogenic TEM (cryo-TEM) for visual conformation of NDLPs, inductively coupled plasma atomic emission spectroscopy (ICP-AES) for the presence of lipids on NDs by analysis of phosphorus on lipids and ELISA for confirming the presence of functional antibody and determining the degree of antibody loading on the NDLPs were performed. In addition, confocal microscopy was used to evaluate the targeting capabilities of anti-EGFR antibody-loaded NDLPs by comparing NDLP localization after incubation of targeted NDLPs with two breast cancer cell lines, such as EGFR-overexpressing MDA-MB-231 and non-EGFR-overexpression MCF-7. Finally, the analysis confirmed NDLPs were ~ 50 nm in size, possessed a nearly neutral zeta potential, as well as 7.74 µg of antibody and 174 µg of epirubicin loading per mg of NDs with great selectivity against targeting moieties.



NDs (Day 25)

Dox (Day 49)

ND-Dox (Day 90)


TUNEL

H & E (200x)

H&E

CON (Day 28)

Figure 5. Nanodiamond--doxorubicin (NDX) was capable of markedly improving glioblastoma therapy efficiency via convection enhanced delivery. While control and drug-only conditions resulted in tumor persistence, NDX administration resulted in tumor regression and enhanced treatment safety where drug distribution was largely confined only to the tumor region and not healthy tissue.

In order to assess the degree of tumor localization in vivo and efficacy of targeted drug delivery of NDLPs to EGFRoverexpressing tumors, MDA-MB-231 implanted mice were studied for 10 days and 7 weeks, respectively. With regard to the degree of tumor localization from the in vivo experiment, targeted NDLPs showed significant differences in tumor localization at 48 and 72 h compared to untargeted NDLPs, which confirmed that the anti-EGFR antibody on NDLPs enhanced the degree of tumor localization of targeted NDLPs. More importantly, to test the efficacy of targeted drug delivery of NDLPs, PBS, epirubicin, untargeted NDLP-Epi and antiEGFR--NDLP-Epi were injected intravenously once a week in tumor-bearing mice, and tumor volumes of each treatment condition were compared over the course of 7 weeks. In this experiment, all mice treated with unmodified epirubicin suffered drug-related mortality in 4 weeks. In contrast, NDLPEpi and anti-EGFR--NDLP-Epi-treated mice survived 7 weeks with markedly smaller tumor sizes in comparison to PBS treatment. In addition, anti-EGFR--NDLP-Epi treatment mediated a > 50% reduction of tumor volume compared to untargeted NDLP-Epi, and nearly complete tumor regression. This study showed that NDLPs could be readily selfassembled and were able to markedly enhance treatment efficacy via improved tumor localization and drug tolerance by reducing drug-induced mortality. All of these features make NDLPs very promising for the future of the field of biomedical imaging and drug delivery. Toward orthopedic and dental applications of nanodiamond drug delivery

6.4

Bone morphogenetic proteins (BMPs) are well-studied regulators of bone and cartilage development and have been shown to promote bone formation by inducing osteoblast differentiation. After the recent Food and Drug Administration approval of InFUSE for both sinus and localized alveolar

ridge augmentation, BMPs are being increasingly applied to oral and maxillofacial procedures. However, in vivo studies indicate that the effectiveness of BMP-2 is largely correlated to its local concentration, signifying the need for a delivery system to prevent the rapid clearance of BMP-2 by diffusion. While BMPs are currently delivered by implanting bulky collagen sponges in large procedures, this approach is not practical for most oral and maxillofacial surgeries that generally deal with small surgical sites. Moore et al. developed novel nanoparticle suspensions as promising injectable alternatives for the delivery of BMP-2 and low doses of basic-fibroblast growth factor (b-FGF) for oral surgical procedures [87]. The effectiveness of ND suspensions for simultaneously delivering both BMP-2 and b-FGF to the desired location and their ability to successfully accelerate localized bone growth were evaluated. The high adsorption capacity of NDs allows BMP-2 and b-FGF to be readily loaded into ND clusters through physisorption. The loading was confirmed by DLS analysis and Fourier transform infrared spectroscopy, and the incorporation of BMP-2 and b-FGF to ND surfaces was quantified using ELISA. After BMP-2 and b-FGF-loaded ND clusters were delivered to C2C12 myoblasts, ELISA analysis and alkaline phosphatase (ALP) activity were used to determine the degree of BMP-2 and b-FGF-induced differentiation of myoblasts into ALP-producing osteoblasts. Moore et al. successfully demonstrate that NDs are indeed capable of simultaneously delivering two functional proteins, BMP-2 and b-FGF, which are both required for effective bone healing in vivo. The capability of NDs to deliver two proteins in one ND cluster offers a remarkable advantage because the combination of BMP-2 and b-FGF induces proliferation of precursor myoblasts in addition to differentiation. The results indicated delayed or pH-triggered release of proteins. This may in fact help surgeons gain more control over the location


11



of protein delivery during operation. The sustained release of proteins limits rapid clearance by diffusion, ultimately maintaining the potent proteins in the desired region for a longer duration. In addition, protein-loaded ND suspensions offer an attractive option as a delivery vehicle for BMP-2 because a liquid medium can be conveniently administered by injection or rinse to surgical sites with various space limitations. As therapeutic delivering agents of potent osteoinductive proteins, ND suspensions clearly serve as an effective alternative to BMP-soaked collagen sponges. NDs loaded with BMP-2 and b-FGF are far more superior due to their ability to readily deliver proteins to a localized area in liquid form and prolong the effect of the proteins through sustained protein release. Thus, NDs are a promising protein and drug delivery platform that can significantly enhance bone healing after oral and maxillofacial surgical procedures. 7.

Conclusion

The first review in Expert Opinion in Drug Delivery devoted to ND particles was published in 2009 [88], where first very promising results demonstrating ND capabilities as vehicles for systemic and localized drug delivery were presented. Since then, significant progress has been achieved in combining targeting, imaging and drug delivery capabilities of NDs into a single platform. This in turn has enabled the ND platform to emerge as a promising agent for both translationally relevant treatment and diagnostics [89]. In the field of NDmediated bioimaging discussed in details in this review, unique imaging modalities had been elaborated based on the inherent properties of diamond and creation of crystallographic defects. A combination of photoluminescence, cathodoluminescence and photoacoustic properties of NDs became available for design of novel multifunctional biolabels with unprecedented signal stability since the associated emitters are well preserved within an ND core. ND particles after proper surface modification had been externally labeled with molecular contrast agents for fluorescence imaging, magnetic resonance imaging and PET. In the field of ND-mediated drug delivery most recent achievements include treatment of drug-resistant tumors using NDs integrated with DOX [43]. NDs not only improve the circulatory lifetime of DOX but also allow lethal doses of DOX drug to be administered systemically while reducing toxicity. NDs adsorbed with DOX had been successfully used for localized drug delivery through CED for glioblastoma therapy [84]. This method of treatment allowed the therapeutic agents to cross the BBB while maintaining limited drug distribution and minimal toxicity. Besides applications in cancer therapy, NDs embedded into drug-eluting contact lenses has been used for localized drug delivery into the eye [82]. NDs also served as therapeutic delivering agents of potent osteoinductive proteins capable of providing significantly enhanced bone healing after oral and maxillofacial surgical procedures [87]. These recent achievements in preclinical studies demonstrate a high level of 12

maturity of the ND-mediated drug delivery approach and warrant future efforts focusing on clinical translation of ND-based imaging and therapeutic agents. 8.

Expert opinion

While NDs were only recently introduced as promising drug delivery and imaging agents, they have already impacted the field of nanomedicine with their unique properties which include the faceted architectures and versatile surface electrostatic and chemistry properties found with detonation NDs, and the bright/photostable and biocompatible properties observed with fluorescent NDs. In examining the spectrum of important advancements that have been reported in the field, including marked improvements in tumor treatment efficacy and safety, as well as the ability for preclinical stem cell tracking, challenges to clinical translation remain. Once these challenges are addressed, the promise of improved treatment outcomes for globally prevalent diseases such as cancer and tissue repair represents a foundation for changes in the way that medicine is practiced. Despite the host of both imaging and therapy applications that have been proposed for the ND platform, the costs of developing novel drugs, both imaging and therapeutic can be prohibitively expensive that can reach into the billions per compound. Therefore, the translational roadmap for ND development must rely on identifying ideal compounds that are conducive toward scalable ND--drug synthesis and indications where ND-therapeutic or ND-imaging agent hybrids display marked improvements over clinical standards with respects to efficacy, safety, cost and other important parameters. Applying these constraints to the ND translational roadmap would be catalytic toward the realization of ND-based agents in the clinic. Among the important considerations that need to be addressed when developing a new material for the clinic are the CMC of the synthesis process, comprehensive absorption, distribution, metabolism, and excretion assessment, and proper toxicological evaluation. Furthermore, global regulatory agency policies (e.g., US FDA, Pharmaceutical and Medical Devices Agency of Japan, and European Medicines Agency, among others) toward nanomedicine are becoming clearer, which should drive new advancements in the field. Once the ND developmental pathway aligns with the requirements for clinical translation and continued validation progresses, the future of ND application in the clinic remains promising.

Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.



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Affiliation V Vaijayanthimala1, Dong Keun Lee2, Sue V Kim2, Albert Yen2, Nathaniel Tsai2, Dean Ho*3, Huan-Cheng Chang*4 & Olga Shenderova†5 †, *Authors for correspondence 1 Indian Institute of Sciences, Department of Materials Engineering, Karnataka, India 2 University of California-Los Angeles, UCLA School of Dentistry, 10833 Le Conte Avenue, Room B3-068A, Los Angeles, CA 90095, USA 3 Professor of Oral Biology and Medicine and Bioengineering, Co-Director, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, UCLA School of Dentistry, Jonsson Comprehensive Cancer Center, Center for Oral, Head and Neck Cancer Research, Los Angeles, CA 90095, USA 4 Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan 106, Republic of China 5 International Technology Center, Ada´mas Nanotechnologies, Inc., 8100 Brownleigh Dr., S.120, Raleigh, NC 27617, USA E-mail: [email protected]

15

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Challenges and Opportunities in Drug Delivery for Wound Healing.

Drug development and discovery: challenges and opportunities.

Challenges and opportunities in epigenetic drug discovery.

Polypharmacology: challenges and opportunities in drug discovery.

Imaging pulmonary arterial thromboembolism: challenges and opportunities.

Drug Delivery Approaches in Addressing Clinical Pharmacology-Related Issues: Opportunities and Challenges.

Opportunities in respiratory drug delivery.

Optical imaging of head and neck cancer: opportunities and challenges.

Drug discovery in rare indications: opportunities and challenges.

Data Challenges and Opportunities in the Evolving Medical Imaging Practice.

Cardiovascular MR imaging at 3 T: opportunities, challenges, and solutions.

Opportunities for Photoacoustic-Guided Drug Delivery.

Smart Nanoparticles for Drug Delivery: Boundaries and Opportunities.

Multivalent polymers for drug delivery and imaging: the challenges of conjugation.

Vascular-homing peptides for targeted drug delivery and molecular imaging: meeting the clinical challenges.

Polyhydroxyalkanoates, challenges and opportunities.

2014: Challenges and opportunities.

Pulmonary drug and vaccine delivery: therapeutic significance and major challenges.

New approaches to antiretroviral drug delivery: challenges and opportunities associated with the use of long-acting injectable agents.

Challenges and solutions in topical ocular drug-delivery systems.

Receptor-targeted drug delivery: current perspective and challenges.

Self-microemulsifying drug delivery system (SMEDDS)--challenges and road ahead.

Advances and Challenges of Liposome Assisted Drug Delivery.