Preparation of Hydroxylated Fullerenes
Hydroxylated fullerenes were prepared using the phase transfer method of Li et al. (1993)
. Sodium hydroxide (NaOH) was mixed with a solution of C60
(SES Research, Houston, TX) and toluene, then added to the phase transfer agent (40% aqueous tetrabutylammonium hydroxide (TBAH)). The purple solution was agitated for 30 min to form a colorless solution with a brown precipitate. The toluene phase was aspirated and residual toluene removed by evaporation under vacuum. Deionized water was then added to the product, and the solution mixed for 24 hr with constant air flow to solubilize the precipitate and allow hydroxylation of the product. Excess CO2
was removed through a 1 M NaOH column and the product washed thrice with methanol to remove residual NaOH and TBAH. The C60
product was solubilized in methanol, centrifuged at 1450 xg for 10 min, and supernatant removed to recover the product. Subsequent washes in 10 ml deionized water dissolved the product, with methanol added to precipitate the product for centrifugation. In the final step, the product was dissolved in water, passed through a 0.2 μm nylon filter, and dried under vacuum. Further hydroxylation was promoted by adding 3% hydrogen peroxide (H2
) after the final methanol treatment. The precipitate was dissolved in 25 ml 3% H2
and stirred in a sealed bottle for 24 hr. Washing and drying steps performed for the H2
treated product were the same as above.
Characterization of Hydroxylated Fullerenes
The degree of fullerene hydroxylation was measured via x-ray photoelectron spectroscopy (XPS) using a Kratos Axis ULTRA system (Shimadzu). The fullerol powder was collected to a thickness greater than 500 μm on double-stick copper tape, with survey and high-resolution scans performed (scan area: 300 μm x 700 μm) to obtain elemental composition and functionality. The spectra were obtained using a pass energy of 160 eV (step size 1 eV), and high-resolution scans were generated with a pass energy of 20 eV (step size 0.1 eV). Binding energies were standardized using the C 1s peak (284.6 eV). The number of hydroxyl groups for the C60 derivative was estimated using the peak areas of high-resolution scans of C 1s and O 1s, with the results averaged from three sample spectra. The results were confirmed using attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). The fullerenol powder was deposited on a germanium crystal, and the spectra collected with a Thermo Electron Nicolet 8700 ATR-FTIR spectrometer at a 4 cm−1 resolution with 256 scans. Background spectra were generated with the blank Ge crystal prior to running the sample. A C60(OH)24 sample (MER Corp, AZ) was used as the standard reference material to validate the XPS and ATR-FTIR results.
The NP size was measured by dynamic light scattering (DLS) at 22°C with a Zetasizer Nano ZS (Malvern Instruments) of a 1 mg/ml dilution of the fullerenol in deionized water. In addition, 10 μl stock fullerenol was placed onto a formvar-coated grid, air-dried overnight, and examined on a FEI/Philips EM208S transmission electron microscope operating at an accelerating voltage of 80 KV.
White, female, 8 week old Sprague-Dawley rats weighing between 200–250 g were obtained from Charles River Laboratories, Inc. (Morrisville, NC) and quarantined for 14 days to ensure they were healthy with no underlying medical conditions. Once removed from quarantine, rats were individually housed in Nalgene Metabolic Cages with a 150–300 g capacity (Nalgene, Rochester, NY) and allowed a one week acclimation period prior to the beginning the study. Animals, exposed to a normal diurnal cycle (12 hr light/dark cycle) with environmental controls maintained at approximately 23°C and 43–47% relative humidity, were treated in accordance with the guidelines prepared by the Institute of Laboratory Animal Resources (1996)
. The rats were watered and fed a ground diet of Purina Lab Diet 5001 (PMI, Richmond, IN) ad libitum.
The rats were divided into 4 groups: 8, 24, and 48 hr treatment (n=4/group) and 48 hr control (n=4). Blood, urine, and feces were collected from the treatment groups prior to dose. The C60(OH)30 dosing solution was prepared under sterile conditions by adding 7.5 ml sterile nonpyrogenic water (USP for injection) (Baxter Healthcare Co., Deerfield, IL) to 75 mg of the hydroxylated fullerene powder in a sterile, nonpyrogenic injection vial (Hospira, Lake Forest, IL). The 10 mg/ml solution was vortexed for 10 min to solubilize the product. The rats were immobilized in a Tailveiner®Rat Restraint System (Braintree Scientific, Inc.; Braintree, MA) with the tail exteriorized for access to the lateral tail veins. The injection site was cleaned with an alcohol pad and a 10 mg/kg IV dose of C60(OH)30 was administered via a lateral tail vein with a 25-gauge needle on a 1 ml syringe. The lateral tail vein of one animal in the 8 hr group collapsed during the injection, preventing a full dose of the fullerol to enter the bloodstream. This animal was removed from the study, leaving three animals in this group.
Following administration of dose, blood and urine were collected from the rats at termination of dosing at 8 and 48 hr. The blood was centrifuged, plasma collected, and both samples stored at −80°C for later analysis. Urine collected at each sample time was placed into glass vials, quantitated, and stored at −80°C. After 8, 24, and 48 hr, each group of rats was euthanized via CO2
asphyxiation according to the AVMA Guidelines on Euthanasia (2007)
. Each animal was necropsied, with 29 target tissues collected and weighed. The tissues included liver (one sample from each of the 4 lobes), kidneys, urinary bladder, thymus, lymph nodes (3 samples – mesenteric, subcutaneous and inguinal), brain, adrenal glands, heart, muscle (2 samples– quadriceps and gluteals), full thickness skin (dorsal and ventral sites), adipose tissue (subcutaneous and intra-abdominal), ovaries, stomach, small intestines (duodenum, jejunum and ileum), pancreas, colon (descending), lungs, spleen and skin surrounding the injection site. The tissues were trimmed and fixed in 10% neutral-buffered formalin. The remaining unfixed tissue was weighed and stored at −80°C.
All tissue samples were processed routinely through graded alcohols, cleared in Clearite, and infiltrated and embedded paraffin in a Tissue-Tek VIP 2000 Tissue Processor (Miles Scientific). Tissue samples were sectioned at 5 μm, mounted on glass slides, stained with hematoxylin and eosin (H&E), and cover-slipped with PermountR. Slides were randomized by tissue type using a random number generator and evaluated blinded for any histopathology changes. All tissue changes were graded on a standard severity scale of minimal, mild, moderate or severe.
Blood and Urine Chemistry
Blood chemistry was performed with a COBAS Integra 400 Plus System chemical analyzer (Roche Diagnostics Corp., Indianapolis, IN) on plasma samples from the 48 hr treatment and control groups to determine chemical, endocrine, or electrolyte fluctuations. Blood chemistry parameters that were measured included blood urea nitrogen (BUN), creatinine, Na, K, Ca, Cl, P, total protein, albumin, globulin, cholesterol, total bilirubin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), and gamma-glutamyl transpeptidase (GGT). Urine protein and creatinine levels were determined with the COBAS Integra 400 Plus System only for the 0 and 24 hr time points in the 24 and 48 hr treatment groups (n=8) to screen for potential renal dysfunction.
The mean values for each treatment were calculated and significant differences (p < 0.05) determined using the Student’s t-test. DLS data was also analyzed for outlying data using analysis software integrated into the Malvern Zetasizer Nano system.