Studies On Preparation And Bioavailability Of Paclitaxel-loaded Poly (D,L-lactide-co-glycolide) Nanoparticles For Oral Administration

Paclitaxel,as a anticancer drug,has been widely used in clinic, especially for the treatment of breast cancer and ovarian cancer significantly.Since it is poor water solubility,clinically used paclitaxel in the paclitaxel injection is stabilized and dissolved by the mixture of polyoxyethylene castor oil(Cremophor EL) and ethanol(1:1,V/V).But Cremophor EL can promote histamine release,and usually cause severe allergic reactions and other adverse reactions.To solve the above problems, the research on the preparation of paclitaxel does not contain Cremophor EL and can improve the bioavailability become the hot spot.Nanoparticles for oral delivery of paclitaxel could not only reduce side effects and increase its stability,but also profit for storage and transport.Hence, paclitaxel nanoparticles(TAX-NPs) were prepared,the prescription and preparation technology of nanoparticles was optimized.In vivo evaluation of TAX-NPs were carried out.Main contents and results are as follows.1.In the study,HPLC determination was developed for paclitaxel in vitro and it was validated by characterization of linearity,precision as well as accuracy.Biodegradable polymer PLGA was used as a drug carrier,and the TAX-NPs were prepared by an emulsification-diffusion method.The effect of formula and technological factors on the quality of preparation was investigated by the estimate index of particle size and encapsulation efficiency of the TAX-NPs.The in vitro stability and release characteristics in vitro of TAX-NPs were investigated,the differential scanning calorimetry(DSC) and X-ray powder diffraction(XRD) were used to analyze existence state of the paclitaxel in the nanoparticles.The average diameter of TAX-NPs was(99±7.6) nm,and Zeta potential was(78.3±5.87) mV.The entrapment efficiency and the content of drug loading of TAX-NPs were(56.99±0.29)%and(7.04±0.13)%,respectively. Freeze-dried TAX-NPs at 4℃for 6 months showed good stability. Differential scanning calorimetry(DSC) and X-ray powder diffraction (XRD) analyses showed TAX was effectively wrapped in the nanoparticles as molecule morphous.TAX-NPs at two different pH values of PBS release medium for in vitro release,to simulate the in vivo environment,the lower the pH value in the condition,the faster the drug release.It is very favorable for TAX-NPs anti-tumor effects for tumor in the acidic environment2.The Caco-2 cell uptake of nanoparticles and its influencing factors were investigated to evaluate the feasibility of nanoparticles as a carrier of oral anticancer drugs.Nanoparticles were labeled by wrapping hydrophobic fluorescent probe coumarin-6(Cou-6) and fluorescence microplate reader was used to detect fluorescence intensity to investigate the particle size, incubation time,the concentration of nanoparticles and the surface properties of nanoparticles on Caco-2 cell uptake of nanoparticles.The laser confocal scanning microscope was used to observed Caco-2 cell uptake of nanoparticles.Compared with the nanoparticles emulsified by PVA,the nanoparticles emulsified by DMAB showed a significant increase in the Caco-2 cells uptake efficiency,and the uptake efficiency of nanoparticles with particle size of 100 nm was topmost.The results indicate that nanoparticles can be administered via the gastrointestinal tract.3.The absorption kinetics of free TAX and TAX-NPs were compared by the intestine of rats cannulated in situ recirculation experiment.The absorption kinetics equation of free TAX was lnX_{剩余}=lnX₀-0.154×t.And the absorption kinetics equation of TAX-NPs was lnX_{剩余}=lnX₀-0.0023×t, the correlation coefficient r were 0.9460 and 0.9102,respectively.The absorption of the two significant difference(P＜0.05) and the absorption speed of TAX-NPs was faster than that of free TAX.4.The analytical method of concentration of paclitaxel in plasma was devoloped.The established chromatographic conditions was as follow:the column was Diamonsil C₁₈(250mm×4.6mm,5μm),a mobile phase was acetonitrile/water(47:53),the temperature of column was 30℃,detective wavelength was 227 nm,flow rate was 1 ml/min and injection volume was 20μl.The recoveries of high,medium and low concentration were 91.23 %,92.68%,89.38%,respectively.Intra-day RSD were 3.81%,3.49% 2.89%,respectively.Inter-day RSD were 5.12%,4.59%,3.48%, respectively.Quantitative minimum concentration was 0.02μg/ml.These results showed that the established method was suitable for determining the plasma concentration of paclitaxel.Bioavailability in animal vivo was studied by a randomized and controlled study.Healthy SD rats were used as the tested subjects and paclitaxel injection was used as the reference formulation.Experimental results showed that the absolute bioavailability of TAX-NPs was 19.5%. These result showed that the nanoparticles drug delivery system promoted the absorption of paclitaxel to improve their oral bioavailability.