Chemicals and cell culture materials
The 6-arm PEG-SG (6-arm polyethylene glycol N-hydroxy succinimidyl glutarate), and 6-arm PEG-AM (6-arm polyethylene glycol amine) were developed by SunBio Inc. (An-yang, Republic of Korea). The 5-fluorouracil (5-FU), NaH2PO4, Na2HPO, methyl alcohol (analytical grade), and diethyl ether were supplied by Sigma (St. Louis, MO, USA). Dulbecco's modified Eagle medium (DMEM), penicillin/streptomycin, and trypsin EDTA were purchased from GIBCO (Carlsbad, CA, USA). Aqueous fetal bovine serum (FBS) was obtained from WelGENE (Daegu, Republic of Korea).
Cell Line and Animals
For the pharmacokinetic study, male Sprague-Dawley rats were obtained from Orient Bio Inc. (Seongnam, Republic of Korea). The A549 human lung carcinoma cell line, a generous gift from Cha Biomedical Center (Seoul, Republic of Korea), was carefully inoculated into the dorsal neck of male nude mice (Balb/c Slc-nu/nu), which were supplied from the Central Lab Animal Inc. (Seoul, Republic of Korea). All in vivo experiments were carried out with the approval of the Institutional Animal Care and Use Committee (IACUC) at Konkuk University and in harmony with the Guide for Laboratory Animal Care and Use (IACUC Approval No. KUV7015, 7016)
Preparation of 5-FU-loaded PEG hydrogel and in vitro Release of 5-FU-loaded PEG-hydrogel
The 5-FU-loaded PEG-hydrogel was designed as shown in Fig 1. The 6-arm PEG-AM was dissolved in 10 mM phosphate buffer (pH 6.0), and 5-FU was added into the solution at a concentration of 1 mg/ml. The 6-arm PEG-SG was also dissolved in 10 mM phosphate buffer (pH 6.0). The 6-arm PEG-AM and 6-arm PEG-SG solutions were prepared at various concentrations (i.e., 7, 10, and 15% w/v). Approximately, 0.5 ml of 6-arm PEG-AM solution containing 5-FU was placed in a 6-well plate, and an equal volume of 6-arm PEG-SG solution (0.5 ml) was added and mixed. The plate was shaken vigorously until the solutions hardened, forming the hydrogel. After hardening, the hydrogel was washed gently with 1 ml of 10 mM phosphate buffer (pH 7.2) to remove the any residual 5-FU left on the hydrogel surface. The in vitro release study was carried out over 8 days. To quantify 5-FU released from the hydrogel, 2.5 ml of 10 mM phosphate buffer (pH 7.2) was added into each well and replaced every day. The plate was sealed and placed at room temperature. The quantity of 5-FU released from the hydrogel was measured by monitoring the absorbance at 265 nm. Since the released NHS or other PEG compounds gave additional signals at this wavelength, we carried out an additional experiment to assess background values without 5-FU. We calculated the net concentration of 5-FU by subtracting the background signals resulting from other compounds from the total signal.
Fig. 1 shows the cross-linking reaction of 6-arm PEG-amine (PEG-AM) and 6-arm PEG-succinimidyl glutarate (PEG-SG). When the PEG-SG is mixed with PEG-AM, PEG-SG separates the N-hydroxy succinimides (NHS) from its arms. Additionally, the terminals of PEG-SG (-CO2) are cross-linked with the amine (-NH2) groups of PEG-AM. This cross-linking between PEG-AM and PEG-SG changes the two PEG solutions into a gelatinous form (US Patent 6858736).
Pharmacokinetics
Rats were divided into two groups: a free 5-FU treatment group and a 5-FU-loaded PEG-hydrogel group. An aqueous solution of free 5-FU and a 5-FU-loaded PEG-hydrogel were administered subcutaneously to the rats at a dose of 100 mg/kg. To formulate the 5-FU-loaded PEG-hydrogel, PEG-SG and PEG-AM containing 5-FU were dissolved in sodium phosphate buffer (pH 8.0) and mixed together in equal volumes. The free 5-FU drug solution was injected into rats using a normal syringe, and the 5-FU-loaded PEG-hydrogel was injected using a mixing syringe device (Doowon Meditec Corp., Youngin-city, Republic of Korea). The aqueous solution of PEG-hydrogel (PEG-SG and PEG-AM) immediately changed into a gel after passing through the injection needle. Blood samples were collected at minutes (0, 5 and 30) and hours (1, 2, 3, 4, 12, and 24) after drug administration. Sampling continued until the PEG-hydrogel could not be palpated under the skin. Blood samples were centrifuged at 8000 rpm for 5 minutes (HANIL Science Industrial Co., Inchon, Republic of Korea), and harvested serum (150-200 μl) was subjected to HPLC analysis [7]. Samples were extracted using ethyl acetate (6~8 ml) and then evaporated to dryness under N2 gas in a water bath adjusted at 60°C. A 1.5 mM sodium phosphate buffer (pH 5.8) was added to reconstitute the residue. Approximately 200-300 μl of the reconstituted solutions were filtered and injected for HPLC (Agilent 1100, Santa Clara, CA, USA). Separation was accomplished via isocratic elution of the mobile phase, which contained methanol and 1.5 mM sodium phosphate buffer (99:1, v/v, pH 5.8), with a flow rate of 1 ml/min. A C18(Capcell Pak UG120, 5 μm, 4.6 μm I.D. × 250 μm, Shiseido, Tokyo, Japan) was used as an analytical column. The analysis was carried out at a column temperature of 40°C. The wavelengths of the FLD (fluorescence detector) were 260 nm and 350 nm for excitation and emission, respectively.
Pharmacokinetic parameters were estimated for each rat by using the program "WinNonlin" to fit the serum concentration versus time data to the following equation: C
p
= (K
a
*F*D)/{V
d
*(k
a
-k
el
)}*(exp
-kel*t- exp
-ka*t), where C
p
is the serum concentration and ka and kel are the absorption and the elimination rate constants, respectively. The F, D, and Vd represent the bioavailability, dose, and volume of distribution, respectively. The lag time was not considered, and the absorption and elimination rates were applied using first-order kinetics. The area under the concentration vursus time curve (AUC0-8 day) was calculated using the trapezoidal rule from time t = 0 to the last measured concentration on Day 8. Serum drug concentrations and the estimated pharmacokinetic parameters were reported as means ± SD.
Antitumor Activity of 5-FU-loaded PEG-hydrogels in Tumor-bearing Nude Mice
A total of 3 × 106 of cells from the A549 line, a human non-small-cell lung adenocarcinoma cell line, were inoculated into the dorsal neck of 4-week-old male nude mice (Balb/c Slc-nu/nu) as described in earlier reports [7, 8]. The tumor mass was monitored every week. When the tumors grew to 100-400 mm3, the mice were randomized into three groups: an untreated control group, a free 5-FU drug control group, and a 5-FU-loaded PEG-hydrogel group. The free 5-FU drug and 5-FU-loaded PEG-hydrogel were prepared as described above was subcutaneously injecting around the tumor mass once per week for 4 weeks at a dose of 85 mg/kg. Body weight, tumor volume, and clinical assessment of the mice were monitored every week until the end of the study. There was no significant difference in body weight changes among the groups. When tumors were palpable and visible, the tumor volume (TV) was measured with a Vernier caliper and calculated using the following formula:
The antitumor effect was estimated by calculating the relative tumor volume and the relative tumor inhibition rate (IR, %). The relative tumor volume (RTV) represents the tumor volume when the drugs are given to the mice.
where V0 is the tumor volume when the drugs are given to the mice and Vt is the tumor volume at each measurement.
The TRTV represents the RTV of treated groups, and the CRTV represents the RTV of the untreated control group.
Histopathological Examination
Sections of A549 taken from subcutaneously transplanted tumor masses were fixed with formalin and embedded in paraffin. Five micrometer tissue sections were prepared and stained with H & E.
Statistical Analysis
Multiple comparison tests for different treatment groups were conducted. An ANOVA multiple comparison test (Dunnett's test) was performed to determine which pairs of groups differed significantly. An unpaired Student's t-test was also used to compare the 5-FU-loaded PEG-hydrogel group versus the 5-FU-treated control group. The level of significance was taken as p < 0.05 or 0.01. Values represent means ± SD. Statistical analyses were performed with Statistical Analysis Systems software (SAS/STAT Version 8.1, Cary, NC, USA).