Diamond as a nanomedical agent for versatile applications in drug delivery, imaging, and sensing

Authors

  • Han B. Man,

    1. Department of Mechanical Engineering, Northwestern University, Evanston, IL 60208, USA
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  • Dean Ho

    Corresponding author
    1. Division of Oral Biology and Medicine, and Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, School of Dentistry, University of California, Los Angeles 90095, USA
    2. The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California, Los Angeles 90095, USA
    • Phone: +1-310-7949851, Fax: (847) 491-3915
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Abstract

A spectrum of materials has been explored as potential platforms for nanomedicine in the areas of drug delivery, imaging, and diagnostics/sensing. Depending upon the application required, the therapeutic compound being delivered, or the imaging agent being modified, certain nanoparticulate materials offer specific advantages that may uniquely enhance efficiency. Among the classes of particles being investigated, diamond-based platforms have emerged as promising vehicles for drug delivery and imaging following several recent studies that demonstrate their ability to enhance therapeutic efficacy, particularly for anthracyclines, mediate markedly improved magnetic resonance imaging contrast and photostable fluorescence, possess scalable processing parameters, and exhibit biocompatibility, among many other important attributes. More specifically, detonation NDs have faceted surfaces that can mediate potent interactions with surrounding water molecules or therapeutic compounds. This attribute can mitigate premature drug release to prevent major side effects such as myelosuppression, while water molecule recruitment can increase the relaxivity of covalently conjugated gadolinium. These properties of NDs and diamond-based devices will be discussed in this article, with a focus on therapeutic delivery in addition to insight on their use in imaging, devices, implants/coatings, and biocompatibility. Key areas of potential, where advances in biology and medicine can be realized through the continued development of this platform as well as requisite future studies, will also be highlighted.

1 Introduction

In the context of biological and medical applications, diamond-based platforms including NDs produced via detonation and high-pressure high temperature (HPHT), ultra-/nanocrystalline diamond films (UNCD/NCD), and single/polycrystalline diamond films have yielded exciting advancements. Initial studies have demonstrated their application towards the delivery of hydrophilic and hydrophobic cancer drugs, therapeutic proteins, and nucleic acids. Studies have also examined their fluorescent properties and applications as sensor devices and implant coatings, among others. Examples of diamond-mediated improvements such as enhanced therapeutic efficacy and/or specificity as well as unique luminescence properties have been demonstrated 1–18. Recently, NDs have also been used to improve the efficacy of magnetic resonance imaging (MRI) agents. Specifically, covalent conjugation of gadolinium (III) (Gd(III)) has resulted in among the highest ever reported per-Gd relaxivity values in the literature 19.

Recent work has continued to utilize NDs towards therapeutic and imaging applications, as well as considerable efforts to examine their biocompatibility both in vitro and in vivo as a precursor towards translational development. With regards to small molecule drug delivery, doxorubicin (Dox), paclitaxel, cisplatin, 4-hydroxytamoxifen (4-OHT), and purvalanol A, are all examples of compounds that have been adsorbed (reversible) or covalently conjugated to NDs 20–25. In addition to small molecule therapy, protein delivery has also been explored 26, 27. Applications have included the delivery of insulin for its potential as a pro-vascularization/anti-infection wound healing agent, as well as the delivery of the therapeutic transforming growth factor beta (TGF-beta) antibody for its potential as an anti-scarring agent. A third class of therapeutic being explored is comprised of nucleic acids, such as DNA plasmids and siRNA. Recent studies have demonstrated a 70-fold enhancement in plasmid transfection efficacy and improved siRNA-mediated gene silencing compared to commercial standards 28, 29. Further studies have demonstrated siRNA delivery for therapy against Ewing's sarcoma cells 30.

In addition to drug delivery, NDs are being widely investigated for imaging applications 11–16, 18. Both detonation NDs (DNDs) and HPHT NDs have offered important advantages in this area because HPHT NDs, which can be implanted with nitrogen vacancy centers (N-V), have been shown to be remarkably photostable while maintaining biocompatibility. Consequently, they have recently been used for imaging in the C. elegans model 16. DNDs can be functionalized with imaging markers such as positron emission tomography labels, near-infrared emitting dyes, or other fluorescent agents which can also enable in vivo imaging 31, 32. A recent gadolinium (III) (Gd(III))-ND hybrid resulted in a per-Gd relaxivity that was among the highest ever reported values for nanoparticle and clinical imaging agents, demonstrating the unique attributes of the ND surface 19.

To continue the progression of NDs towards clinically relevant applications in drug delivery, biosensing, and implant coating, enhancing their scalability as disperse particles while simultaneously accounting for the uniformity of their surface functionality have also been widely studied. Many advances reported in the area of deagglomeration and surface chemistry processing have been in bacterial detection and neuronal stimulation 33–50. Furthermore, significant progress has been made in the area of diamond-based films as coatings for retinal implants, as well as UNCD-based sensors for DNA-based diagnostics. This feature article will highlight many of the recent findings in biological and medical applications of DND, HPHT ND, and UNCD/NCD/single/polycrystalline platforms and briefly discuss requisite studies to serve as precursors for clinical translation.

2 Drug delivery

Current objectives in drug delivery and nanomedicine research often involve the use of nanoparticles to improve properties that range from drug retention, to therapeutic efficacy and safety. NDs have recently been harnessed for the delivery of many classes of compounds including small molecules, therapeutic proteins, and nucleic acids.

2.1 Cancer therapy via nanodiamonds

Among the earlier studies of ND drug delivery was their use in Dox release 20, 21. One of the most common drugs used for treatment, Dox is also highly toxic and can cause major complications in the form of myelosuppression and cardiotoxicity, among others. Through a study which introduced the concept of ND drug delivery, NDs have been shown to potently sequester Dox on their surface, delaying drug release and activity (Fig. 1) 20, 21. Maintained drug efficacy against multiple cell lines, as well as quantitative real-time polymerase chain reaction studies, were conducted to assess ND safety. The results did not show the increase in expression of inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) 21. More recent studies have examined the preclinical safety of NDs in murine and worm models have demonstrated initial potential, warranting continued development of the platform. Subsequent studies utilized NDs to deliver cisplatin, where pH-dependent drug release was observed 24. Additionally, the water-insoluble drugs 4-OHT and purvalanol A which are being studied as breast cancer and liver cancer therapies respectively, were delivered by NDs with preserved efficacy following an efficient synthesis process 25.

Figure 1.

(online color at: www.pss-a.com) (A) Nanodiamond (ND)-doxorubicin (Dox) complexes (NDX) were capable of markedly improving drug treatment efficacy. Lethal dosages of Dox, when delivered alone, significantly accelerated animal mortality. NDX complexes of the same dosages resulted in improved drug tolerance, where animals survived the study and drug efficacy was even further enhanced, resulting in the smallest tumors in the study. (B) Nanodiamond-doxorubicin complexes result from the potent adsorption of the Dox to the ND surface, which can reduce drug toxicity and mediate sustained release. (C) NDX treatment results in significantly smaller tumors compared to unmodified drug administration. Figure reprinted from Ref. 20.

2.2 Nanodiamond-mediated protein delivery

In addition to small molecule delivery, NDs have also been used for therapeutic protein release, with insulin and the TGF-beta antibody serving as the model therapeutics 26, 27. Insulin is being explored as a potential wound healing and vascularization promoting agent following severe burns and other conditions. ND delivery of insulin was shown to be pH-triggered at basic pH values, which are commonly observed following bacterial infections that accompany serious wounds. In addition to insulin therapy, NDs have been used to deliver the TGF-beta antibody, which is being utilized as a potential anti-scarring agent. ND-antibody complexes were shown to be stored stably in phosphate buffered saline (PBS) while incubation in serum-containing media resulted in triggered release. The preservation of protein structure after release was confirmed via enzyme linked immunosorbent assay (ELISA) 27. While many ND applications have focused on cancer drug delivery, these two studies serve as examples of how NDs can be potentially used for wound healing and anti-scarring applications. Furthermore, the regenerative medicine space, which covers domains such as bone, cardiovascular, and neuro tissue engineering, often rely on controlled growth factor release. The capacity for NDs to mediate protein elution to minimize burst release, as well as the recent synthesis of polymeric matrices or planar deposition of NDs, may be precursors to their extension towards regenerative medicine applications.

2.3 Nanodiamond gene delivery

The combination of gene delivery and nanomedicine has been widely investigated because of challenges with combining efficacy and safety into a single platform 28, 29. Conventional approaches are often highly efficient and more toxic, or less efficient and less toxic. NDs have been utilized as gene delivery platforms when combined with a conventional polyethylenimine (PEI) 800 polymer (PEI800) that is often less efficient and less toxic than its counterparts. Using the NIH 3T3 fibroblast line, transfection of the green fluorescent protein (GFP) and luciferase plasmids was assessed. Compared to PEI800 alone, ND-PEI800 mediated a 70-fold increase in transfection efficacy with maintained biocompatibility for plasmid DNA. Additionally, ND-PEI800s mediated a 400- and 800-fold increase in transfection efficacy when compared to amine and carboxylate-terminated NDs 29. This complex also successfully silenced GFP expression via siRNA delivery with enhanced efficacy and reduced toxicity compared to lipofectamine in serum-containing media (Fig. 2) 28. Subsequent studies have utilized ND-PEI complexes to address specific disease models such as Ewing's sarcoma, a devastating pediatric bone cancer. In this study, siRNA delivery was used to inhibit EWS-Fli1 expression, which has been shown to reduce the capacity for cancer cell proliferation. Both the presence of the NDs as well as siRNA was shown via fluorescence readings which was confirmed via colocalization and phase contrast imaging (Fig. 3) 30.

Figure 2.

(online color at: www.pss-a.com) (Above) A schematic of polyethylenimine 800(PEI800)-ND synthesis is shown. (Below): Gel electrophoresis demonstrates the degree of siRNA loading onto ND-PEI800 complexes for various loading ratios. The ND-PEI800 complexes resulted in significantly improved siRNA delivery efficacy as compared with other polymer transfection agents in serum. Figure reprinted from Ref. 28.

Figure 3.

(online color at: www.pss-a.com) Fluorescent images show the cellular localization of fluorescent nanodiamond-siRNA complexes for Ewing's sarcoma treatment via EWS-Fli1 inhibition. (a) The red emission represents ND fluorescence, (b) siRNA fluorescence is visible via FITC labeling shown in green, (c) a corresponding colocalization graph is shown, and (d) a cellular phase contrast image is shown. Figure reprinted from Ref. 30.

2.4 Preclinical validation of nanodiamond drug delivery efficacy and safety for cancer treatment

Perhaps the most comprehensive pre-clinical validation of ND efficacy and safety in the literature is the synthesis of an ND-Dox compound (NDX) towards the treatment of a drug resistant liver and breast cancer in a murine model (Fig. 1) 20. Drug resistant tumors can often efflux or remove cancer drugs before they have had a chance to function, markedly decreasing, or precluding treatment efficacy. In addition to efficacy analysis, this safety study demonstrated that NDX does not elicit myelosuppression, which is the dose limiting side effect of chemotherapy attributed to superinfections and patient mortality. This finding was particularly important because it confirmed that adsorbed Dox does not appear to release early in the bloodstream following intravenous administration, preventing signifcant toxicity even though the Dox was not conjugated to the ND surface. This successfully demonstrated that reversible drug adsorption not only mediated improvements in drug efficacy, but also improved treatment safety. NDX shows promise as a scalably synthesized therapeutic without the need for drug conjugation. Additional distribution and liver/systemic toxicity studies illustrated that the serum ALT level, an indicator of liver toxicity, and serum IL-level, an indicator of systemic toxicity, did not change significantly following ND administration. NDX was also shown to increase the circulation half-life 10-fold compared to unmodified Dox while also increasing drug retention in both the breast and liver cancer models. In tumor treatment, NDX increased drug efficacy in both the liver and breast models. Even while drug was potently bound to the ND surface to reduce toxicity, the drug was still able to release from the ND surface, confirming preserved drug activity. Most notably, the treatment efficacy towards the highly resistant breast cancer model, which was virtually unaffected by the unmodified drug, was markedly increased following NDX administration. Furthermore, when the unmodified drug dose was doubled, it became fatal, resulting in complete animal mortality prior to study completion. However, NDX administration at the lethal dose prevented animal mortality by increasing drug tolerance, and further increasing treatment efficacy. This study serves as a promising precursor for ND translation given the demonstration of both increased treatment efficiency as well as safety.

3 Imaging with nanodiamonds

A multitude of studies have examined the use of NDs as in vitro agents for fluorescence monitoring. As an example, stable fluorescence from N-V centers allows NDs to be utilized for long-term tracking studies in cells, which is particularly applicable given the requisite photostability [−16, 18]. Several additional studies have used fluorescent NDs to deliver Paclitaxel and track diamond localization following administration. In addition, the aforementioned exciting advances in ND treatment of Ewing's sarcoma cell lines were accomplished using fluorescent NDs, which enabled the simulaneous monitoring of siRNA release in the cytosol 30.

3.1 In vitro tracking studies

Fundamental study of N-V center properties and photostability as well as their applications as imaging agents is a promising area of fluorescent ND research. One platform possessing these combined characteristics is highly favorable particularly when designing probes for cellular processes. Loss of signal can limit the acquisition of important cellular/biomolecular processes since they take place within extremely protracted timescales. Furthermore, NDs conjugated with surface labels, such as protein analogues or antibodies, can further increase their specificity. For example, folate-coated NDs were capable of enhanced targeting efficacy 51.

The high yield production of fluorescent NDs has also been demonstrated. Prior work has examined the mass fabrication NDs with N-V centers and their intracellular transport dynamics have been examined. Bombardment via 40-keV He beam produced 25 nm diameter photostable fluorescent NDs with a nearly 2 orders-of-magnitude improvement in yield 11. Subsequent work has demonstrated the presence of color centers in NDs with diameters on the order of 20 nm 15. NDs with radii of under 4 nm, with stable luminescence and high-contrast optically detected electron spin resonance (ODESR), have also been synthesized with ∼35% of the NDs containing a defect center. These fluorescent NDs have been suggested as effective quantum probes in the interrogation of ion channel function towards potential biosensing and drug discovery applications 52. Mechanistic uptake studies have also been conducted using fluorescent NDs, with initial studies indicating that they are capable of endosomal escape. Various imaging studies were conducted, where smaller ND particles and their internalization were subsequently observed via photoluminescence confocal imaging. Transmission electron microscopy (TEM) was also utilized, revealing the localization of single digit fluorescent NDs with diameters between 5 and 10 nm and their colocalization with intracellular vesicles within the cytoplasm. Blocking agent studies showed that particle uptake can be attributed to clathrin-mediated endocytosis since caveolae blocking agents revealed no effect on uptake. Possible mechanisms of cytoplasmic localization included passive transport uptake and endosomal escape 18.

3.2 C. elegans validation of nanodiamond safety

Among the studies that are evaluating the biocompatibility of NDs, the use of fluorescent NDs within the C. elegans model has produced relevant findings. To evaluate the impact of feeding and microinjecting NDs on the worm, resulting stress response, and reproduction performance has recently been examined 16 120 nm diameter NDs were examined with both acute and long-term analysis. Unmodified NDs typically localized within the worm lumen. However, dextran and bovine serum albumin (BSA)-modified NDs adsorbed against the intestinal cells. ND microinjection into the gonads resulted in the larvae containing NDs. Regarding fluorescent ND biocompatibility, reproductive capabilities, and survival of the worms were unchanged. Nuclear translocation of GFP-tagged cytoplasmic DAF-16 (DAF-1:GFP) served as a readout for stress response. Dextran and BSA addition did not cause translocation, while heat shock caused DAF-16:GFP translocation. These studies indicate that fluorescent NDs do not appear to cause stress in the C. elegans model, and serve as an important precursor for the continued development of ND imaging agents 16.

3.3 Clinically relevant imaging with nanodiamonds

In addition to the innate fluorescence of HPHT NDs, the detonation-based NDs can also be harnessed as substrates for the covalent conjugation of various imaging or labeling agents, including near infrared (NIR)-emitting dyes, radiolabels for pre-clinical studies, and fluorescein. These additions make it possible to track ND localization and distribution. Outside of cellular and animal model tracking, the development of medical imaging agents through the covalent conjugation of gadolinium(III) (Gd(III)) to the ND surface, has also garnered increased attention. While the addition of a nanoparticle can certainly be used to improve circulation times and other properties, the ability for the particle to markedly improve contrast efficiency would represent a significant advancement. A recent study demonstrated a 12-fold increase in per-Gd relaxivity, from 5.42 ± 0.20 to 58.82 ± 1.18 mM−1 s−1 at 1.5 T (60 MHz), representing among the highest ever reported values (Fig. 4) 19. In the context of potential medical applications, another milestone following the demonstration of an order-of-magnitude increase in relaxivity would be to reduce the Gd dose to the patient by an order of magnitude. As a photostable fluorescent agent or covalently labeled imaging compound, NDs have the potential to serve several versatile roles.

Figure 4.

(A) Various magnetic resonance images of nanodiamond-gadolinium(III) (Gd(III)) samples are shown, with the brightest represented by 48 µM Gd(III). (B) The covalent conjugation of Gd(III) to the ND surface mediated among the highest ever reported per-Gd values. Figure reprinted from Ref. 19.

3.4 Octadecylamine-functionalized nanodiamonds

In addition to NDs containing N-V centers, the covalent conjugation of octadecylamine (ODA) to the ND surface has resulted in fluorescent (blue) particles. ND-ODA clusters ranged from 100 to 300 nm in diameter. Furthermore, while dispersed in solvents including benzene, toluene or chloroform, they were stable for one week without requiring sonication. After drying and storage for 2 years, no altered properties or loss of blue fluorescence emission was reported 32.

4 Diamond-based films

Ultra-/nanocrystalline diamond/single/polycrystalline diamond films possess unique properties that make them suitable for a broad array of applications that range from biosensor devices to implant coatings 35–40, 44–46, 76–78. In particular, diamond thin film platforms excel in their biocompatibility, wide electrochemical potential windows, and chemical and mechanical stability.

4.1 Bioinert coatings for retinal implants

The benefits of diamond thin films as biocompatible materials are demonstrated by their application on implantable retinal microchips 45. Implantable devices have become increasingly effective, such as retinal microchip implants that can potentially help restore lost vision. But the key factor of packaging, that protects this device from corrosive biological environments and protects the eye from the implant, is not a simple procedure without the proper material for coatings. UNCD is a potential solution that is both chemically inert and biocompatible. UNCD films can be grown at low temperatures (400 °C) which is suitable to be deposited on silicon microchips. Furthermore, these films are deposited smoothly compared with normal diamond films, where the highly ordered coating can potentially enable improvements in implant lifetime. Cyclic voltammetry and scanning electron microscopy (SEM) studies demonstrated the biocompatibility of the UNCD films following implantation in rabbit eyes (Fig. 5). Following 6 months of implantation, little evidence of inflammation caused by the implants was discovered. Additionally, the UNCD coating was not degraded while exposed silicon was significantly corroded after contact with corrosive liquids from the eye.

Figure 5.

A scanning electron microscope (SEM) image of an ultrananocrystalline diamond (UNCD) film coating a silicon chip is shown. The UNCD films were developed as coatings for retinal implants and evaluated in vitro and in vivo for safety characteristics. Films were approximately 1 µm thick and deposited at 400 °C. Figure reprinted from Ref. 45.

4.2 Diamond films for sensing applications

With the development of effective and economical techniques in nanocrystalline diamond thin film deposition, diamond surfaces have become promising candidates for a variety of sensing applications. An early application of a diamond-based sensor took advantage of diamond as an active substrate able to immobilize/anchor biomolecules 48. Its superior properties as an electrode material enabled diamond to be used as the foundation for an amperometric sensor. NCD was deposited on silicon substrates at a thickness of 2 µm using a vapor deposition modality. GFP molecules were then coupled to the surface through the formation of peptide bonds. Not only was GFP still actively detected by fluorescence microscopy, but they also retained a strong signal after physical agitation, confirming their covalent attachment. An enzyme-based amperometric sensor was also constructed on an NCD surface. In this study it was functionalized with catalase to detect the presence of hydrogen peroxide. A similar type of biosensor, also equipped to detect hydrogen peroxide, was developed using the horseradish peroxidase (HRP) enzyme instead 47. The protein was also covalently bound to the NCD film using peptide bonds. The resulting sensor was able to detect hydrogen peroxide over a large linear range (0.1–45 mM) at a voltage setting of 0.5 V.

Different versions of diamond-based sensors have been developed to detect other molecules such as penicillin and nucleic acids with high levels of sensitivity 49, 50. The enzyme penicillinase was immobilized within a field-effect capacitive electrolyte–diamond–insulator–semiconductor (EDIS) structure 49. This sensor exhibited very low detection limits for penicillin (5 µM) and high sensitivity (60–70 mV decade−1) owing to the electrochemical response of NCD. Diamond in the form of nanowires have also been effectively integrated into a DNA sensor 50. Vertically arranged diamond nanowires were fabricated by etching boron-doped diamond via reactive ion etching. Using diamond nanoparticles as a hard mask, a carpet of upright wires, measuring 3–10 nm long and spaced around 11 nm apart, was produced. Functionalization of the nanowire tips with phenyl groups made it possible to bind with oligonucleotide strands. After a set of probe DNA strands was immobilized onto the nanowire surface, cyclic voltammograms made it possible to differentiate readings between target DNA strands and single-base-mismatched DNA towards a potential clinically relevant diagnostic application. Furthermore, a sensitivity limit of around 2 pM was achieved, with little DNA degradation after 30+ cycles of DNA hybridization. Diamond film-based materials have shown promise in the area of biochemical sensors after successfully serving as robust platforms for the detection of a spectrum of biomolecules.

5 De-agglomeration and functionalization of nanodiamonds

As NDs continue to be developed for potential biological and clinical applications, the ability to preserve their dispersability following surface functionalization is an important requirement, as uncontrolled agglomeration can preclude definable surface properties, reduce cellular uptake, and induce toxicity. Previous methodologies have included the use of zirconia beads coupled with 400 W sonication, which were shown to markedly reduce the particle size, and cause pH-mediated changes to particle dispersability and precipitation (Fig. 6). The collection of primary nanoparticle dispersal techniques includes high shear mixing, ultrasonication, and milling, among others. Routes utilized for ND processing can result in the formation of a “hard gel” or “soft gel,” which is associated with the degree of a water nanophase surrounding the ND particles following bulk water removal (Fig. 7). Of these methodologies, ceramic beads can disperse nanoparticle aggregates while simultaneously chemically functionalizing their surfaces, through a process called beads-assisted sonication, or BASD 43. BASD has allowed both silane and aryl groups to be homogeneously functionalized across the ND surface. Condensation between ND–OH groups and (3-acryl-oxypropyl) trimethoxysilane was used for ND silanization. Arylation was accomplished using diazonium salts at high temperatures. For both processes, sonically propelled ceramic beads broke up agglomerates via impact and shear forces between the beads. BASD-arylated particle diameters were approximately 4.8 nm while parallel bead-less sonication coupled with magnetically based stirring resulted in diameters of approximately 45 and 80 nm, respectively. Therefore, BASD represents a promising approach for dual ND functionalization and de-agglomeration, which are two increasingly important attributes of scalable ND synthesis.

Figure 6.

(online color at: www.pss-a.com) The pH-dependent dispersal of nanodiamond agglutinates is shown here. It can be observed that the modulation of pH levels from acidic to basic results in varying levels of precipitation. Figure reprinted from Ref. 34.

Figure 7.

(online color at: www.pss-a.com) Bead milling-dependent deagglutination of NDs and processing routes to yield ND soft gels and hard gels based upon water evaporation (bulk or nanophase) are shown. Figure reprinted from Ref. 63.

In addition to bead-based deagglomeration methods, salt and sucrose-assisted milling was demonstrated as a scalable and cost-effective approach to produce uncontaminated NDs with sub-10 nm diameters 53. Salt and sucrose possess the necessary hardness to reduce ND agglomerates into primary particles. Using an optimized ND to salt ratio, 500 rpm fixed rotation speed, and increasing the duration of salt-assisted milling resulted in smaller particles. Rapid milling times of as few as 5 min resulted in substantial decreases in ND diameter, from approximately 1000 nm to approximately 150–200 nm. Combining the milling process with basic pH levels mediated further de-aggregation due to the carboxylated ND surfaces. Longer duration milling times of 5 h produced ND particles with diameters of approximately 10 nm and below. To process the post-milled NDs, straightforward washing steps could thoroughly remove salt/sugar while simultaneously preventing re-aggregation. As such, salt/sugar-assisted milling serves as a powerful methodology in that non-toxic and easily removable agents could be used to produce sub-10 nm diameter particles. This may represent a safe and promising approach for the processing of NDs for downstream biological applications which require rapid/biocompatible ND milling 53.

6 Safety, biocompatibility, and future development

The field of nanomedicine has realized significant advancements by improving therapies for some of the our generation's most challenging physiological disorders 54–56. Among the many approaches being considered by the nanomedicine community, diamond represents a material that unites a multitude of essential drug delivery and imaging properties into one platform. As a result, it has also been shown to be applicable towards sensing and implant coatings 57–61. Regarding ND biocompatibility, in vitro and in vivo studies have been conducted to examine characteristics ranging from cell viability and gene program activity, to in vivo mechanisms, physiological behavior, and localization/distribution 62. The continued development of the ND and diamond film platforms will involve the combination of modeling/simulation studies to better understand unique diamond properties. This knowledge will inform production scale-up and further improvements in the efficacy and safety of drug delivery, imaging, and development of novel devices for sensing using UNCD/NCD, and single/polycrystalline diamond 63–78. In addition, continued advancements in the versatility of surface functionality can mediate new applications in ND particle-based detection. This is exemplified by the recent use of carbohydrate-modified ND particles for the rapid optical detection of E. coli, where a facile mixing/shaking process resulted in cell-mediated particle clustering and the rapid detection of bacteria (Fig. 8). Furthermore, NDs can be harnessed within polymeric matrices, while UNCD coatings can be further functionalized for localized drug delivery applications (Fig. 9). With all of the promising findings resulting from diamond platform research, continued investigations pertaining to material safety will be needed. A recent study, for example, examined DNA damage in embryonic stem cells after exposure to ND particles, which warrants further investigation into the varying impact that NDs may have depending on the biological system used for safety studies 73. Furthermore, there is a need to better understand the long-term effects of biodistribution, clearance, and the identification of optimal applications for initial large animal validation. More specifically, the route and length of time needed for ND clearance from multiple animal models (e.g., rodent, non-rodent mammal, non-human primates), will serve as a key precursor to clinical trials. Emerging studies using the zebrafish model are underway which will also contribute to a better understanding of the in vivo effects of nanodiamonds 74. Important ND properties such as NV center photostability, utilized in long-term cell tracking through multiple cell division cycles and exocytosis monitoring, may be useful during the course of ND localization, distribution, and safety studies 75. Therefore, continued investigation into this crucial aspect of ND safety will serve as a foundation for the future of this exciting material.

Figure 8.

(online color at: www.pss-a.com) Carbohydrate (mannose)-coated nanodiamonds can be used for bacterial agglutination to result in optically detectable bacterial presence following a rapid mixing process between E. coli and carbohydrate-functionalized ND particles. This may serve as a rapid sensing technique by harnessing ND surface chemistry. Figure reprinted from Ref. 42.

Figure 9.

(online color at: www.pss-a.com) An uncoated ultrananocrystalline diamond (UNCD) film (top), and collagen/anti-inflammatory therapeutic film functionalizing a UNCD film (below) are shown. Drug releasing UNCD coatings may further prolong device functionality by suppressing adverse effects following implantation. Figure reprinted from Ref. 70.

Acknowledgements

D.H. gratefully acknowledges support from the National Science Foundation CAREER Award (CMMI-0846323), Mechanics of Materials grant CMMI-0856492, Center for Scalable and Integrated NanoManufacturing (DMI-0327077), DMR-1105060, V Foundation for Cancer Research Scholars Award, Wallace H. Coulter Foundation Translational Research Award, American Chemical Society Petroleum Research Fund Grant 47121-G10, and National Cancer Institute grant U54CA151880 (The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health), and European Commission funding program FP7-KBBE-2009-3.

Biographical Information

Han Bin Man is a fourth year PhD candidate in Mechanical Engineering at Northwestern University. He is currently working in Professor Dean Ho's laboratory studying the applications of nanodiamonds (NDs) for cancer therapy. In addition to investigating the use of NDs as drug delivery vehicles to treat various chemo-resistant cancers, he is studying the use of polymer-functionalized NDs as therapeutic gene-delivery agents via both experiments and simulations. He obtained a BS from the California Institute of Technology in 2009 with a wide range of previous research experience from modeling wind flow on Saturn's moon, titan, to testing carbon nanotube electrical properties.

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Biographical Information

Dr. Dean Ho is currently a Professor in the Division of Oral Biology and Medicine, Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, and Co-Director of the Jane and Jerry Weintraub Center for Reconstructive Biotechnology at the UCLA School of Dentistry. His research team is focused on the development of nanodiamond platforms for targeted drug delivery and imaging. He is a recipient of the National Science Foundation CAREER Award, Wallace H. Coulter Foundation Translational Research Award, V Foundation for Cancer Research V Scholars Award, John G. Bollinger Outstanding Young Manufacturing Engineer Award of the Society of Manufacturing Engineers, Distinguished Young Alumnus Award from the UCLA School of Engineering and Applied Science and an invited attendee of the National Academy of Engineering Frontiers of Engineering Symposium.

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