Ethyl acetate (analytical reagent), dichloromethane (DCM; chemical reagent) and 5-FU were purchased from Sigma-Aldrich. Poly(dl-lactide-co-glycolic acid) (PLGA; L:G = 50:50, MW 47 kDa) was purchased from Lakeshore Biomaterials, Inc. (USA).

Purebred male rabbits, 2–3 months of age, weighing 2.0–2.5 kg were used for the efficacy study in vivo. Adult male Sprague Dawley (SD) rats (200.2±25.1 g) were used for determining the in vivo sustained-release of the 5-FU concentration in the blood. SD rats and rabbits were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd. Animals were kept under standardized rodent conditions at 22.2±1.2°C.

The 5-FU microsphere delivery system was placed in dichloromethane solution of PLGA. After stirring for 1 min, the suspension was transferred into the hydrophilic oil phase (hO; containing 73% w/w ethylene glycol, 9% w/w PVA and 18% w/w glycerol) to become embryonic microspheres. The embryonic microsphere emulsion was then placed into sodium chloride solution (10%, w/w) and stirred for 2 h. The microspheres were gathered through centrifugation and freeze drying before usage. The control microspheres were prepared using a water-in-oil-in-water (W/O/W) method.

The size and surface morphology of the 5-FU microsphere delivery system were determined using a Hitachi S-4700 scanning electronic microscope (SEM). These microspheres were coated with gold in a vacuum before being scanned by SEM and the coating procedure was repeated three times.

5-FU microspheres (12 mg) were weighed and suspended in 5 ml DCM, and then the suspension was applied to centrifugation at 12,000 rpm for 6 min to remove PLGA. The remaining 5-FU microparticles were gathered by washing and centrifugation for an additional three times. Finally, the DCM solvent residues were removed by using evaporation in vacuum drying. The amount of 5-FU in the microsphere delivery system was determined by HPLC. The encapsulation efficiency (EE) was calculated using the formula as below:

The release tests were performed at 37°C. Microspheres (12 mg) weighed were re-suspended in 1 ml PBS (pH 7.4) release medium. Then the samples were incubated in a simulating biological fluid incubator at 37°C under a shaking rate of 150±10 rpm. The release medium containing the microspheres was centrifuged, and then the supernatant of the samples was completely drawn for each day, and another 1 ml of fresh release medium was then placed. The concentration of the 5-FU drug was measured using HPLC. Average and standard deviation of the data were obtained from three repeats of the same experiment.

After the subcutaneous injection of the 5-FU microsphere delivery system and administration of the 5-FU solution, the rats were initially anesthetized using halothane before performing the experiments. Blood (0.85 ml) was gathered from each rat at specific intervals following injection using puncture of the eye vein and collected in heparinized (15 μl containing 75 units) polypropylene tubes. The samples were centrifuged at 12,000 × g for 14 min in an Eppendorf AG (5414 D) centrifuge, and the samples were then collected to gain plasma samples. Proteins in the plasma were removed through precipitation by adding 2 M trichloroacetic acid (6 μl TCA/100 μl plasma), and then centrifuged (12,000 × g, 5 min), and finally the plasma was placed at −20°C. 5-FU was extracted from this plasma using 6 ml ethyl acetate (41) and 100 μl PBS (0.5 M, pH 8.0) was placed into 500 μl plasma. The samples were then shaken for 7 min and centrifuged (4,200 × g, 3 min), and the ethyl acetate phase was gathered. The ethyl acetate phase was removed by nitrogen gas at 55°C, and then the dried samples were re-dissolved with 100 μl (0.01 M pH 4.0 of potassium dihydrogen phosphate (KH₂PO₄), and the 5-FU drug concentration in the plasma was measured by HPLC (Shimadzu LC-10ATVP, Japan). The reverse phase (Waters) chromatographic C18 column was used. The mobile phase was 0.01 M pH 4.0 of KH₂PO₄. The flow speed of the mobile phase was 1 ml/min, and a wavelength of 266 nm of the detector was used for measurement of the drug concentration. To determine the content of 5-FU, 5-FU standards of 0.1–100 μg/ml in PBS (1 mM, pH 7.4) were used. Drug-free plasma samples pooled with a known content of 5-FU were used for samples containing plasma. In both cases, 5-FU standard profiles were determined using an external standardization method and linear profiles with a correlation coefficient of 0.990 obtained from the area under the UV absorption peak determinations. The retention time of 5-FU drug was 10.5±0.5 min. From the area under the absorption peak determinations of the plasma 5-FU concentration and retention time, a decreased rate of 5-FU drug concentration in the rat plasma was calculated through the slope of the straight line from the time of maximum 5-FU drug concentration, the absence of drug in rat plasma, and the circulating SD rat blood volume. For calculating the circulating SD rat blood volume, the suggested average value of 64 ml/kg body weight was calculated (41).

The local antitumor efficacy studies were carried out in rabbits with colon tumors. All rabbit experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Shanghai JiaoTong University. Purebred male rabbits, 2–3 months of age, weighing 2.0–2.5 kg were used. After anesthesia, the proximal lateral side of the thigh skin was routinely shaved, disinfected, and the medial side of the thigh muscle of each rabbit was explanted with 1 mm³ fresh tumor tissue blocks. Continuous observation of tumor growth was carried out. Tumors ~2 cm in diameter were noted in all rabbits two weeks after the transplantation. Eighteen rabbits were separated into three groups, namely: i) no therapy group (blank group); ii) single intravenous 5-FU treatment group; and iii) microsphere administered group. All rabbits after anesthesia underwent CT scanning, three-dimensional outline tumor on CT image processor workstations, and measurement of tumor size.

The data are expressed as the average ± standard deviation, and statistically significant differences were determined using one-way ANOVA test. P<0.05 was considered to indicate a statistically significant difference.

Size and surface morphology of the microsphere delivery system showed smooth surfaces, spherical shape, and sizes range from 100 to 150 μm (Fig. 1).

The in vitro release 5-FU drug profile of the microsphere delivery system in the PBS release medium is shown in Fig. 2. The in vitro drug released from the microspheres using the S/O/hO method was gradual and steady which lasted for one month. The cumulative sustained-release of 5-FU amounted to 92±4.5% of the total loading in the microspheres. The burst release amount of 5-FU was 11±2.6% within the first day. The 5-FU encapsulation efficiency of the microspheres using the novel method was 89.85±3.63%. Considering the loss of the microsphere delivery system in the preparation process, the real encapsulation efficiency would have been much higher.

The release of the 5-FU microsphere delivery system showed that the profile of the drug concentration in the rat plasma changed with time (Fig. 3). Mild burst release was observed from the microsphere delivery system at the initial stage of release, and the drug concentration reached a maximum of 2.4±0.22 μg/ml (Fig. 3A) from the microsphere delivery system at 24 h after injection and then more stable concentrations were maintained from day 2 to day 20 compared with the 5-FU solution subcutaneous injection. A decreased release speed of the 5-FU drug concentration in rat plasma was determined from the straight line slope from the time of free 5-FU up to the time of maximum 5-FU drug concentration, and the circulating blood volumes for rats. Circulating blood volume in the rat was 58–70 ml/kg in terms of body weight (42), and 64 ml/kg was the suggested average value. Therefore, the speed of 5-FU drug concentration in rat plasma was 2.41±0.28 μg/day. When the 5-FU solution at 35 mg/kg was administered through subcutaneous injection, the maximum 5-FU concentration was 18.11±1.02 μg/ml, half an hour after the subcutaneous injection with 5-FU being determined for 5 h (Fig. 3B). The AUC of 5-FU injected by the 5-FU microsphere delivery system was 12 times more than that of the subcutaneous injected drug in solution. This was possibly due to the fact that the microsphere delivery system released 5-FU drug in a sustained profile, which can prolong the time of drug concentration in blood and decrease the clearance speed of 5-FU.

In order to evaluate the therapeutic efficacy of the microsphere delivery system and 5-FU solution injection, rabbits with colon cancer were intra-tumorally administered with the 5-FU sustained-release microspheres, or intra-tumororally injected with the 5-FU solution, 0.5% sodium chloride (30 mg/kg 5-FU drug continuous infusion once as control group). Changes in tumor volume and the tumor growth inhibition rate in the rabbits were determined for 10 days. As shown in Fig. 4 the microsphere delivery system and the drug solution inhibited the tumor growth rate and achieved a significant increase in the antitumor efficacy of rabbits compared to that of the blank group. Yet, the inhibition ratio of tumor volume and tumor growth rate for the microsphere delivery system was better than that of the solution group. The toxicity related to the microsphere delivery system was determined by measurement of changes in body weight. Changes in the rabbit body weight in the 5-FU solution and 5-FU sustained-release microsphere groups were monitored every two days throughout this study. However, approximately similar results were found in regards to the changes in body weight between the treated groups using 5-FU solution and 5-FU sustained-release (27) microspheres (P>0.05).

The intra-tumor administration of the drug delivery system to mice (43,44) had an enhanced antitumor effect. The antitumor efficacy of the microsphere delivery system was also studied in rats which indicated much more significantly reduction in the mortality rate than the group administered with the water solution (45).

To study the pathological effect of the microsphere delivery system, tumor samples from rabbits on day 10 were embedded, fixed, sectioned and analyzed using hematoxylin and eosin (H&E) staining. As shown in Fig. 5 tumor tissues that received the 5-FU microspheres (Fig. 5C) exhibited certain significant necrotic areas, and had poorly defined borders and weak staining, while tumors tissues in the blank group (Fig. 5D) showed whole regions of proliferating tumor cells with a regular array and strong staining. At the same time, less necrosis was also shown in the 5-FU solution group (Fig. 5B).

In conclusion, in the present study, the 5-FU sustained-release microspheres using a novel S/O/hO method was developed for the 5-FU sustained-release, and local targeting in tumor tissues. 5-FU sustained-release microspheres were fabricated with biocompatible and biodegradable PLGA. According to our test data, the 5-FU drug sustained-release effect from the 5-FU sustained-release microspheres in vitro and in vivo was for ~30 and 21 days.

In addition, the antitumor effects were also studied in rabbits, which demonstrated an enhanced antitumor effect by administering 5-FU sustained-release microsphere delivery system compared to the intra-tumoral administration of the 5-FU water solution. Local administration of the 5-FU microsphere delivery system enhanced the drug concentration in the cancer tissues. Therefore, the microsphere delivery system may improve the antitumor effects and reduce the 5-FU toxicity in normal tissues. As a result, intra-tumoral administration of the microsphere delivery system may provide better local treatment for cancer chemotherapy against solid tumors.