Extraction Of Paclitaxel From Taxus Cuspidata And Construction Of Bacterial Cellulose-based Oral Micelles

Taxus cuspidate Siebold & Zucc.is mainly distributed in Japan,North Korea,Russia and northeastern China,and its branches and leaves are one of the main raw materials for paclitaxel(PTX)extraction.The commonly used extraction methods of PTX from Taxus chinensis often have shortcomings such as long time,high energy consumption,low safety and large amount of organic solvents,which bring great difficulties to the large-scale production of PTX.In addition,as a natural anticancer drug,PTX has a good inhibitory effect on breast cancer,ovarian cancer and lung cancer.At present,PTX is mainly administered in the form of injection in clinical application,and injection administration has defects such as large toxic and side effects,instability and short efficacy.Compared with injection,oral administration is safer and more suitable for long-term administration.However,the poor water solubility and low oral bioavailability of paclitaxel severely limit the application of its oral preparations in clinical treatment.In view of the above problems,this study selected the branches and leaves of Taxus chinensis with strong regeneration ability as the extraction raw materials,and established an extraction process with high efficiency,high safety and little damage to the structure of paclitaxel.In addition,this study developed a bacterial cellulose-based oral micellar drug delivery system with P-glycoprotein(P-gp)inhibition.The anticancer activity in vitro and in vivo was evaluated.The specific research results are as follows:(1)In this study,the probe-type ultrasound method was used to assisted Sapindus saponin to extract PTX from the branches and leaves of Taxus chinensis,and the extraction process of PTX was optimized by combining single factor and response surface methodology: the concentration of sapindus saponin was 7.4 mg/m L,the solid-liquid ratio was 1:32.6,the ultrasonic time was 15.6 min,the ultrasonic power was 106.6 W,and the predicted PTX extraction rate was 90.95%.The actual PTX extraction rate was 90.68% ± 1.2%.It is basically consistent with the predicted extraction rate.It shows that the extraction method has good stability and high repeatability.After purification,a high-purity PTX product with a purity of97.11%±0.35% was obtained.(2)In this study,the amphiphilic polymer BC-QT was obtained by coupling bacterial cellulose(BC)and quercetin(QT)through succinic anhydride.FTIR and 1H NMR results indicated that BC was successfully linked to QT.Different ratios of BC and QT were synthesized,and the critical micelle concentrations were tested to be 0.091 mg/m L(1:1),0.052mg/m L(1:2)and 0.043 mg/m L(1:3),respectively.In the safety evaluation,after co-incubating BC-QT polymer with a concentration of 1-200 μg/m L with hepatocyte LO2 for 48 h,the cell viability was above 95%.When the concentration was 10 mg/m L,the development of zebrafish embryos was adversely affected,resulting in abnormal embryo development and embryonic death.Below this concentration,the zebrafish embryos developed well and the heart rate of the incubated zebrafish was normal,indicating that this amphiphilic compound is less toxic and suitable for the development of drug delivery systems.(3)After the safety evaluation of the BC-QT polymer,it will be used to develop a PTX oral formulation.Considering factors such as particle size,drug loading and encapsulation efficiency,the BC-QT polymer with a ratio of 1:2 was finally selected to load PTX in this study.The optimal drug loading conditions determined by single factor optimization are: the ratio of water and ethanol(v/v)is 10:1,the concentration of carrier is 1mg/m L,and the ratio of carrier to drug(m:m)is 10:3,the ultrasonic power was 270 W,the ultrasonic time was 20 min,the drug loading of the sample was finally measured to be 25.10%,and the encapsulation efficiency was 98.32%.Before and after drug loading,the zata potentials of BC-QT blank micelles and BC-QT-PTX were-20±2.5 m V and-22±1.2 m V,respectively,and the PDIs were0.13±0.04 and 0.15±0.03,respectively.In addition,it can be seen from the DSC and XRD results that the loaded PTX is in an amorphous state.It can be seen from the in vitro release results that only about 25% of the BC-QT-PTX nanomicelles are released in the simulated gastrointestinal medium,which indicates that the nanomicelles will remain stable in the intestinal tract and will not be destroyed by digestive juices.It is beneficial to the complete uptake of micelles by intestinal cells into the body fluid circulation system.Transmission electron microscopy of the nanomicelles treated with gastrointestinal media further confirmed the stability of the nanomicelles.Caco-2 cellular uptake studies have shown that the main way that nanomicelles are taken up by cells is the endocytic pathway mediated by clathrin,caveolin and energy consumption.And the Nile red fluorescent dye loaded in nanomicelles is easier to be taken up by cells in a time-dependent manner.Compared with free PTX,the apparent permeability coefficient of BC-QT-PTX nanomicelles was 8.3 times that of its original drug.Importantly,there was no significant change in the apparent permeability coefficient of PTX after addition of the P-gp inhibitor verapamil.The bioavailability of BC-QT-PTX nanomicelles was 4.16 times that of the original PTX drug.(4)This study investigated the antitumor effect of BC-QT-PTX nanomicelles in vitro and in vivo.In the MTT experiment,due to the hydrophobicity of PTX,the toxic effect of the original drug of PTX on LLC cells did not change significantly with the concentration,while the nanomicelle group showed an obvious concentration dependence.The higher the concentration of nanomicelles,the inhibition of LLC cells.the higher the rate.In the results of the ability to induce apoptosis,the apoptosis rates of PTX,PTX+VRP and BC-QT-PTX nanomicelles after co-incubating with cells for 24 h were 23.52%,22.81% and 84.89%,respectively.The apoptosis rates after 36 h were 47.24%,54.5% and 98.43%,respectively,and the nanomicelles showed an absolute advantage in inducing apoptosis.Using in vivo imaging technology,we observed that nanomicelles can persist in the intestine for a long time,and most of them are absorbed by intestinal cells into the body fluid circulation as intact micelle particles.High fluorescence intensity,while the fluorescence intensity of other visceral tissues is weak.It shows that the loaded paclitaxel avoids the efflux of P-gp in the intestine due to its hydrophilic shell and the presence of QT,and also reduces the efflux of P-gp on tumor cells,coupled with the EPR effect of tumor tissue,which prolongs the circulation time of BC-QT-PTX nanomicelles in tumor-bearing mice,and accumulates in tumor tissues continuously,which improves the anti-tumor activity of PTX.In the solid tumor treatment experiment,the normal saline group,BC-QT blank micelle group,PTX low-dose + VRP(PTX/L + VRP,5 mg/kg PTX+ 25 mg/kg VRP),and PTX low-dose group were investigated respectively.(PTX/L,5 mg/kg PTX),PTX high-dose group(PTX/H 20 mg/kg PTX),PTX-loaded BC-QT-PTX micelle lowdose group(BC-QT-PTX/L,5 mg/kg PTX),tumor growth trend of PTX-loaded BC-QT-PTX micelle high-dose group(BC-QT-PTX/H,20 mg/kg PTX).The results found that the tumor inhibition rates of the BC-QT-PTX/L and BC-QT-PTX/H groups were 68.06% and 93.54% in the nanomicelle group while maintaining the weight of the mice,while the PTX/L+VRP,The tumor inhibition rates of the PTX/L and PTX-H groups were 64.27%,47.94%,and 58.67%,respectively,and had certain toxicity to mice.The weight of the mice in the oral PTX/L+VRP and PTX-H groups varied at different times.decreased within the segment.In the HE staining study,the heart,liver,spleen,lung and kidney in the nanomicelle group showed no obvious signs of damage,but in the original drug paclitaxel group,there were different degrees of damage to the liver and spleen.This is due to the damage to the internal organs caused by the unutilized PTX during metabolism.On the contrary,in the tumor tissue sections,the treatment groups all had a killing effect on tumor cells,and the nanomicelle group showed outstanding performance,especially the high-dose nanomicelle group.