Construction And Targeting Evaluation Of Tumor Cell Cycle-Specific Targeting Mixed Micelles

The treatment of malignant tumors has become a major issue in the scientific community nowadays.One of the most effective ways to solve this problem is to develop targeted agents.Preparations of cell cycle-specific drugs such as paclitaxel,cytarabine,and vinblastine have been used in clinical treatment.However,the agent is not designed for a specific phase of tumor cells,resulting in a limited amount of drug entering in tumor cells,thus the anti-tumor effect was limited.Therefore,this project was proposed to design a multi-functional cell cycle-specific and pH sensitive drug delivery system.Poly(ethyleneglycol)-b-Poly(L-histidine)(PEG-PHIS)was a type of pH-sensitive material with high biocompatibility.Duramycin was used to modify poly(ethylene glycol)-b-poly(D,L-lactide)(Dur-PEG-PDLLA).A mixed micelle system was formed by Dur-PEG-PDLLA and PEG-PHIS,carrying paclitaxel(PTX),a cell cycle specific model drug,entering into tumor sites.In vivo study showed that the mixed micelle system could enhance the targeting effect,prolonging the circulation time of the drug and enhancing the anti-tumor effects.This experiment is divided into the following four parts:Part 1.Synthesis and characterization of copolymers.PEG-PHIS was synthesized by ring-opening polymerization using Boc-His(Dnp)-OH?isopropanolas.Duramycin was grafted to HCOO-PEG-PDLLA by EDC/NHS-mediated acylation reaction.The chemical structures of PEG-PHIS and Dur-PEG-PDLLA were confirmed by ~1H-NMR and FTIR.The results confirmed the successful synthesis of PEG-PHIS and Dur-PEG-PDLLA.Part 2.Preparation of the paclitaxel mixed micelle and investigation of its characteristics.The effects of dialysis,film dispersion and dissolution method on the drug loading and particle size distribution of Dur-PEG-PDLLA/PEG-PHIS micelles were investigated.The results showed that the dialysis method was the best preparation method.The obtained preparation had a relative high drug-loading content of 14.7±0.3%and an encapsulation efficiency of 89.1±2.5%.Dynamic light scattering(DLS)showed that particle size was188.6±5.3 nm.Transmission electron microscopy(TEM)showed that the mixed micelles had a regular spheroidal structure with uniform size and good dispersion.The critical micelle concentration(CMC)of the mixed micelles was 0.09?g/mL,which indicated that the micelles had good anti-blood dilution ability.The particle size and drug release behavior of micelles at different pH were also studied.The results showed that the particle size and drug release of the micelles changed significantly under different pH conditions,especially at pH 5.0,indicating that the mixed micelles are pH sensitive.Part 3.Cell cycle-specific uptake and in vitro anti-tumor activity of paclitaxel mixed micellesFITC was conjugated to HCOO-PEG-PDLLA for fluorescence studies.The uptake of PEG-PDLLA,Dur-PEG-PDLLA,PEG-PDLLA/PEG-PHIS,and Dur-PEG-PDLLA/PEG-PHIS by HeLa cells was studied by confocal laser confocal microscopy(CLSM)and flow cytometry(FCM).The results showed that in the Dur-PEG-PDLLA and Dur-PEG-PDLLA/PEG-PHIS group,the intracellular fluorescence intensity increased significantly,indicating that Dur modification can increase the uptake of nanoparticles by cells.To further study the cell cycle-specific uptake,G1,S and G2/M phase cells were obtained by cell-synchronization method,and normal tumor cells were used as control.The results showed that Dur modification can significantly increase the uptake of nanoparticles by G2/M phase cells,which is 1.9 times higher than that of unmodified cells.The safety of blank micelles and the anti-tumor activity of drug-loaded preparations were studied by cytotoxicity test(MTT).The results showed that the blank micelles were basically nontoxic,while the drug-loaded micelles showed higher toxicity comparing with the commercial preparation of paclitaxel(Taxol).Part 4.In vivo pharmacokinetic study and targeting evaluation of mixed micelles.The drugl-loaded micelles was injected through the tail vein and the commercial paclitaxel preparation Taxol was tested as control.The half-life of the mixed micelles was4.144±0.548 h,while Taxol was 0.866±0.016 h.The mixed micelles prolonged the circulation time of the drug in the body and achieved a sustained release effects.In vivo imaging of small animals preliminarily studied the distribution of micelles in mice.Compared with Taxol,micelles accumulated at the tumor site and reached the peak at 8h.This indicated that paclitaxel mixed micelles had targeting effect.