Biocompatibility Assessment of Detonation Nanodiamond in Non

Biocompatibility Assessment of Detonation Nanodiamond in Non-Human Primates and Rats Using Histological, Hematologic, and Urine Analysis Laura Moore,†,⬡ Junyu Yang,‡,⬡ Thanh T. Ha Lan,§ Eiji Osawa,∥ Dong-Keun Lee,⊥ William D. Johnson,# Jianzhong Xi,*,‡ Edward Kai-Hua Chow,*,¶,△ and Dean Ho*,⊥,▲,○,●,□ †

Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 60208, United States Department of Biomedical Engineering, Peking University, Beijing, China 100871 § Alverno Clinical Laboratories, Hammond, Indiana 46324, United States ∥ NanoCarbon Research Institute, Asama Research Extension Centre, Shinshu University, Ueda, Nagano 386-8567, Japan ⊥ Division of Oral Biology and Medicine, UCLA School of Dentistry, ▲Department of Bioengineering, UCLA School of Engineering, ○ The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, ●California NanoSystems Institute, and □Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095, United States # Life Sciences Group, IIT Research Institute, Chicago, Illinois 60616, United States ¶ Cancer Science Institute of Singapore, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599 △ Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117600 ‡

ABSTRACT: Detonation nanodiamonds (DNDs) have been widely explored for biomedical applications ranging from cancer therapy to magnetic resonance imaging due to several promising properties. These include faceted surfaces that mediate potent drug binding and water coordination that have resulted in marked enhancements to the efficacy and safety of drug delivery and imaging. In addition, scalable processing of DNDs yields uniform particles. Furthermore, a broad spectrum of biocompatibility studies has shown that DNDs appear to be well-tolerated. Prior to the clinical translation of DNDs for indications that are addressed via intravenous administration, comprehensive assessment of DND safety in both small and large animal preclinical models is needed. This article reports the results of a DND biocompatibility study in both non-human primates and rats. The rat study was performed as a multiple dose subacute investigation in two cohorts that lasted for 2 weeks and included histological, serum, and urine analysis. The non-human primate study was performed as a dual gender, multiple dose, and long-term investigation in both standard/clinically relevant and elevated dosing cohorts that lasted for 6 months and included comprehensive serum, urine, histological, and body weight analysis. The results from these studies indicate that NDs are well-tolerated at clinically relevant doses. Examination of dose-dependent changes in biomarker levels provides important guidance for the downstream in-human validation of DNDs for clinical drug delivery and imaging. KEYWORDS: nanodiamond, biocompatibility, nanomedicine, biomaterial, carbon he field of nanomedicine has made substantial strides toward the improved treatment and diagnosis of a broad spectrum of disease processes ranging from cancer to infectious diseases.1,2 However, progress has been limited due to the substantial costs associated with drug development. Therefore, in the context of the field of drug development, it is vital to select specific carriers, drugs, and indications where a nanoparticle formulation will mediate improvements over clinical standards. Among the many promising carriers being used for

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applications in nanomedicine, nanodiamonds (NDs) in the form of fluorescent NDs (fNDs) and detonation NDs (DNDs) have recently received increasing attention due to their broad capabilities in photostable cell labeling, drug delivery, and medically relevant imaging, among others.3−38 Received: February 2, 2016 Accepted: July 20, 2016 Published: July 20, 2016 7385

DOI: 10.1021/acsnano.6b00839 ACS Nano 2016, 10, 7385−7400

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Figure 1. Nanodiamond−drug synthesis and characterization. (A) Optical transparency of NDX (2.5 mg of NDs per 1 mL of water) with corresponding final drug concentrations of the NDs in 1 mL of the ND−drug solutions. The final drug concentrations of 1, 2.5, and 5 mg of NDX solutions are, respectively, 919 ± 3.3, 2421 ± 3.3, and 4937 ± 1.8 μg/mL (n = 3). The values are the initial amounts of doxorubicin for attempted incorporation onto 5 mg of NDs. (B) Dynamic light scattering analyses for the ND and NDXs. The blue plots represent analysis on day 0 after synthesis was completed for each sample. The red plots represent analysis on day 3 after the synthesis of NDX. The green plots represent analysis on day 7 after the synthesis of NDX. (C) Fourier transform infrared spectra of (a) nanodiamond (ND), (b) doxorubicin (DOX), (c) 1 mg of NDX, (d) 2.5 mg of NDX, and (e) 5 mg of NDX.

largely performed using in vitro, ex vivo, or murine models. Therefore, the continued development of NDs for clinical administration requires the assessment of their tolerance in large animal models with comprehensive blood and urine analysis, among other indicators. In addition, the implementation of a dual gender and long-term large animal study is important due to the fact that substantial differences in normal serum and urine reference levels can exist between male and female subjects. This study reports the results from contract research organization (CRO)-partnered rat DND and non-human primate biocompatibility studies. For the rat study, male CD-1:IGS rats were administered a bolus intravenous injection once weekly for 2 weeks; three cohorts were each administered a standard (1 mg), higher (2 mg) DND dose or vehicle control dose. A blinded histopathology analysis was subsequently conducted on isolated rat tissue sections. For the non-human primate study, male and female cynomolgus monkeys were administered an intravenous injection once monthly for a duration of 6 months; two cohorts were each administered either a standard (15 mg/kg) or elevated (25 mg/kg) DND dose, and blood and urine draws were

As NDs continue to progress toward clinical translation, DNDs, in particular, have emerged as particularly promising carriers for drug delivery and magnetic resonance imaging due to their faceted surfaces and uniform particle architecture. In particular, DNDs are capable of potently binding anthracyclines and sustaining their release without the need to permanently modify either the DND surface or the drug itself.13,16 These DND−anthracycline complexes were capable of markedly improving drug tolerance and mediating maximally efficacious tumor reduction using drug dosages that were lethal when delivered on their own. In the area of medically relevant imaging, DNDs were used to carry gadolinium(III) (Gd(III)), resulting in a 1 order of magnitude increase in per-Gd(III) relaxivity for magnetic resonance imaging (MRI) applications.14 This was among the largest increase ever reported for per-gadolinium relaxivity, compared to all nanoparticle and clinical MRI contrast agents. In order to evaluate ND biocompatibility, numerous cell lines and preclinical models have been used as testing platforms for several informative studies.11,13,24,36,38−42 These studies were 7386


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Figure 2. Complete hematological and coagulation profile of rats treated with detonation nanodiamonds. Rats treated twice weekly with vehicle control (n = 5), 1 mg of ND (n = 5), or 2 mg of ND (n = 5) were subjected to a complete hematological analysis on days 3 and 10. No evidence of systemic toxicity, inflammation, or coagulopathy was observed.

nanopure water (non-human primates) using a probe sonicator. For both studies, subject dosing was calculated based on the human equivalent dose of doxorubicin delivered as nanodiamond−doxorubicin for chemotherapy.43 Of note, this dosing does not take into account the improved efficacy that has been observed with nanodiamond−doxorubicin and may therefore overestimate the human dose.16 Nanodiamond characterization was carried out using unmodified NDs, which were tested for biocompatibility in vivo in this study, as well as ND−doxorubicin complexes (NDX), which will serve as the first ND-based therapeutic complex for clinical validation (Figure 1). This analysis was conducted to compare the properties of unmodified NDs with drug-modified NDs to provide proper context when considering the translational pathway of NDX and the selection of the first disease indication for NDX therapy. In order to examine the dispersibility of NDX

systematically acquired for comprehensive toxicity analysis. At the conclusion of the dosing protocol, the animals were subsequently entered into an observation phase to assess visible changes to their health and monitor possible weight changes. The results indicated that the DNDs were well-tolerated for the duration of both studies and also provided important insight as to the maximum tolerated dose (MTD) and the “no observed adverse effect level” (NOAEL). Therefore, these studies provide an important foundation for the nonclinical risk assessment of DND-based therapies and imaging agents as they progress toward potential in-human validation.

RESULTS AND DISCUSSION Nanodiamond Preparation and Characterization. DNDs were suspended in either phosphate-buffered saline containing 5% bovine serum albumin (BSA) (rodents) or 7387


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many applications in biomedical imaging and drug delivery will require that DND complexes are dosed multiple times. For example, standard treatment regimens for anthracycline chemotherapeutics require dosing every 1−2 weeks for 4−8 weeks.44 Therefore, these preclinical safety studies were conducted to evaluate a more chronic response to serial treatment with DNDs at both standard and elevated dosages. Three cohorts of male CD-1:IGS rats were administered either 1 or 2 mg of DNDs in saline containing 5% BSA or vehicle control weekly for 2 weeks. Rats were subjected to complete serum chemistry, hematology, and urine analysis 3 days following each DND treatment. Upon study completion, blood samples were assessed for variations in serum chemistry parameters, and selected organs were sectioned for histological analysis. The DNDs appeared to be well-tolerated in both dosing cohorts and at all time points. All of the hematologic and urinalysis parameters measured indicated that the DNDs did not cause systemic inflammatory, toxic, or pro-coagulant responses and that all major organ systems maintained normal function. Hematologic testing indicated that DND treatment did not cause any changes to white blood cell and platelet counts or white blood cell differential counts (Figure 2). These results collectively demonstrated that DND treatment did not cause subacute inflammation. We observed a modest depression in the red blood cell count, hemoglobin, and hematocrit on day 3 that was accompanied by a 5% increase in reticulocyte count in both DND-treated groups (Figure 2). These results suggested that the DND-treated animals were mildly anemic, which may have been caused by an extra blood draw that the treated animals received on day 1. It should be noted that all of these indices normalized by day 10, suggesting they were not treatment-dependent effects. The variation in red blood cell indices was the only substantial change in hematologic parameters that was observed. In addition to assessing the effect on the hematologic profile of the rats, this study also addressed the impact DNDs have on the coagulation system. For any injectable molecule or material, it is vital to address the function of the coagulation system because any variation can rapidly cause mortality. To address this issue, this study measured clotting times and fibrinogen levels after DND treatment (Figure 2). Prothrombin time, which measures the ability to initiate the coagulation cascade, was within normal limits for all subjects tested and not substantially different between the treatment groups. Additionally, we observed that the fibrinogen concentrations were within normal limits and not substantially different between treatment groups (Figure 2). Fibrinogen is a protein necessary for mediating clot formation and consolidation. However, its systemic concentration is strongly correlated with systemic inflammation and coagulation disorders. The observation of normal serum fibrinogen levels in the treated subjects confirmed the absence of systemic inflammation or evidence of disruption of the coagulation system. The hematological and coagulation studies demonstrated that the DND-treated rats did not experience any catastrophic responses, such as severe inflammation or massive coagulation that required early removal from the study. We were also able to assess the response of several major organ systems through the analysis of multiple serum chemistry markers. Liver function testing was conducted because DNDs, like many other nanoparticles, appear to accumulate in the liver.13,16 A conventional clinical liver function panel includes the measurement of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline

in water, vials containing varying concentrations of NDX were prepared against background, and optical transparencies were assessed. All NDX samples were optically transparent and dispersed in water. The amount of DOX loaded onto the ND surface was determined by measuring the absorbance of unbound DOX at 550 nm by UV spectroscopy and subtracting from the initial loading amount. As shown in Figure 1A, the loading efficiencies of 1, 2.5, and 5 mg of NDX were 919 ± 3.3, 2421 ± 3.3, and 4937 ± 1.8 μg/mL, respectively. The confirmation of DOX loading onto the NDs was determined by dynamic light scattering (DLS) and ζ-potential analysis at days 0, 3, and 7. There was no substantial change in the ND cluster size during this time period. The hydrodynamic size and ζ-potential of DND were measured to be 59.0 ± 0.82 nm (0 days), 54.3 ± 0.24 nm (3 days), and 52.7 ± 0.10 nm (7 days) and 50.9 ± 0.82 mV (0 days), 52.5 ± 0.66 mV (3 days), and 51.6 ± 0.81 mV (7 days), respectively (Figure 1B). After incorporation of DOX onto the NDs, the NDX complex sizes increased and the ζ-potential was decreased. The hydrodynamic size and ζpotential of NDX complexes (1 mg) were measured to be 107.5 ± 0.22 nm (0 days), 107.9 ± 0.67 nm (3 days), and 112.1 ± 2.41 nm (7 days) and 43.4 ± 0.57 mV (0 days), 42.3 ± 0.48 mV (3 days), and 42.1 ± 0.50 mV (7 days), while 2.5 and 5 mg of NDX exhibited hydrodynamic sizes of 124.1 ± 1.03 nm (0 days), 126.6.9 ± 1.41 nm (3 days), and 127.2.1 ± 0.70 nm (7 days) and 187.4 ± 1.20 nm (0 days), 188.1 ± 0.28 nm (3 days), and 189.6 ± 0.63 nm (7 days), respectively. In addition, 2.5 and 5 mg of NDX exhibited a ζ-potential of 41.4 ± 0.67 nm (0 days), 39.5 ± 0.80 nm (3 days), and 36.4 ± 0.33 nm (7 days) and 30.6 ± 0.58 nm (0 days), 29.6 ± 0.48 nm (3 days), and 28.4 ± 0.58 nm (7 days), respectively (Figure 1B). In order to confirm drug loading onto the NDs, Fourier transform infrared (FTIR) spectroscopy was performed. The characteristic vibrational peaks of DOX were shown in the spectra of three different concentrations of NDX in Figure 1C. The ND spectrum showed the peaks for CO stretching vibration at 1700−1800 cm−1 and O−H bending vibration peak from 1632 cm−1, which correspond to ketones, esters, lactones, and carboxylic acid functional groups on the ND surface. Furthermore, CH3 asymmetric stretching vibration peaks are also represented at 2940 cm−1. All of these peaks were overlapped with the peaks of the NDX spectra. The NDX spectra were also compared to the peaks associated with the DOX functional groups. Out-of-plane bending vibration of CC−H (780−850 cm−1), out-of-plane bending of N−H (804 cm−1), C−O stretching of the alcohol group (970−1300 cm−1), two stretching vibration of C−O−C (1200−1300 and 890−1000 cm−1), two stretching vibration bands of CC (1560−1590 and 1603− 1616 cm−1), N−H+ in-plane bending vibrations (1614 and 1578 cm−1), and the stretching vibration band of CO (1620−1650 cm−1) were clearly shown on the NDX spectra but not on the ND-only spectrum, indicating the successful incorporation of DOX onto the ND surface. In addition, increasing DOX concentrations were reflected in the vibration peaks that showed corresponding increases in signal intensity in the spectra in Figure 1C. Rodent Safety Study. Previous studies have demonstrated that DNDs and DND-based complexes are well-tolerated during the acute phase of treatment.13 Although these studies provide essential information necessary for clinical translation, they incompletely characterize the response to DND treatment. Additional work has shown that NDs continue to be excreted into the bladder 10 days after initial treatment.16 Additionally, 7388


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Figure 3. Complete serum chemistry analysis of rats treated with detonation nanodiamonds. Rats treated twice weekly with vehicle control (n = 5), 1 mg of ND (n = 5), or 2 mg of ND (n = 5) were subjected to a complete serum chemistry analysis on days 3, 10, and 15. No evidence of abnormal liver or kidney function was observed.

phosphatase (ALP), bilirubin, and γ-glutamyl transpeptidase (GGT). AST and ALT are strong indicators of hepatocyte function and health, whereas LDH and ALP are less specific indicators of liver health that are often increased with systemic inflammation. We observed that ALT was within normal limits for all animals tested (Figure 3). We observed a modest increase in AST levels in 3 out of 10 treated rats. These included one treated with 1 mg of DNDs and two treated with 2 mg of DNDs. However, no statistically significant difference in group means was seen in either of the DND-treated groups compared to the control group. Therefore, the increases were not considered treatment-related or indicative of liver damage. Serum LDH and ALP were also within normal limits, confirming that ND treatment did not induce apparent systemic inflammation or severe liver damage (Figure 3). The observation of normal serum bilirubin and triglyceride levels indicated that the liver’s

metabolic capacity remained intact, while the absence of elevated GGT indicated that the biliary tract was healthy. Furthermore, the lack of depression in albumin and total protein levels and observation of normal prothrombin time indicated that the liver’s synthetic capacity remained intact (Figure 3). Collectively, these results indicated that although the DNDs may accumulate in the liver, they do not cause liver damage or impair function. Renal system function was assessed due to its central role in blood filtration. The standard panel for renal function testing included serum blood urea nitrogen (BUN) and creatinine measurement in combination with a complete urinalysis. Both BUN and creatinine levels were unaffected across the treatment groups, suggesting that the ND treatment did not impair renal filtration functions (Figure 3). The kidney also plays a central role in the regulation of electrolyte balance, which is crucial to virtually all body functions. 7389


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Figure 4. Serum electrolyte analysis of rats treated with detonation nanodiamonds. Rats treated twice weekly with vehicle control (n = 5), 1 mg of ND (n = 5), or 2 mg ND (n = 5) were subjected to a serum chemistry analysis on days 3, 10, and 15. No evidence of alteration in serum electrolytes or glucose was observed.

Overall, we found few changes between the ND-treated groups and controls (Figure 5). The spleen pathology was consistent across the treatment groups, with no evidence of hemorrhage, necrosis, hyperplasia, atrophy, hypertrophy, fibrosis, foreignbody materials, or other significant morphologic alterations. Similar results were observed for the kidney, which was without evidence of atrophy, hypertrophy, hemorrhage, necrosis, inflammation, glomerular sclerosis, ischemic changes, vacuolization, tubular dilation, foreign-body materials, or other significant morphologic alterations. Evaluation of heart sections also showed no evidence of hypertrophy, foreign-body materials, inflammation, necrosis, ischemia, or fibrosis across groups. Lung histopathology showed scattered foci of peribronchial lymphoid aggregates across treated and control subjects. Two treated rats (one at 1 mg of DND, one at 2 mg of DND) showed single, larger foci of chronic inflammation. However, there was no evidence of consolidation, thromboemboli, necrosis, fibrosis, or foreign-body materials. Because of the relatively short study duration and the presence of the chronic findings in only one animal per group, it was unlikely that the DND treatment induced these indicators of chronic changes. The analysis of liver histopathology showed evidence of minor changes in treated groups compared to controls. All groups, including the control rats, showed evidence of mild, patchy microvesicular steatosis and extramedullary hematopoiesis. In the treated groups, we observed increased microvesicular steatosis progressing to steatohepatitis, occasional single-cell necrosis, and cholestasis. These changes were observed in the absence of infarction, hemorrhage, regional necrosis, fibrosis, portal inflammation, and cirrhosis. It is important to note that the changes observed were relatively mild and potentially reversible. Furthermore, the alterations in histopathology were not accompanied by substantial deviations in liver function testing parameters, indicating that the liver continued to function normally. Therefore, the collective hematologic, urine, and histological analysis supported the continued preclinical evaluation of sustained DND administration in a non-human primate model.

We evaluated the standard panel of serum electrolytes and found no substantial differences between the treatment groups (Figure 4). Sodium, potassium, and chloride were consistent across the treatment groups, indicating that the kidneys were maintaining electrolyte balance. Additionally, calcium and phosphate were also within the normal range, which confirmed that the hormonal systems for calcium and phosphate regulation were functioning properly. Additionally, the lack of elevation in calcium or phosphate levels suggested that there were no ongoing processes causing bone degeneration. Furthermore, because serum glucose was not substantially different between the treatment groups, we could infer that DND treatment did not adversely affect the systems that regulate blood glucose. Consistent with standard clinical studies, comprehensive urinalysis was performed. The urine was also assessed microscopically for casts, epithelial cells, phosphate crystals, bacteria, red blood cells, and white blood cells. The only significant difference found between the treatment groups was a modest decrease in specific gravity and refractive index in the 1 mg cohort on day 10. Because this effect was not dose-dependent, it was not considered to be treatment-related. It is important to note that all of the other measured parameters were within normal limits and did not vary between the treatment groups. Overall, the results from the hematologic and serum chemistry analyses indicated that the DNDs were well-tolerated by all treatment groups. Rodent Histological Analysis. In addition to assessing the blood and urine indicators of disease, we also conducted a histological evaluation of several organs, including the liver, spleen, kidney, lungs, and heart. The liver and spleen were chosen because of the role of the reticuloendothelial system in removing nanoparticles from circulation. We also assessed kidney samples due to the renal system’s role in blood filtration and homeostatic maintenance. The lungs were assessed because of the potential for nanoparticle-based emboli formation following intravenous injections. Finally, we selected the heart because of its interaction with more blood, and therefore more intravenously injected DNDs, than any other organ in the body. 7390


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Figure 5. Histopathology of rats treated with detonation nanodiamonds. Representative images of hematoxylin and eosin stain of liver, spleen, kidney, lung, and heart obtained on day 15 from rats treated twice weekly with vehicle control (n = 5), 1 mg of ND (n = 5), or 2 mg ND (n = 5). Images were taken at 10× (liver, spleen, lung) or 20× (kidney, heart).

Body Weight Analysis Following Nanodiamond Administration in a Non-Human Primate Model. During the course of the study, all of the treatment groups (male and female, control, 15 and 25 mg/kg DND) were monitored for body weight and appetite. There were no apparent acute or sustained fluctuations in animal body weight during the entire study period, and no apparent changes to animal appetite (Figure 6). This served as an indicator that DND administration did not appear to cause severe systemic toxicity or adversely affect the nutrient transport or digestive systems, which are critically important for their clinical implementation. Complete Blood Count (CBC) Analysis Following Detonation Nanodiamond Administration in a NonHuman Primate Model. As is standard clinical practice, a complete blood count was obtained on all subjects in order to assess the overall health and screen for inflammation, hemolysis, and systemic toxicity and any other effects (Figure 6). Overall,

Detonation Nanodiamond Administration in a NonHuman Primate Model. Non-human primate test subjects were treated monthly for a period of 6 months, with scheduled serum draws taken before and 1 week after each DND dosing event in order to monitor the acute and long-term effects of DND administration. Test subjects were randomized into control and two treatment groups, a standard dose of 15 mg/ kg and an elevated dose of 25 mg/kg. For the purposes of discussing animal responses to treatment, they will be referred to by an identifier (e.g., 080118) followed by gender (M/F) and standard or elevated dosing (S/E) in parentheses. Reference values for non-human primate serum and urine analysis were identified through prior studies and provided where applicable following the abbreviation of each assayed marker.45−49 It should be noted that these reference values can vary substantially between studies based on subject gender, age, diet, baseline health status, and other factors. 7391


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Figure 6. Hematologic parameter analysis and weights of monkeys treated with detonation nanodiamonds. Monkeys were treated monthly with either a standard dose (15 mg/kg) or a high dose (25 mg/kg) of NDs. Blood was sampled prior to each treatment and 1 week after treatment. Statistically significant differences in platelet count were noted at multiple time points (*p < 0.05, **p < 0.01). All other parameters were nonsignificant.

levels. The difference between groups appears to be caused by an increase in total platelets in the control group rather than a reduction in the treated groups as the treated groups maintained values consistent with their pretreatment baseline values. Therefore, this finding was not attributed to DND administration.

the only statistically significant variation in hematological parameters was in the platelet count (Figure 6, PLT). Samples taken 1 week after doses 2, 5, and 6 revealed higher platelet counts in the control group compared to both treatment groups (reference 382.13 ± 91.97 × 109/L). Of note, PLT readings had large fluctuations in all test subjects, which is consistent with the large standard deviations observed with the normal reference 7392


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Figure 7. Complete serum chemistry analysis of monkeys treated with detonation nanodiamonds. Monkeys were treated monthly with either a standard dose (15 mg/kg) or a high dose (25 mg/kg) of NDs. Blood was sampled prior to each treatment and 1 week after treatment. No statistically significant variations in parameters were observed.

(RDW-CV, 14.75 ± 1.28%) were assessed (Figure 6). Aside from test subject 070473(M/S) who had one elevated RDW-CV

As a part of the standard CBC, red blood cell counts (RBC, 6.22 ± 0.45 × 1012/L) and red blood cell distribution width 7393


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However, both subjects settled into acceptable LDH levels relative to their respective normal ranges. No apparent trends or sustained fluctuations in LDH were observed with the other test subjects. Control subject 071837(M) and treatment subjects 071079(M/S) and 080410(F/E) showed transient elevations in ALP (reference 623 ± 297 U/L) prior to the fourth injection; however, they all returned to their normal range within 1 week. Given that these fluctuations in inflammatory markers were all transient and occurred across control and treatment groups, it is unlikely that they were due to DND treatment. Overall, DNDs do not appear to cause systemic inflammation or substantial cell death at either the standard or elevated treatment doses. As discussed above, it was important to assess both liver and kidney function due to their roles in metabolism and excretion. The liver is particularly important, as virtually all nanoparticles, including DNDs, appear to accumulate in the liver. Multiple serum markers were utilized to assess liver function (Figure 7). Serum ALT and AST were used to assess hepatocyte health and function. Subject 070696(F/E) and control subject 071691(M) exhibited temporarily elevated ALT levels (reference 48.06 ± 20.00 U/L) following the initial dosing, but both subjects completed the study with normal ALT readings relative to their respective starting ALT levels. No apparent adverse events were observed. AST levels (reference 50.60 ± 22.63 U/L) were also assessed, and no apparent abnormalities in AST levels were observed outside of normal physiologic fluctuation. The liver’s metabolic capacity was assessed through total bilirubin (TBIL), triglycerides (TG), and cholesterol (CHOL). Analysis of TBIL levels (reference 3 ± 1 μmol/L) showed that subject 071805(M/E) and control subject 071669(M) exhibited temporary increases in TBIL levels that settled back into their respective normal ranges. No apparent trends or adverse events were observed with the test subjects (Figure 7). CHOL levels (reference 3.28 ± 0.67 mmol/L) were assessed in all subjects, and aside from subject 080118(F/S) exhibiting elevated levels from the beginning through the end of the study and control subject 071837(M) displaying a transient increase in CHOL levels, no adverse events or trends were reported. Similar results were obtained for serum TG levels (reference 0.32 ± 0.17 mmol/ L), where two control subjects exhibited elevated levels, but no adverse trends were reported in DND-treated subjects (Figure 7). The liver’s synthetic capacity was assessed using total protein (TP) and albumin levels (ALB). Of note, TP and ALB may also be used as indicators of the kidney’s filtration capacity. Analysis of TP levels (reference 81.49 ± 4.16 g/L) showed that test subject 071805(M/E) and control subject 071837(M) exhibited one elevated reading; however, TP levels returned to normal ranges in both animals with no apparent fluctuation trends. Measurement of ALB levels (reference 43.72 ± 3.39 g/L) showed no apparent fluctuation trends or adverse events aside from transiently elevated levels in subjects 080118(F/S) and 071805(M/E). These findings provide an indication that no apparent damage to the liver was induced in both the standard and elevated DND dosing cohorts for both genders (Figure 7). Kidney function was assessed using an array of serum chemistry markers including creatinine (CREA), BUN, and serum electrolytes (Figure 7). CREA (reference 63.82 ± 9.31 μmol/L) analysis revealed no apparent trends or substantial decreases or increases in CREA serum levels with the exception of one elevated reading in control subject 071837(M) that rapidly decreased to normal levels. Of note, BUN (reference 8.19 ± 1.44 mmol/L) analysis did show substantial variability in blood

reading that settled back to a normal level relative to the subject’s starting and ending RDW-CV levels, no substantial or sustained deviations were observed with all of the test subjects. Hemoglobin (HGB, 141.88 ± 10.53 g/L) analysis revealed fluctuations in all test subjects that were consistent with the normal reference level standard deviations. Test subjects 071805(M/E) and 070473(M/S) temporarily exhibited higher than normal HGB levels, although both subjects ended the study with normal HGB levels relative to their respective baseline values. Hematocrit (HCT, 48.98 ± 2.94%) levels showed negligible fluctuations, while mean corpuscular volume (MCV, 78.84 ± 3.34 fL) readings revealed higher values at the end of treatment. Mean corpuscular hemoglobin concentration (MCHC, 289.49 ± 10.46 g/L) analysis showed that the test subjects generally exhibited decreasing trends that converged toward the normal range, while mean corpuscular hemoglobin (MHC, 22.81 ± 1.11 pg) readings showed very little fluctuation within each test subject. Combined, these findings indicate that the test subjects were not anemic and did not experience a hemolytic response to the nanoparticle infusions. This is consistent with the subjective observation that none of the animals appeared to be anemic and exhibited normal overall health. White blood cell counts (WBC, 13.31 ± 4.41 × 109/L) and neutrophil counts (NEU, 6.43 ± 3.72 × 109/L, 46.66 ± 14.37%) were also measured as a part of the CBC (Figure 6). The substantial standard deviation in the reference levels was reflected in the measurements in the test subjects, with the control, standard dosing, and elevated dosing subjects all exhibiting substantial fluctuations. However, no apparent escalation or depression trends in WBC or NEU levels were observed during the course of the study. Lymphocyte counts (LYM, 6.00 ± 2.38 × 109/L, 45.96 ± 13.20%) reflected the large standard deviations that were observed with the reference level standards. While some subjects exhibited WBC, NEU, and LYM counts that were below the expected normal range, no trends in the values were observed. These findings are unlikely to be related to DND administration as the values were observed in all groups over the entire course of treatment. The collective CBC panel provided important insight into the overall health of the test subjects. Despite occasional fluctuations in CBC marker levels during the study, there were no trends that were directly attributable to DND administration. Furthermore, all subjects maintained their weight throughout the study. The absence of a clear inflammatory response, anemia, or systemic toxicity indicated that DND administration was well-tolerated. Serum Chemistry Analysis Following Detonation Nanodiamond Administration in a Non-Human Primate Model. In addition to the hematological analysis, this study included a complete serum chemistry assessment to determine if DND administration had any adverse effects on specific organ systems (Figures 6 and 7). C-reactive protein (CRP), LDH, and ALP were used as markers for systemic inflammation and cell death (Figure 6). There were no treatment related variations in CRP, LDH, or ALP. Subjects 070473(M/S) and 080118(F/S) as well as control-treated subject 071669(M) exhibited temporary increases in CRP levels (reference 0−8.3 mg/L); however, levels returned to baseline ranges. Additionally, CRP levels remained consistently low in all elevated dosing subjects throughout the course of treatment, suggesting minimal to no effect of DNDs on CRP-related inflammation. LDH (reference 604.72 ± 218.04 U/ L) testing showed that subject 070476(F/S) and control subject 071691(M) exhibited spikes in their respective LDH levels. 7394


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Figure 8. Urinalysis of monkeys treated with detonation nanodiamonds. Monkeys were treated monthly with either a standard dose (15 mg/kg) or a high dose (25 mg/kg) of NDs. Blood was sampled prior to each treatment and 1 week after treatment. No statistically significant variations in parameters were observed.

but no apparent trends with regard to sustained increases or decreases. Overall, while fluctuations in serum markers for kidney function were observed, virtually all of these fluctuations converged back within a reasonable range relative to each subject’s initial levels. There were no apparent trends observed with regard to long-term/sustained effects that were dependent upon DND administration, indicating that the fluctuations may have been due to transient kidney activity during DND processing. Overall, while fluctuations in serum electrolytes, markers for kidney function, were observed, they were not sustained. Virtually all of these fluctuations returned to a level that was within a normal range relative to each subject’s baseline measurement. There were no apparent trends to suggest DND-mediated long-term/sustained effects, indicating that the fluctuations may have been due to transient kidney activity during DND processing. Prior studies have shown that there are no apparent adverse histological effects following DND administration as well as the administration of other classes of nanomaterials in non-human primates.16,50,51 Coupled with the longer-term analysis of kidney function presented in this study, it appears that DNDs do not cause sustained disruption to renal activity during a 6 month DND dosing period. With regard to the

levels at each reading. However, this was observed for all test subjects, including the controls. The same response was observed with blood glucose (GLU, reference 3.49 ± 1.05 mmol/L) with no apparent trends or deviations that were clearly DND administration-dependent. As the kidney is the main regulator of electrolyte balance, assessment of serum electrolyte concentrations is a key step in assessing kidney function. Perhaps, the most important electrolyte to test is phosphate (P), as levels can rise dangerously high in kidney failure. P level testing (reference 2.27 ± 0.39 mmol/L) showed that test subject 080118(F/S) and control subject 071691 each had one noticeable drop in P levels, but both subjects converged within reference levels comparable to all of the other test subjects. Despite the fluctuations that were observed during the assessment of these serum chemistry markers, the fluctuations were not sustained, and no adverse events were reported. In addition, if any of the fluctuations observed were dose-dependent, there was no evidence of persistent deviations from the respective normal ranges of the test subjects. The analysis of potassium (K, reference 4.2 ± 0.8 mEq/L), calcium (Ca, reference 9.7 ± 0.10 mEq/L), chloride (Cl, reference 107.9 ± 5.7 mEq/L), and sodium (Na, reference 149.2 ± 5.7 mEq/L) revealed transient elevations in serum levels 7395


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Figure 9. Histologic analysis of monkeys treated with detonation nanodiamonds. Monkeys were treated monthly with either a standard dose (15 mg/kg) or a high dose (25 mg/kg) of NDs. Tissue samples from the liver, kidney, lung, heart, and spleen were obtained at 6 months from control, standard dose (both genders), and elevated dose (both genders) for analysis.

(Figure 8) levels were normal for all subjects. All subjects tested negative for urinary bilirubin, with the exception of treatment subject 071805(M/E), in which positive readings were present at two time points but completed the study with consistently negative BIL tests. There was substantial variability in urine protein levels (uPRO) for all subjects. Testing revealed transiently positive uPRO readings for all subjects, including control subjects, indicating that the protein presence was not likely due to treatment. All subjects tested positive for urine leukocytes and erythrocytes (uLEU, uERY, Figure 8) at least once over the course of the study. Low levels of uLEU are considered normal for healthy subjects. Although the average uLEU levels were higher in the standard treatment group compared to the control group, the difference was not statistically significant. Of note, all subjects except one (080410(F/S)) completed the study with uLEU values less than or equal to their pretreatment levels. Similar to the uLEU testing, uERY testing showed substantial variability in all subjects without any significant trends over the course of the study. Although there were no significant trends in uLEU or uERY levels, the variability in both merits further evaluation. Finally, there was substantial variability in all urine electrolytes (sodium, potassium, chloride, and calcium, Figure 8). Electrolyte levels may vary based on diet and hydration status. However, there were no obvious trends in any of the electrolytes based on treatment group or time points. Additionally, there was no evidence of kidney damage or infection during the course of the study. Collectively, the urinalysis and serum chemistry data confirm that both the liver and kidneys of all study subjects were

impact of elevated DND dosing (25 mg/kg) on serum chemistry compared to standard DND dosing (15 mg/kg), no obvious sustained dosage-dependent fluctuation trends were observed. However, some acute instances of elevated serum marker readings (e.g., ALT, TBIL, LDH) following elevated DND dosing may provide important information toward the determination of MTD and NOAEL. While the 25 mg/kg dose may potentially reside above the NOAEL threshold, it is below the MTD as there was no mortality or even dose-limiting toxicities identified at that dose. Further tests will help to confirm the MTD dose prior to clinical validation. Urinalysis Following Detonation Nanodiamond Administration in a Non-Human Primate Model. Urine from all subjects was assessed at the same time points that hematologic and serum chemistry analyses were completed (Figure 8). Reference values for urinalysis in cynomolgus monkeys were based on prior reference point establishment studies.52 Specific gravity (reference 1.002−1.03) measurements were normal for all subjects during the course of treatment. Urine pH (reference 4.6−8.2) readings showed that subjects 071425(M/E), 071079(M/S), 070473(M/S), and 080118(F/S) each reached a urine pH level of 9, which is above the normal range, at one time point (Figure 8). However, all of these subjects completed the study with normal urine pH levels. Urine nitrite tests came back negative for all subjects, indicating that none of the subjects had urinary tract infections. Urine glucose (uGLU) readings were negative for nearly all test subjects. One control subject (071691(M)) and one treatment subject (070508(F/E)) tested positive for uGLU at one time point each. However, both subjects completed the study with negative uGLU. Urobilinogen 7396


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and 25 mg/kg utilized in this study serves as a foundation for clinical translation of DND-based therapeutic and contrast agents. Future work will include the completion of non-human primate histology to further confirm material tolerance. Additionally, comprehensive pharmacokinetic analysis and the establishment of chemistry, manufacturing, and controls compliance are required for the establishment of in-human validation protocols.

functioning normally. In addition, there were no substantial differences in outcomes between the standard and elevated DND administration cohorts, providing strong support for the implementation of the estimated clinical DND dosages that are between 6.75 and 13.5 mg/kg.16 Non-Human Primate Histopathology Analysis. Tissue analysis was conducted for one control (071691(M)), two standard dose monkeys (080118(F) and 071079(M)), and two elevated dose monkeys (070696(F) and 071425(M)). Tissue from the liver, kidney, lung, heart, and spleen were stained with hematoxylin and eosin (Figure 9). No gross abnormality including hemorrhage was noted in the standard and elevated ND dose monkeys compared to the controls. Histologic examination of heart sections taken from monkeys given the elevated ND dose demonstrated increased nucleomegaly and muscle fiber hypertrophy compared to the controls. However, heart sections taken from the standard ND dose monkeys showed only mild changes not substantially different from the controls. The liver parenchyma from the elevated ND dose monkeys showed evidence of prominent capillary congestion and dilatation compared to the control animals. Morphologically similar but less advanced changes were also seen in the standard ND dose monkeys. However, hematologic analysis for liver function did not reveal any apparent organ dysfunction. Lung sections from the standard and elevated ND dose monkeys showed evidence of alveolar capillary congestion and dilatation. Control monkeys, however, also displayed similar morphology, suggesting morphological effects related to all groups of monkeys used in this study. There are no appreciable morphologic alternations in the kidneys and spleens from the standard dose and elevated ND dose monkeys compared to the control group. As previously noted, the estimated DND dosage will be between 6.75 and 13.5 mg/kg, which are both below the standard DND dose administered in this study and substantially below the 50 mg/kg elevated dose. Furthermore, it should be noted that DND-based therapies will likely be administered in combination with other drugs, further reducing their dosage. The emergence of powerful combination therapy optimization technologies will result in multidrug, nanomedicine-based therapeutics with drugdose ratios that result in maximally efficacious and safe treatment.10,15,21,53,54

MATERIALS AND METHODS Materials. Detonation nanodiamonds were purchased from the NanoCarbon Research Institute Ltd. (Nagano, Japan), and 10 mg/mL concentrations were probe sonicated in an ice bath prior to intravenous administration (30 s up to 42 Hz). Doxorubicin hydrochloride was obtained from Sigma-Aldrich (Milwaukee, WI, USA). Nanodiamonds, solvents, and all reagents were sterilized prior to use. Unless otherwise specified, all materials were purchased from VWR (Radnor, PA). Synthesis and Characterization of Nanodiamond-Modified Drugs. In order to synthesize the NDX complexes with various concentrations of doxorubicin, 5 mg of doxorubicin was prepared by mixing with 1 mL of sterilized water. This was followed with the addition of autoclaved NDs (5 mg/mL) at a ratio of 1:5 (doxorubicin/ND, w/w), 1:2 (w/w), and 1:1 (w/w). NaOH solution (1 M) was then added to the NDX complexes to induce interactions between NDs and doxorubicin until the final concentration of NaOH was 2.5 mM. The NDX complexes were mixed homogeneously and incubated at room temperature in the dark. After 3 days of incubation, ND−drug suspensions were centrifuged and desalinated by deionized water until transparent ND−drug solutions were obtained. The resulting final NDX complexes were resuspended in water with concentrations of 2.5 mg/ mL (ND/water, w/v). To verify the presence of DOX on the ND surface, FTIR was performed (PerkinElmer FTIR Spectrum 2000, Waltham, MA, USA). For the FTIR analysis, 1 mg of ND, DOX, and NDXs was mixed with 100 mg of potassium bromide (KBr) and pelletized by pressing the mixture of sample and KBr. The FTIR spectra were subsequently recorded with a resolution of 1 cm−1 and an accumulation of 128 scans over the wavenumber range of 500−4000 cm−1. To determine the amount of drug loaded onto the NDs, the supernatants collected during the washing/centrifuging steps were analyzed by UV absorption spectroscopy (Synergy/H1, Biotek, Winooski, VT, USA). The hydrodynamic diameter and ζ-potential of the ND and NDXs were measured by using a Zetasizer Nano ZS (Malvern Instruments, UK). Nanoparticle sizes were measured at a 173° backscattering angle with more than three runs at 25 °C, and they were determined from the average of the more than triplicate runs of the particles’ z-average values. The ζ-potential was also determined with automatic mode at 25 °C in water using DTS-1070C clear zeta cells. Multidose ND Toxicity in Rats. Toxicity studies were performed in conjunction with the FDA-compliant CRO at the Illinois Institute of Technology Research Institute (IITRI). Male CD-1:IGS rats (139−155 g; Charles River Laboratories, Wilmington, MA) were administered 1 mg of ND, 2 mg of ND, or vehicle control (n = 5) once weekly (days 1 and 8) for 2 weeks. Autoclave-sterilized NDs were suspended at 5 mg/ mL in phosphate-buffered saline containing 5% BSA (Sigma-Aldrich, St. Louis, MO) and administered as a bolus. Fasting blood samples for clinical chemistry, hematology, and coagulation were acquired on days 3, 10, and 15 by retro-orbital sinus puncture. Overnight urine samples were also collected on days 3 and 10 and subjected to microscopy and urine chemistry. On day 15, all animals were sacrificed and underwent a complete necropsy. Additional blood was drawn from rats treated with NDs by retro-orbital puncture on day 1 in order to obtain a baseline for pharmacokinetic studies. Rat Histology. Liver, spleen, heart, lungs, and kidney sections were fixed in 10% neutral buffered formalin at necropsy, embedded in paraffin, and sectioned prior to staining with hematoxylin and eosin using standard protocols. Analysis was conducted using an Olympus BX41 microscope.

CONCLUSION This study provided key insight into the biocompatibility of preclinical DND administration in a non-human primate and a rat model. To our knowledge, the findings reported in this work are comprehensive with regard to the extent of DND safety characterization, particularly given the substantial testing parameters, long study duration, and repeated dosing scheme. This study evaluated both male and female cynomolgus monkeys with two DND dosing levels, a standard dose of 15 mg/kg (n = 5) and an elevated dose of 25 mg/kg (n = 4), in addition to controls (n = 3). These results provide important insight into downstream maximum tolerated dose and no apparent adverse effect level of DND administration that will be confirmed in future studies. The collective findings from the multidose rodent and non-human primate studies, with subacute and chronic durations, indicate that DNDs are well-tolerated. The estimated clinical DND monotherapy dosage will be between 6.75 and 13.5 mg/kg. In addition, the implementation of powerful combination therapy optimization technologies to optimize DND-containing multidrug treatment will likely further reduce the DND and drug dosage. Therefore, the absence of organ dysfunction at both 15 7397


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Non-Human Primate Care and Identification. Cynomolgus monkeys of both genders with ages ranging from 3 to 6 years were used for the study. Three control subjects were utilized for the study and maintained by Kunming Biomed International (KBI), a CRO accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). They were assigned identifier numbers 071691(M), 071837(M), and 071669(M). Test subjects given a standard dose of detonation nanodiamonds (15 mg/kg) were assigned identifier numbers 080118(F), 071079(M), 070473(M), 070476(F), and 080410(F). Test subjects given an elevated dose of detonation nanodiamonds (25 mg/kg) were assigned identifier numbers 071425(M), 071805(M), 070696(F), and 070508(F). Subjects were provided with controlled rooms compliant with animal welfare guidelines with rigorous environmental controls to maintain temperature (18−26 °C), humidity (30−70%), air circulation, and light−dark cycle (12/12 h). Monkeys were fed a welfare guidelinecompliant commercial monkey diet and had water available whenever needed. The testing protocol was reviewed and approved by the KBI Institutional Animal Care and Use Committee (IACUC). Adverse events were defined as conditions where the subjects displayed any indication of significant toxicity or intolerance of detonation nanodiamonds. Study directives stipulated that the observation of adverse events was to result in immediate removal from the study with immediate efforts made to ensure property care for the animal in compliance with established guidelines for animal welfare. Criteria for subject removal from the study included weight loss greater than 25%, anorexia for >5 days, the inability to ambulate to feed or obtain water, a moribund state, infection not responding to antibiotic therapy, and indications of specific organ system dysfunction including cyanosis, dyspnea, seizure, or nonhealing wounds. Non-Human Primate Dosing Protocol. Each monkey was given a total of 6 doses of DNDs at either 15 or 25 mg/kg. Fasting blood and urine samples were obtained prior to each DND administration event to provide necessary baseline information. Three weeks after the last DND dose, fasting blood and urine samples were obtained. The study subjects in each treatment group were treated for a duration of 6 months. Blood and urine were collected for serum chemistry, complete blood count, and urinalysis. Monkeys in this dual gender multidose study were deemed healthy enough to complete the study based on initial serum and urine tests prior to the first DND dose. Of note, one subject that was initially randomized to the elevated dose treatment group succumbed to illness that was deemed unrelated to DND administration. Due to the confounding variable of the subject’s illness, it was not included in any of the final analyses. Non-Human Primate Serum and Urine Analysis. Serum and urine samples were obtained using conventional equipment and procedures that have been previously outlined.49 Serum chemistry, complete blood count, urinalysis, weight monitoring, and observation period assessment were conducted to comprehensively characterize test subject health. Non-Human Primate Histopathology Analysis. Immediately following euthanasia, harvested tissues were fixed in 10% neutralbuffered formalin for a minimum of 48 h. Samples were subsequently dehydrated in increasing concentrations of ethanol and embedded in paraffin wax. Tissue sections that were 4−5 μm thick were adhered to positively charged glass slides. Sections were routinely stained with hematoxylin and eosin for evaluation. All slides were evaluated using a Nikon Eclipse 80i upright microscope (LHS-N100C-1, Nikon Instruments Inc., Japan). Statistics. All analyses were performed in GraphPad Prism using two-way ANOVA using the Bonferroni correction for multiple comparisons. A repeated measures analysis was used for the nonhuman primate studies to take into account between subject variation. Data were considered significant at p < 0.05.

Author Contributions ⬡

L.M. and J.Y. contributed equally to this work.

Notes

The authors declare the following competing financial interest(s): E.O., D.-K.L., E.K.-H.C., and D.H. are co-inventors of multiple patents pertaining to nanodiamond processing, functionalization, and biomedical applications.

ACKNOWLEDGMENTS L.M. gratefully acknowledges Drs. Andrew Mazar and Irawati Kandela of the Northwestern University Center for Developmental Therapeutics, the Mouse Histology and Phenotyping Core at Northwestern University, and Dr. Bill Johnson of the Illinois Institute of Technology Research Institute (IITRI) for helpful discussions. L.M. also gratefully acknowledges funding support from National Cancer Institute Grant 1F30CA17415601. J.X. acknowledges funding support from the College of Engineering, Peking University, as well as the NSFC (Grant No. 81325010, 81421004, and 31371443). E.K.-H.C. gratefully acknowledges support from the National Research Foundation Cancer Science Institute of Singapore RCE Main Grant, National Medical Research Council (NMRC CBRGNIG NMRC/BNIG/ 2012/2013), and Ministry of Education Academic Research Fund (MOE AcRF Tier 1 T1-2012 Oct-11 and Seed Fund Grant T1-BSRG 2014-05). This work is funded by the NCIS Yong Siew Yoon Research Grant through donations from the Yong Loo Lin Trust. D.H. gratefully acknowledges support from the National Science Foundation CAREER Award (CMMI-1350197), Center for Scalable and Integrated NanoManufacturing (DMI0327077), CMMI-0856492, DMR-1343991, OISE-1444100, V Foundation for Cancer Research Scholars Award, Wallace H. Coulter Foundation Translational Research Award, 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), Society for Laboratory Automation and Screening Endowed Fellowship, Beckman Coulter Life Sciences and the American Academy of Implant Dentistry Research Foundation under Grant No. 20150460. D.H. also gratefully acknowledges the Departments of Biomedical Engineering and Mechanical Engineering at Northwestern University for their extensive support during the administration of this study. REFERENCES (1) Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Hydrogels in Biology and Medicine: From Molecular Principles to Bionanotechnology. Adv. Mater. 2006, 18, 1345−1360. (2) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751−760. (3) Faklaris, O.; Joshi, V.; Irinopoulou, T.; Tauc, P.; Sennour, M.; Girard, H.; Gesset, C.; Arnault, J.-C.; Thorel, A.; Boudou, J.-P.; Curmi, P. A.; Treussart, F. Photoluminescent Diamond Nanoparticles for Cell Labeling: Study of the Uptake Mechanism in Mammalian Cells. ACS Nano 2009, 3, 3955−3962. (4) Wu, T.-J.; Tzeng, Y.-K.; Chang, W.-W.; Cheng, C.-A.; Kuo, Y.; Chien, C.-H.; Chang, H.-C.; Yu, J. Tracking the Engraftment and Regenerative Capabilities of Transplanted Lung Stem Cells Using Fluorescent Nanodiamonds. Nat. Nanotechnol. 2013, 8, 682−689. (5) Huang, H.; Pierstorff, E.; Osawa, E.; Ho, D. Active Nanodiamond Hydrogels for Chemotherapeutic Delivery. Nano Lett. 2007, 7, 3305− 3314.

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. 7398


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ACS Nano (6) Mochalin, V. N.; Pentecost, A.; Li, X.-M.; Neitzel, I.; Nelson, M.; Wei, C.; He, T.; Guo, F.; Gogotsi, Y. Adsorption of Drugs on Nanodiamond: Toward Development of a Drug Delivery Platform. Mol. Pharmaceutics 2013, 10, 3728−3735. (7) Zhang, Q.; Mochalin, V. N.; Neitzel, I.; Knoke, I. Y.; Han, J.; Klug, C. A.; Zhou, J. G.; Lelkes, P. I.; Gogotsi, Y. Fluorescent PllaNanodiamond Composites for Bone Tissue Engineering. Biomaterials 2011, 32, 87−94. (8) Mochalin, V. N.; Shenderova, O.; Ho, D.; Gogotsi, Y. The Properties and Applications of Nanodiamonds. Nat. Nanotechnol. 2012, 7, 11−23. (9) Adnan, A.; Lam, R.; Chen, H.; Lee, J.; Schaffer, D. J.; Barnard, A. S.; Schatz, G. C.; Ho, D.; Liu, W. K. Atomistic Simulation and Measurement of Ph Dependent Cancer Therapeutic Interactions with Nanodiamond Carrier. Mol. Pharmaceutics 2011, 8, 368−374. (10) Chow, E. K.-H.; Ho, D. Cancer Nanomedicine: From Drug Delivery to Imaging. Sci. Transl. Med. 2013, 5, 216rv4. (11) Wang, X.; Low, X. C.; Hou, W.; Abdullah, L. N.; Toh, T. B.; Mohd Abdul Rashid, M.; Ho, D.; Chow, E. K.-H. Epirubicin-Adsorbed Nanodiamonds Kill Chemoresistant Hepatic Cancer Stem Cells. ACS Nano 2014, 8, 12151−12166. (12) Kim, H.-J.; Zhang, K.; Moore, L.; Ho, D. Diamond NanogelEmbedded Contact Lenses Mediate Lysozyme-Dependent Therapeutic Release. ACS Nano 2014, 8, 2998−3005. (13) Moore, L.; Chow, E. K.-H.; Osawa, E.; Bishop, J. M.; Ho, D. Diamond-Lipid Hybrids Enhance Chemotherapeutic Tolerance and Mediate Tumor Regression. Adv. Mater. 2013, 25, 3532−3541. (14) Manus, L. M.; Mastarone, D. J.; Waters, E. A.; Zhang, X.-Q.; Schultz-Sikma, E. A.; MacRenaris, K. W.; Ho, D.; Meade, T. J. Gd(III)Nanodiamond Conjugates for MRI Contrast Enhancement. Nano Lett. 2010, 10, 484−489. (15) Wang, H.; Lee, D.-K.; Chen, K.-Y.; Chen, J.-Y.; Zhang, K.; Silva, A.; Ho, C.-M.; Ho, D. Mechanism-Independent Optimization of Combinatorial Nanodiamond and Unmodified Drug Delivery Using a Phenotypically Driven Platform Technology. ACS Nano 2015, 9, 3332− 3344. (16) Chow, E. K.; Zhang, X.-Q.; Chen, M.; Lam, R.; Robinson, E.; Huang, H.; Schaffer, D.; Osawa, E.; Goga, A.; Ho, D. Nanodiamond Therapeutic Delivery Agents Mediate Enhanced Chemoresistant Tumor Treatment. Sci. Transl. Med. 2011, 3, 73ra21. (17) Lam, R.; Chen, M.; Pierstorff, E.; Huang, H.; Osawa, E.; Ho, D. Nanodiamond-Embedded Microfilm Devices for Localized Chemotherapeutic Elution. ACS Nano 2008, 2, 2095−2102. (18) Chen, M.; Pierstorff, E. D.; Lam, R.; Li, S.-Y.; Huang, H.; Osawa, E.; Ho, D. Nanodiamond-Mediated Delivery of Water-Insoluble Therapeutics. ACS Nano 2009, 3, 2016−2022. (19) Lee, D.-K.; Kim, S. V.; Limansubroto, A. N.; Yen, A.; Soundia, A.; Wang, C.-Y.; Shi, W.; Hong, C.; Tetradis, S.; Kim, Y.; Park, N.-H.; Kang, M. K.; Ho, D. Nanodiamond−Gutta Percha Composite Biomaterials for Root Canal Therapy. ACS Nano 2015, 9, 11490−11501. (20) Toh, T.-B.; Lee, D.-K.; Hou, W.; Abdullah, L. N.; Nguyen, J.; Ho, D.; Chow, E. K.-H. Nanodiamond−Mitoxantrone Complexes Enhance Drug Retention in Chemoresistant Breast Cancer Cells. Mol. Pharmaceutics 2014, 11, 2683−2691. (21) Ho, D.; Wang, C.-H. K.; Chow, E. K.-H. Nanodiamonds: The Intersection of Nanotechnology, Drug Development, and Personalized Medicine. Sci. Advances 2015, 1, e1500439. (22) Huang, H.; Pierstorff, E.; Osawa, E.; Ho, D. Protein-Mediated Assembly of Nanodiamond Hydrogels into a Biocompatible and Biofunctional Multilayer Nanofilm. ACS Nano 2008, 2, 203−212. (23) Smith, A. H.; Robinson, E. M.; Zhang, X.-Q.; Chow, E. K.; Lin, Y.; Osawa, E.; Xi, J.; Ho, D. Triggered Release of Therapeutic Antibodies from Nanodiamond Complexes. Nanoscale 2011, 3, 2844−2848. (24) Mohan, N.; Chen, C.-S.; Hsieh, H.-H.; Wu, Y.-C.; Chang, H.-C. In Vivo Imaging and Toxicity Assessments of Fluorescent Nanodiamonds in Caenorhabditis Elegans. Nano Lett. 2010, 10, 3692−3699. (25) Hui, Y. Y.; Cheng, C.-L.; Chang, H.-C. Nanodiamonds for Optical Bioimaging. J. Phys. D: Appl. Phys. 2010, 43, 374021.

(26) Havlik, J.; Petrakova, V.; Rehor, I.; Petrak, V.; Gulka, M.; Stursa, J.; Kucka, J.; Ralis, J.; Rendler, T.; Lee, S.-Y.; Reuter, R.; Wrachtrup, J.; Ledvina, M.; Nesladek, M.; Cigler, P. Boosting Nanodiamond Fluorescence: Towards Development of Brighter Probes. Nanoscale 2013, 5, 3208−3211. (27) Tisler, J.; Balasubramanian, G.; Naydenov, B.; Kolesov, R.; Grotz, B.; Reuter, R.; Boudou, J.-P.; Curmi, P. A.; Sennour, M.; Thorel, A.; Börsch, M.; Aulenbacher, K.; Erdmann, R.; Hemmer, P. R.; Jelezko, F.; Wrachtrup, J. Fluorescence and Spin Properties of Defects in Single Digit Nanodiamonds. ACS Nano 2009, 3, 1959−1965. (28) McGuinness, L. P.; Yan, Y.; Stacey, A.; Simpson, D. A.; Hall, L. T.; Maclaurin, D.; Prawer, S.; Mulvaney, P.; Wrachtrup, J.; Caruso, F.; Scholten, R. E.; Hollenberg, L. C. L. Quantum Measurement and Orientation Tracking of Fluorescent Nanodiamonds inside Living Cells. Nat. Nanotechnol. 2011, 6, 358−363. (29) Barnard, A. S. Diamond Standard in Diagnostics: Nanodiamond Biolabels Make Their Mark. Analyst 2009, 134, 1751−1764. (30) Barnard, A. S. Self-Assembly in Nanodiamond Agglutinates. J. Mater. Chem. 2008, 18, 4038−4041. (31) Xing, Z.; Pedersen, T. O.; Wu, X.; Xue, Y.; Sun, Y.; FinneWistrand, A.; Kloss, F. R.; Waag, T.; Krueger, A.; Steinmüller-Nethl, D.; Mustafa, K. Biological Effects of Functionalizing Copolymer Scaffolds with Nanodiamond Particles. Tissue Eng., Part A 2013, 19, 1783−1791. (32) Liang, Y.; Ozawa, M.; Krueger, A. A General Procedure to Functionalize Agglomerating Nanoparticles Demonstrated on Nanodiamond. ACS Nano 2009, 3, 2288−2296. (33) Heyer, S.; Janssen, W.; Turner, S.; Lu, Y.-G.; Yeap, W. S.; Verbeeck, J.; Haenen, K.; Krueger, A. Toward Deep Blue Nano Hope Diamonds: Heavily Boron-Doped Diamond Nanoparticles. ACS Nano 2014, 8, 5757−5764. (34) Perevedentseva, E.; Lin, Y.-C.; Jani, M.; Cheng, C.-L. Biomedical Applications of Nanodiamonds in Imaging and Therapy. Nanomedicine 2013, 8, 2041−2060. (35) Liu, K.-K.; Zheng, W.-W.; Wang, C.-C.; Chiu, Y.-C.; Cheng, C.-L.; Lo, Y.-S.; Chen, C.; Chao, J.-I. Covalent Linkage of NanodiamondPaclitaxel for Drug Delivery and Cancer Therapy. Nanotechnology 2010, 21, 315106. (36) Yuan, Y.; Chen, Y.; Liu, J.-H.; Wang, H.; Liu, Y. Biodistribution and Fate of Nanodiamonds. Diamond Relat. Mater. 2009, 18, 95−100. (37) Zhang, X.; Hu, W.; Li, J.; Tao, L.; Wei, Y. A Comparative Study of Cellular Uptake and Cytotoxicity of Multi-Walled Carbon Nanotubes, Graphene Oxide, and Nanodiamond. Toxicol. Res. 2012, 1, 62−68. (38) Moore, L.; Grobarova, V.; Shen, H.; Man, H. B.; Micova, J.; Ledvina, M.; Stursa, J.; Nesladek, M.; Fiserova, A.; Ho, D. Comprehensive Interrogation of the Cellular Response to Fluorescent, Detonation and Functionalized Nanodiamonds. Nanoscale 2014, 6, 11712−11721. (39) Yuan, Y.; Wang, X.; Jia, G.; Liu, J.-H.; Wang, T.; Gu, Y.; Yang, S.T.; Zhen, S.; Wang, H.; Liu, Y. Pulmonary Toxicity and Translocation of Nanodiamonds in Mice. Diamond Relat. Mater. 2010, 19, 291−299. (40) Kumari, S.; Singh, M. K.; Singh, S. K.; Grácio, J. J. A.; Dash, D. Nanodiamonds Activate Blood Platelets and Induce Thromboembolism. Nanomedicine 2014, 9, 427−440. (41) Mona, J.; Kuo, C. J.; Perevedentseva, E.; Priezzhev, A. V.; Cheng, C. L. Adsorption of Human Blood Plasma on Nanodiamond and Its Influence on Activated Partial Thromboplastin Time. Diamond Relat. Mater. 2013, 39, 73−77. (42) Xing, Y.; Xiong, W.; Zhu, L.; O̅ sawa, E.; Hussin, S.; Dai, L. DNA Damage in Embryonic Stem Cells Caused by Nanodiamonds. ACS Nano 2011, 5, 2376−2384. (43) Reagan-Shaw, S.; Nihal, M.; Ahmad, N. Dose Translation from Animal to Human Studies Revisited. FASEB J. 2008, 22, 659−661. (44) Fisher, B.; Redmond, C.; Wickerham, D. L.; Bowman, D.; Schipper, H.; Wolmark, N.; Sass, R.; Fisher, E. R.; Jochimsen, P.; Legault-Poisson, S. Doxorubicin-Containing Regimens for the Treatment of Stage II Breast Cancer: The National Surgical Adjuvant Breast and Bowel Project Experience. J. Clin. Oncol. 1989, 7, 572−582. 7399


Article

ACS Nano (45) Aouidet, A.; Bouissou, H.; de La Farge, F.; Valdiguie, P. Serum Reference Values of the Cynomolgus Monkey, a Model for the Study of Atherosclerosis. J. Clin. Chem. Clin. Biochem. 1990, 28, 251−252. (46) Jinbo, T.; Ami, Y.; Suzaki, Y.; Kobune, F.; Ro, S.; Naiki, M.; Iguchi, K.; Yamamoto, S. Concentrations of C-Reactive Protein in Normal Monkeys (Macaca Irus) and in Monkeys Inoculated with Bordetella Bronchiseptica R-5 and Measles Virus. Vet. Res. Commun. 1999, 23, 265−274. (47) Chen, Y.; Qin, S.; Ding, Y.; Wei, L.; Zhang, J.; Li, H.; Bu, H.; Lu, Y.; Cheng, J. Reference Values of Clinical Chemistry and Hematology Parameters in Rhesus Monkeys (Macaca Mulatta). Xenotransplantation 2009, 16, 496−501. (48) Matsuzawa, T.; Nagai, Y. Comparative Haematological and Plasma Chemistry Values in Purpose-Bred Squirrel, Cynomolgus and Rhesus Monkeys. Comp. Haematol. Int. 1994, 4, 43−48. (49) Wang, H.; Niu, Y. Y.; Si, W.; Li, J. Y.; Yan, Y. Reference Data of Clinical Chemistry, Haematology and Blood Coagulation Parameters in Juvenile Cynomolgus Monkeys (Macaca Fascicularis). Vet. Med. 2012, 57, 233−238. (50) Hrkach, J.; Von Hoff, D.; Ali, M. M.; Andrianova, E.; Auer, J.; Campbell, T.; De Witt, D.; Figa, M.; Figueiredo, M.; Horhota, A.; Low, S.; McDonnell, K.; Peeke, E.; Retnarajan, B.; Sabnis, A.; Schnipper, E.; Song, J. J.; Song, Y. H.; Summa, J.; Tompsett, D.; et al. Preclinical Development and Clinical Translation of a Psma-Targeted Docetaxel Nanoparticle with a Differentiated Pharmacological Profile. Sci. Transl. Med. 2012, 4, 128ra39. (51) Liu, J.; Erogbogbo, F.; Yong, K.-T.; Ye, L.; Liu, J.; Hu, R.; Chen, H.; Hu, Y.; Yang, Y.; Yang, J.; Roy, I.; Karker, N. A.; Swihart, M. T.; Prasad, P. N. Assessing Clinical Prospects of Silicon Quantum Dots: Studies in Mice and Monkeys. ACS Nano 2013, 7, 7303−7310. (52) Weekley, B. L.; Deldar, A.; Tapp, E. Development of Renal Function Tests for Measurement of Urine Concentrating Ability, Urine Acidification, and Glomerular Filtration Rate in Female Cynomolgus Monkeys. J. Am. Assoc. Lab. Anim. Sci. 2003, 42, 22−25. (53) Mohd Abdul Rashid, M. B.; Toh, T. B.; Silva, A.; Nurrul Abdullah, L.; Ho, C.-M.; Ho, D.; Chow, E. K.-H. Identification and Optimization of Combinatorial Glucose Metabolism Inhibitors in Hepatocellular Carcinomas. J. Lab. Automat 2015, 20, 423−437. (54) Zarrinpar, A.; Lee, D.-K.; Silva, A.; Datta, N.; Kee, T.; Eriksen, C.; Weigle, K.; Agopian, V.; Kaldas, F.; Farmer, D.; Wang, S. E.; Busuttil, R.; Ho, C.-M.; Ho, D. Individualizing Liver Transplant Immunosuppression Using a Phenotypic Personalized Medicine Platform. Sci. Transl. Med. 2016, 8, 333ra49.

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