Pilot study on biocompatibility of fluorescent nanodiamond-(NV)-Z~800 particles in rats: safety, pharmacokinetics, and bio-distribution (part III)
Introduction:We hereby report on studies aimed to characterize safety, pharmacokinetics, and bio-distribution of fluorescent nanodiamond particles (NV)-Z~800 (FNDP-(NV)) administered to rats by intravenous infusion in a single high dose.
Methods: Broad scale biological variables were monitored following acute (90 minutes) and subacute (5 or 14 days) exposure to FNDP-(NV). Primary endpoints included morbidity and mor- tality, while secondary endpoints focused on hematology and clinical biochemistry biomarkers. Particle distribution (liver, spleen, lung, heart, and kidney) was assessed by whole organ near infrared imaging using an in vivo imaging system. This was validated by the quantification of particles extracted from the same organs and visualized by fluorescent and scanning electron microscopy. FNDP-(NV)-treated rats showed no change in morbidity or mortality and preserved normal motor and sensory function, as assessed by six different tests.
Results: Blood cell counts and plasma biochemistry remained normal. The particles were principally distributed in the liver and spleen. The liver particle load accounted for 51%, 24%, and 18% at 90 minutes, 5 days, and 14 days, respectively. A pilot study of particle clearance from blood indicated 50% clearance 33 minutes following the end of particle infusion.
Conclusion:We concluded that systemic exposure of rats to a single high dose of FDNP- (NV)-Z~800 (60 mg/kg) appeared to be safe and well tolerated over at least 2 weeks. These data suggest that FNDP-(NV) should proceed to preclinical development in the near future.
Recent years have ushered in a strong and growing interest in the bioengineering of nanodiamond particles (NDP).1–5 Certain strains of NDP, such as the fluorescent NDP (FNDP), have been prospected for multiple medical diagnostic tests, drug delivery vehicles, imaging biomarkers, cell tracking, and translational biomarkers.6–10 The highly diverse options available for chemical modifications of NDP enable co-junction with materials that offer opportunities for imaging targeted pathologies such as tumors11,12 or blood clots.13 NDP are also being extensively studied as carri- ers of pharmacological agents for delivery of cytotoxic chemicals into drug-resistant tumors.14–17 Furthermore, new frontiers were recently broached that demonstrate potential medical application in immune enhancement (vaccine),18,19 adjuvant,20 and genetic neurological conditions.21 Pharmacokinetic (PK) advantages gained by the co-junction of tumor-toxic compounds to NDP have shown preservation of efficacy.
Materials and methods
Antibodies and other reagents
Bitistatin was purified from the venom of Bitis arietans (Latoxan Serpentarium, Valence, France) using two steps of reverse-phase high-performance liquid chromatography, as described previously.4 F-NDP, chemically surface- functionalized with carboxyl groups (-COOH), were purchased from Adamas Nanotechnologies (Raleigh, NC, purchased from Adamas Nanotechnologies (Raleigh, NC, USA). Two strains of F-NDP were used: green fluorescent F-NDP at 700 nm (2×108 particles/mg) and red fluores- NVN cent F-NDP with N-V color centers (F-NDPNV) at 100 nm (5×1011 particles/mg), 700 nm (2×108 particles/mg), and 10,000 nm (5×105 particles/mg). Isoflurane (IF; B34C16A) was purchased from Henry Schein (Melville, NY, USA). Ethyl alcohol (70% denatured) and PE-10 tubing were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Roboz SUT-15-1 5-0 silk suture was purchased from Roboz Surgical Instrument Co. (Gaithersburg, MD, USA). Parafilm and FeCl3 were purchased from Sigma-Aldrich (St Louis, MO, USA).
Coupling of bitistatin to F-NDP
Bitistatin was coupled to the F-NDP of all types using 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochlo- ride (EDC) as a hetero-bifunctional cross-linker,11 according to the methodology described previously.4 Coupling effi- ciency and preservation of bitistatin activity on the various functionalized nanodiamond particles (F-NDP-Bit) were verified using a semiquantitative enzyme-linked immuno- sorbent assay, as described previously.4
Caracterization of NIr emission of F-NDPNV and F-NDP
Bit was purified from B. arietans venom according to a previously described procedure21 with some minor modifi- cations. Briefly, lyophilized snake venom was dissolved in 0.1% TFA (30 mg/mL). Insoluble material was pelleted by centrifugation at 14,000 rpm for 5 minutes at room tempera- ture (RT). The supernatant was fractionated by reverse-phase high-performance liquid chromatography (HPLC) on a C18 column (250×10 mm; Vydac, Hesperia, CA, USA). The col- umn was eluted with a linear acetonitrile gradient 0%–80% over 45 minutes at a flow rate of 2 mL/min (Figure S2). Separation was monitored at 230 nm; fractions containing Bit (~22 minutes retention time) were collected manually and lyophilized in a Speed-Vac system. The lyophilized fractions were dissolved in water, and protein concentra- tions determined using the BCA assay (Pierce, Rockford, IL, USA). The crude Bit preparation (5 mg protein) was re-chromatographed using the same HPLC system but eluting the column with a shallower acetonitrile gradient (20%–80% over 120 minutes). The main peak containing purified Bit (Figure S3) was lyophilized and dissolved in deionized water to prepare the stock solution (8–10 mg/mL) used for coupling to F-NDPs. The purity of Bit, as tested by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, was found to be 98%, based on the digitalized intensity (HP ScanJet G3110 and software UN-scan-It gel version 6.1 by Silk Scientific Corp., Orem, UT, USA) of the major bands at maximal concentration (Figure S4).
Characterization of NIr emission of F-NDPNV and F-NDPNVN
NNIR fluorescence profiles of F-NDP were characterized using a Tecan Infinite 200 PRO (Tecan AG, Männedorf, Switzerland). One hundred microliters of 3 mg/mL of 700 nm F-NDP or F-NDP suspended in de-ionized NV NVN (DI) water was loaded into 96-well polystyrene Fluores- 8472 submit your manuscript | www.dovepress.com Dovepress cence was scanned for all wells with excitations from 230 to 850 nm and emissions from 290 to 850 nm (Figure 1A) at 20 nm intervals. Data were processed in MATLAB 2015b (Mathworks, Natick, MA, USA). Background fluorescence was subtracted from empty wells without F-NDP, and the resulting net fluorescence value was log10-transformed for visualization.
Glass capillaries (40 mm length, 1 mm internal diameter; Thermo Fisher Scientific) were filled with equal volumes (30 μL) of suspensions of F-NDP at concentrations from 0.06 mg/mL and up to 4 mg/mL (1.8–120 μg total par- ticle mass) and sealed at each end by plasticine (Hasbro, Pawtucket, RI, USA). The NIR fluorescence intensity of the various suspensions in the capillaries was analyzed using an in vivo imaging system (IVIS; IVIS 50 Imaging System;