Synthesis and Characterization of Nanodiamond–Growth Factor Complexes Toward Applications in Oral Implantation and Regenerative Medicine

During the past 2 decades, regenerative medicine has emerged as a promising field that is progressing toward tissue regeneration for a broad range of indications.^1,2  However, a current challenge of the field is the need to identify biocompatible and scalable delivery agents to enhance treatment efficacy and safety. Several approaches have attempted to use biomaterials as growth factor carriers for possible sustained delivery and controlled molecular release. For example, incorporation of platelet-derived growth factor into biomaterials (Regranex) has demonstrated successful wound healing and morphogenic proteins 2 and 7 (Infuse, Stryker's OP-1) for bone formation.³  Additional approaches have sought to improve bone graft treatment outcomes.^4,5  Previous studies on the use of nanofeatured titanium surfaces and nanodiamond (ND)–mediated delivery of estrogen in oral implantology demonstrated a stimulation in bone formation, cleft lip palatal expansion, and resistance to chemical corrosion in oral implants.^6–8  In perspective, the application of nanotechnology positively influences oral implantology and improves clinical practice such as osseointegration.

Nanoscale drug delivery vehicles have emerged as a promising strategy for tissue regeneration and controlled release of biomolecules. Many studies of nanoparticle drug delivery platforms have validated the use of nanotechnology for tissue repair.^6–15  Additional studies emphasizing the role of nanoparticle size, shape, charge, and surface chemistry on their resulting toxicity have also been conducted.^16,17  Among the many promising nanoscale drug delivery vehicles that are being explored, NDs have emerged as promising, clinically relevant candidates.^9–15,18  Nanodiamonds are carbon nanoparticles with truncated octahedral structures that are approximately 5 nm in diameter. They are biocompatible products that can be economically processed by ultrasonication, centrifugation, and milling methodologies.^16,19  The surface chemistries of NDs, which are suitable for both electrostatic physisorption and chemical conjugation of a broad spectrum of therapeutics, mediate both high-affinity drug binding and sustained release.^20–40  These characteristics may enable the NDs to serve as translationally relevant growth factor delivery vehicles. In this study, NDs were complexed with epidermal growth factor (EGF) functionalized with Alexa Fluor 488. Epidermal growth factor has previously been explored as a potential agent to promote osteogenic differentiation.^17,41  Therefore, ND-EGF may serve as an agent to enhance osteogenesis. In this study, EGF binding and release studies confirmed that the successful loading of EGF onto the NDs, as well as their prolonged elution, served as an indicator for the prospective applicability of the NDs as drug delivery biomaterials in the field of regenerative medicine.

ND-EGF was synthesized by diluting autoclaved ND solution (NanoCarbon Research Institute Co, Ltd, Nagano, Japan) with H₂O (autoclaved). Then, 0.2 mL (20 μg) of EGF streptavidin biotin-modified Alexa Fluor 488 (Thermo Fisher Scientific, Canoga Park, Calif) was added to the solution and mixed, along with 1M NaOH (Sigma-Aldrich, St Louis, Mo) for aggregation of ND-EGF complexes. The resulting solution was incubated for a minimum of 3 days, at a temperature of 22°C, and was protected from any source of light. After centrifugation, the first supernatant sample was collected for further analysis, and the resulting pellet was diluted and centrifuged further. A second supernatant sample was collected and also saved for further analysis, while the pellet was resuspended in H₂O with a probe sonicator to generate a 5-mg/mL ND solution. The EGF complexed with Alexa Fluor 488 was used to conduct 1:1 serial dilutions, which were analyzed to generate a standard curve.

Two supernatants with unknown Alexa 488 concentrations were collected from the synthesis of ND-EGF. Serial dilutions were analyzed using a BioTek Synergy H1 microplate reader (BioTek Instruments, Inc, Winooski, Vt) and Gen5 2.03 computer software for fluorescence spectroscopy at an excitation wavelength of 350 nm and an emission wavelength of 520 nm.^42–45  Using samples of known concentration and resulting fluorescent intensity obtained from light spectroscopy, a linear trend with its respective equation was generated from the data. From this equation, the Alexa 488 concentrations of the 2 supernatant solutions were calculated using the measured intensity of each sample and plotting the measured intensities along the previously generated standard curve, which gave indications of the amount of EGF functionalized onto NDs. Characterization of the ND-EGF by dynamic light scattering (DLS) and ζ-potential analysis was conducted using the Zetasizer Nano ZS (Malvern, Worcestershire, UK).

Two solutions of biological media were synthesized to provide the ND-EGF compound a simulated biological environment. The first (1:1 biological medium) being a Dulbecco's Modified Eagle Media (DMEM; Thermo Fisher Scientific) and streptomycin (Thermo Fisher Scientific) with 10% fetal bovine serum:phosphate-buffered saline (FBS:PBS) mixture in 1:1 ratio (FBS from Gemini Bio Products, West Sacramento, Calif; PBS from Thermo Fisher Scientific). The second solution (1:10 biological medium) was a DMEM and streptomycin with 10% FBS:PBS mixture in 1:10 ratio.⁴⁶  A sample containing 5 μg of EGF-conjugated Alexa 488 was taken from the ND-EGF solution and centrifuged for 20 minutes at 14 000g. The solution was removed, and 1 mL of biological media was added to the ND-EGF sample for the releasing experiment. Two sets of media were made, with one containing 1:1 and the second containing 1:10 ratios of biological medium with 10% FBS:PBS. The ND-EGF was dispersed within the media, incubated at 36.6°C for 1 hour, and then centrifuged for 20 minutes at 14 000g. Supernatant was extracted from both tubes, and the pellet within each tube was gently detached from the bottom. One milliliter of fresh new media was added back into their respective tubes containing ND-EGF. This procedure was repeated every hour for the next 4 hours and then again at hours 18, 30, 50, 100, 170, and 240, each resulting in a new pair of supernatant solutions ready for fluorescent analysis. All supernatant samples were loaded onto a 96-well plate and analyzed for fluorescence at excitation wavelength 350 nm and emission wavelength 520 nm. The ND-EGF solution containing 1:1 biological medium was then diluted to a 1:10 biological medium solution for further fluorescence spectroscopy analysis.

A schematic of ND-EGF is shown in Figure 1. As shown in Figure 2a and b, EGF was readily loaded onto the ND surface, which was also observed after multiple rounds of incubation and centrifugation. This was further demonstrated after the measured fluorescence intensity was compared with the standard curve of background Alexa Fluor 488 fluorescence intensity (Figure 2a). Furthermore, characterization performed through DLS and ζ-potential analysis of the ND-EGF complexes exhibited substantially increased particle diameters compared with those of unmodified NDs, from 46.6 ± 0.17 to 184.3 ± 1.03 nm (Figure 3). Additional analysis revealed decreased ζ-potentials for ND-EGF complexes compared with those of unmodified NDs, from 55.8 ± 0.37 to 41.4 ± 0.89 mV, respectively (Figure 3).

From the standard curve plotted with 1:10 biological medium as a background (Figure 4a), the cumulative EGF conjugated with Alexa-488 release profiles for ND-EGF samples in 1:1 and 1:10 biological media (DMEM with 10% FBS:PBS) were determined. For both samples, sustained EGF functionalized with Alexa-488 release was observed for 11 days, while relatively higher release was observed during the first 5 hours (Figure 4b). The ND-EGF sample in 1:1 diluted biological medium presented greater total fluorescence release (1.83 ± 0.20 μg) than that of the sample in 1:10 biological medium (0.53 ± 0.05 μg) over the same duration.

EGF functionalized with Alexa Fluor 488 was used to quantify the rate of drug release when bound to ND particles. To quantify ND-EGF binding efficiency, fluorescent spectroscopy analysis was performed to determine sufficient binding.^47–50  Furthermore, characterization through DLS and ζ-potential analysis of the formation of ND-EGF complex exhibited substantially increased diameters compared with the diameter of unmodified NDs. However, its relatively small size distribution still makes it a viable method for drug delivery.^45–48  The potential of NDs as a viable drug binding platform is attributed to their unique chemical surface properties, which allow for a variety of electrostatic interactions to occur. Sodium hydroxide (NaOH) was used to induce an electrostatic charge on the surface chemistry of the NDs.^48,51–57  The slight decrease in ζ-potentials in ND-EGF complexes compared with unmodified NDs (Figure 2b) indicates that the ND-EGF complexes are able to form efficient electrostatic interactions with other molecules.

Culture media solution containing various proteins, carbohydrates, and antibiotics was produced with the intention of simulating a physiologically relevant system to test the sustained growth factor release capabilities of NDs. As shown in Figure 4b, the ND-EGF complexes experience sustained drug release in both the 1:1 and the 1:10 biological media over a nearly 2-week-long period. However, the ND-EGF sample within the 1:1 biological medium released larger amounts of EGF over the same period of time compared with when the sample was within the 1:10 biological medium. This can potentially be explained by the higher concentration of proteins within the 1:1 solution compared with the 1:10 solution. Previous studies have demonstrated that charged proteins are able to competitively bind to the surface of NDs, which enhances drug release.^53,54  Similar to EGF, epirubicin chemotherapy drugs, when bound to NDs, displayed an increasing rate of drug release proportional to increases in concentrations of the FBS within the biological media.⁵⁸ 

Of note, during the EGF release analysis for the 1:1 solution, the concentration of proteins generated a higher than expected intensity background. As a result, the 1:1 ratio solution was diluted to a 1:10 biological medium, resulting in a lower intensity background. Using the diluted solution, the intensity of EGF-Alexa Fluor 488 was measured. Comparing the diluted 1:1 solution with the original 1:10 solution, a consistency in drug release was apparent. After further calculations of the common ratio, the concentrations of EGF–Alexa Fluor 488 released within a 1:1 solution were determined. Data acquired through fluorescent intensity analysis further demonstrated the potential applicability of ND-EGF toward downstream preclinical validation.

In summary, this work demonstrated the synthesis and characterization of ND-EGF toward potential applications in regenerative medicine. Through fluorescence analysis of the supernatant from each solution, the concentration of the released protein was determined. Sustained EGF elution from the ND complexes was confirmed in physiologically relevant solutions with 2 different ratios of proteins and carbohydrates. Thus, this work confirms the sustained release capabilities of NDs and provides valuable support for the potential applications of NDs to deliver growth factors and other therapeutics in a variety of medical applications.

Abbreviations

DMEM

Dulbecco's Modified Eagle's Medium

EGF

epidermal growth factor

FBS

fetal bovine serum

ND

nanodiamond

PBS

phosphate-buffered saline

The authors gratefully acknowledge the American Academy of Implant Dentistry (AAID) Foundation for the support of this work under award 20150460.

D.H. and D.-K.L. are co-inventors of pending patents pertaining to nanodiamond drug delivery.