Construction And Mechanism Of Action Of Tumor Microenvironment Cascade Response Nanoparticle

Paclitaxel is a commonly used chemotherapy drug in clinical practice,but it has many disadvantages such as low solubility,poor stability,large toxic and side effects,and narrow therapeutic window.In recent years,the widespread application of prodrugs and nanotechnology in drug delivery systems have greatly improved the efficacy of paclitaxel and expanded the application of paclitaxel.At present,prodrug nanomaterials,combining the advantages of small-molecule prodrugs and nanotechnology,have the advantages of high drug loading,good stability,strong targeting,intelligent release and low toxic and side effects,which can better improve the efficacy of drugs.It is a hot topic in the researches of chemotherapeutic drugs delivery.In addition,the selective release of intelligently triggered drugs at target sites,which is important for the efficacy and safety of paclitaxel,tumor cells have higher levels of reactive oxygen species(ROS)and reduced glutathione(GSH)than normal cells,and this particular redoxl microenvironment has been widely used to design various stimulus-responsive drug delivery systems.Therefore,based on the characteristics of tumor microenvironment and the advantages of prodrug nanoparticles,the purpose and significance of this paper aims at constructing prodrug nanoparticles that respond to microenvironment and improving the physical and chemical properties of paclitaxel,which can better increase its efficacy.Therefore,based on the highly expressed CD206 receptor on the surface of tumor cells and redox microenvironment formed by intracellular concentration of ROS and GSH.The ROS sensitive paclitaxel prodrugs were constructed and combined with active targeting and GSH sensitive nanomaterials.The multi-stimulus responsive biodegradable prodrug nanomaterials were designed to improve the antitumor efficacy of paclitaxel.This paper is mainly divided into three parts:1.Three paclitaxel prodrugs with H2O2 sensitivity were designed and synthesized,the H2O2 sensitivity of the three prodrugs were verified in vitro,the best prodrug with H2O2 sensitivity were screened out.2.mPEG5k-SS-PLGA60k and Mannose-PEG5k-SS-PLGA60k were used as carrier materials,and self-synthesized paclitaxel prodrug was used as model drug,Mannose targeting and GSH sensitive prodrug nanoparticles were prepared by self-assembly,the formulation and preparation process of prodrug nanoparticles were optimized,and their pharmaceutical properties(particle size,stability,in vitro release,etc.)were systematically evaluated.3.this paper systematically evaluated the antitumor activity of the prodrug nanoparticles,including cytotoxicity,cell uptake and uptake mechanism,cell apoptosis and cellular reactive oxygen species.Firstly,three kinds of H2O2 sensitive paclitaxel prodrugs(ProPTX-1,ProPTX-2,ProPTX-3)were synthesized with paclitaxel and phenylborate,and their structures were tested by NMR and HR-ESI-MS.Stability and cytotoxicity experiments were used to evaluate the H2O2 sensitivity of three prodrugs in vitro.The results showed that ProPTX-3 could rapidly hydrolyze to release paclitaxel under the action of H2O2 and have high cytotoxicity.Therefore,ProPTX-3 was selected as the best H2O2 sensitive prodrug for subsequent experiments.Secondly,ProPTX-3 was used as the model drug and GSH sensitive mPEG5k-SS-PLGA60k and Mannose-PEG5k-SS-PLGA60k were used as the carrier materials.The paper prepared Man-ProPTX-SS-NPs by probe ultrasound method and investigated the pharmaceutical properties of prodrug nanoparticles.The results showed that the prodrug nanoparticles had good appearance and shape,the drug loading was about 14%,the encapsulation rate was more than 93%,the particle size was about 130 nm and the distribution was uniform,the Zeta potential was about-8 mV,the particle size and encapsulation rate have no significantly change after being stored at room temperature for 30 days,this suggests that the nanoparticles have good stability.The GSH and H2O2 sensitivity of ProPTX-SS-NPs drug release were investigated in vitro,and the sensitivity was proportional to GSH and H2O2 concentration.The results showed that the prodrug nanoparticles could keep stable in the systemic circulation and release the drug rapidly in tumor cells to reduce toxicity.Finally,the antitumor activity of the prepared prodrug nanoparticles was evaluated at the cell level.First,the result of MTT cytotoxicity experiments showed that prodrug and prodrug nanoparticles both improved the paclitaxel resistance of MDA-MB-231 cells and enhanced the antitumor activity of paclitaxel;At the same time,the blank preparation has certain cytotoxicity,which can cooperate with prodrug to play an antitumor effect.The three prodrug nanoparticles all increased the toxicity of prodrug to MDA-MB-231 cells,among which the tumor inhibition of Man-ProPTX-SS-NPs was the best;The uptake of PTX-Sol,ProPTX-Sol and three prodrug nanoparticles on MDA-MB-231 cells was further investigated qualitatively and quantitatively.The result was consistent with the result of cytotoxicity,Man-ProPTX-SS-NPs can significantly improve the cellular uptake of ProPTX or PTX.Then,the uptake mechanism of Man-ProPTX-SS-NPs on MDA-MB-231 cells was investigated using endocytosis inhibitors.The result showed that Man-ProPTX-SS-NPs is an energy-dependent,co-mediated uptake of tumor cells by non-clathrin and non-fossa,lipid raft/fossa,and cyclooxygenase(COX)/fossa.In addition,MDA-MB-231 cells with high expression of CD206 receptor were used as the cell model to investigate the affinity of prodrug nanoparticles to CD206 receptor through competitive inhibition experiments.The results showed that the prepared prodrug nanoparticles could specifically bind to the CD206 receptor on the surface of MDA-MB-231 cells and actively enter the cells,thus increasing the accumulation of drugs in the cells.In addition to this,compared with PTX-Sol,ProPTX-Sol and prodrug nanoparticles can significantly promote cell apoptosis(P<0.01),and Man-ProPTX-SS-NPs can significantly induce cell apoptosis,which is consistent with the result of cytotoxicity.Finally,the effects of PTX-Sol,PTX-NPs,ProPTX-Sol and three prodrug nanoparticles on the intracellular ROS levels of MDA-MB-231 were quantitatively determined by flow cytometry.The result showed that prodrug and prodrug nanoparticle could significantly reduce the intracellular ROS(P<0.01),among which,Man-ProPTX-SS-NPs was the best to reduce the intracellular ROS level of MDA-MB-231 cells,which was consistent with the result of cell uptake and cytotoxicity.In summary,the constructed Man-ProPTX-SS-NPs have Mannose targeting,GSH sensitivity and ROS sensitivity,which can significantly improve the intracellular accumulation and release of drugs,it can better play the antitumor effect.