Intratumoral Delivery Of Paclitaxel-loaded PLGA Microspheres In Nude Mice Bearing Laryngeal Squamous Cell Carcinoma Xenografts

OBJECTIVE The polymeric drug system delivered locally provides a novel modality of increasing therapeutic concentrations of drug for a prolonged period while decreasing systemic levels. In the current study, the cytotoxic activity of paclitaxel on human laryngeal cancer cell line Hep-2 was investigated in vitro. On the basis of the evidence that the Hep-2 cell line was sensitive to paclitaxel, the anti-cancer agent was incorporated into the biodegradable polymer PLGA as controlled release microspheres. The characterization, in vitro release profile and the efficacy of the polymer delivered intratumorally against Hep-2 laryngeal squamous cell carcinoma xenografts in nude mice were studied. The level of angiogenic factors and microvessel density in xenografts were examined to explore the pharmacological effect of paclitaxel microspheres.METHODS 1. Hep-2 cells were exposed to concentrations of paclitaxel for 12-72h in vitro. Morphological changes were observed by phase contrast microscopy and anti-proliferation effect was measured with MTT assay. Cell cycle kinetics was analyzed with flow cytometry. Apoptosis was determined by Annexin V-FITC/PI double staining and Confocal laser scanning microscopy. 2. PLGA microspheres containing paclitaxel were prepared by modified solvent evaporation method. Central composite design/response surface methodology was applied to optimize the formulation. The in vitro characteristics and the effect of ⁶⁰Co gamma radiation dose (10, 20, 25 kGy) were investigated. 3. BALB/c nude mice bearing Hep-2 laryngeal squamous cell carcinoma were randomly assigned to one of the six treatment groups consisting of 6 mice each: (1) i.p. paclitaxel injection, 15 mg/kg, twice a week for 3 weeks; (2) i.t. saline; (3) i.t. blank micropheres; (4) i.t. paclitaxel injection, 15 mg/kg; (5) i.t. paclitaxel-loaded PLGA microspheres, 15 mg/kg; (6) i.t. paclitaxel-loaded PLGA microspheres, 25 mg/kg. The tumor size and weight of the mice were measured twice a week. Systemic toxic reactions and skins around the injection sites were examined. The tumor inhibition rate (IR) and tumor volume tripling times (TT) were calculated. 4. The expression of basic fibroblast growth factor (bFGF) andvascular endothelial growth factor (VEGF) and the counts of microvessel density (MVD) in xenografts treated with different schedules were examined by Envision? immunohistological technique.RESULTS 1. Paclitaxel inhibited cell growth in a dose- and time-dependent manner. 24h IC₅₀ was 10. 6nmol/L and 48h IC₅₀ was 6. 23nmol/L. Flow cytometry showed a G2/M arrest by paclitaxel in a dose-dependent manner. G2/M arrest reached peak at 24h and returned to pre-treatment level at 72h. Paclitaxel induced apoptosis in Hep-2 cells. Cells treated with higher concentration of paclitaxel showed faster occurrence of apoptosis. 2. Spherical microspheres with smooth surface were obtained. The drug loading, encapsulation efficiency, mean diameter and span of dispersity were 1.53 %, 97.29%, 42.72μm and 0.95, respectively. Bias between observed and predicted values was 4.08%, 1.91%, 6.48% and -4.90%, respectively. The cumulative release from microspheres was 56.19% for 30 days. After 30 days of in vitro degradation the microspheres lost much of their structural integrity and showed rough surface. The irradiated microspheres were sterile and three doses of ⁶⁰Co gamma radiation did not influence the in vitro characteristics. 3. Compared with the saline control group the tumor size and the tumor weight of the groups treated with paclitaxel solution or microspheres were significantly reduced (P < 0.05) and the tumor volume tripling time (TT) was significantly prolonged (P < 0.01). The TT of the group receiving intratumoral injection of paclitaxel microspheres at low dose was significantly increased relative to the paclitaxel solution groups by intratumoral or intraperitoneal administration (P < 0.05) . The TT of the group treated with paclitaxel microspheres at high dose was significantly increased compared to other paclitaxel treated groups (P < 0.01) . The tumor inhibition rates of groups treated with i.p. paclitaxel injection, i.t. paclitaxel injection, i.t. paclitaxel-loaded PLGA microspheres at low and high doses were 35.99%, 39.37%, 47.83% and 59.90%, respectively. PLGA blank microspheres had no effect on the tumor growth. No significant toxic reactions were observed in the experiment. 4. The immunoreactivity of bFGF and VEGF and the counts of MVD in Hep-2 laryngeal squamous cell xenografts treated with paclitaxel injection or paclitaxel-loaded PLGA microspheres were significantly decreased compared with saline control group (P < 0.05). The i.t. administration of paclitaxel microspheres produced a marked reduction in bFGF level and showed stronger anti-angiogenic activity in comparison with i.p. and i.t. administration of paclitaxel injection (P< 0.01).CONCLUSION 1. Paclitaxel inhibited the growth of Hep-2 cells and apoptosis was one of the main anti-tumor mechanisms. 2. Paclitaxel loaded sustained-release microspheres were successfully prepared and the optimum mathematic model was highly predictive. 3. Intratumoral administration of the polymer delivery system enhances the efficacy of paclitaxel against laryngeal squamous cell carcinoma in nude mice. 4. Paclitaxel exhibited anti-angiogenic activity by inhibition the expression of bFGF and VEGF. This effect could be better exploited via local delivery in the formulation of controlled release microspheres. These findings suggested that paclitaxel-loaded polymeric delivery system was promising for interstitial chemotherapy of laryngeal squamous cell carcinoma.