Study On Multifunctional Paclitaxel Targeting Polymeric Micelles

Purpose:Non-small-cell lung cancer?NSCLC?accounts for 80%of the total patients with lung cancer.It has the characteristics of complex pathogenesis,short proliferation cycle,strong metastatic activity and high fatality rate.Ninety percent of patients with non-small cell lung cancer have suffered tumor metastasis before the diagnosis,thus the best period of surgical resection and chemotherapy was missed.Eventually,they died of the side effects of radiation and chemotherapy and complications caused widespread tumor metastasis.Studies have shown that lung cancer cells not only can metastasize,but also can mutate themselves to generate vasculogenic mimicry?VM?channels under hypoxia,so as to supply nutrients to accelerate the growth of tumor tissue.The less differentiated lung cancer cells are,the more likely they are to generate VM channels,and tumors that have formed blood vessels are less sensitive to conventional chemotherapy,which is also an important reason for ineffective chemotherapy,poor prognosis and even recurrence after treatment.Therefore,a kind of multifunctional paclitaxel-targeted polymeric micelle was established by membrane hydration method in this study to induce apoptosis of lung cancer cells and destroy the VM channels while inhibiting their metastasis activity,so as to improve the clinical chemotherapy effect of non-small cell lung cancer.Method:The target molecule dequimium was modified on the micelle surface to prepare the multifunctional paclitaxel-targeted polymeric micelles,and the optimum preparation process was optimized by using the star-design-response surface method.The synthetic product DSPE-PEG₂₀₀₀-DQA was verified by MALDI-TOF-MS.The physical and chemical properties such as morphology,particle size,PDI,encapsulation rate,in vitro release and stability of the multifunctional paclitaxel-targeted polymeric micelles with the optimal formulation were investigated.At the same time,the in vitro content determination methods of paclitaxel and honokiol were established and the methodology was investigated.The cell uptake and targeting of multifunctional paclitaxel-targeted polymeric micelles in LLT cells were investigated by flow cytometry and inverted confocal microscope.SRB staining was used to investigate the cytotoxicity of multifunctional paclitaxel-targeted polymeric micelles on LLT cells,and the cytotoxicity mechanism of polymeric micelles was studied by the measurement of caspase apoptosis protease content and apoptosis kit.LLT cells were used to establish in vitro lung cancer adhesion model,VM channel model,scratch repair model and invasion model,so as to evaluate the ability of multifunctional paclitaxel-targeted polymeric micelles to inhibit tumor metastasis,and the expression of related proteins was investigated to further study the relevant mechanism.Multispectral in vivo imaging system was used to study the distribution of multifunctional paclitaxel-targeted polymeric micelles in tumor-bearing mice,and pharmacodynamic evaluation was conducted to investigate the antitumor effect of micelles.The safety of micelles was evaluated by measuring the change rate of mice weight and routine blood tests.Results:1.The optimum preparation process was optimized by using the star-design-response surface method.The optimal prescription is as following:hydration temperature was 25?,Soluplus/paclitaxel=150:1?mass ratio?,Soluplus/TPGS₁₀₀₀=8?mass ratio?.The encapsulation rate of paclitaxel and honokiol was 96.11%.2.The multifunctional paclitaxel-targeted polymeric micelles were smooth spheres with regular structures,and had no significant differences among the particles.The average particle size was 91.22±2.19 nm,and the PDI was 0.17±0.01.The encapsulation rates of paclitaxel and honokiol in multifunctional paclitaxel-targeted polymeric micelles were 90.27±2.88%and 92.75±2.62%,respectively.The content determination method was accurate.The release rate of paclitaxel and honokiol within 48 h were 16.10±1.83%and 15.83±2.46%,respectively.The stability of micelles stored in 4?after 60 days was perfect,and leakage did not occur.3.The multifunctional paclitaxel-targeted polymeric micelles can significantly improve the targeting and cell uptake of the drug.4.The IC₅₀0 of multifunctional paclitaxel-targeted polymeric micelles was 0.16±0.07?M,which had a significant killing effect on LLT cells,and its induced apoptosis was associated with the activation of caspase 3 and caspase 9.5.The multifunctional paclitaxel-targeted polymeric micelles can significantly reduce the adhesion between LLT cells and the matrix,and effectively inhibit the ability of scratch repair,invasion,metastasis and VM channel.The mechanism of action was related to the reduced expression activities of related proteins FAK,PI3K,MMP-2 and MMP-9.6.In vivo imaging results showed that multifunctional paclitaxel-targeted polymeric micelles could effectively accumulate in the tumor site of tumor-bearing mice,significantly extend the survival time of tumor-bearing mice and effectively inhibit tumor volume.Blood routine test showed that the blood components of mice were within a reasonable range,and the body weight was normal.Conclusion:In this study,the optimum preparation process was optimized by using the star-design-response surface method.Thin-film hydration method was used to prepare multifunctional paclitaxel-targeted polymeric micelles with small particle size,concentrated PDI,high encapsulation rate,perfect release and good storage stability.The cell uptake and targeting property of polymeric micelles were excellent.The polymeric micelles have significant cytotoxic effects on LLT cells,and the mechanism is related to the expression of apoptotic protease.The polymeric micelles have an obvious inhibitory effect on matrix adhesion,scratch repair,invasion,metastasis and VM channel of LLT cells,and the mechanism is related to the expression of activated metastasis-related proteins.In tumor-bearing mice,the multifunctional paclitaxel-targeted polymeric micelles show strong targeting and can effectively accumulate in tumor sites,significantly inhibit tumor volume and prolong survival time of mice.It also had good safety.