Study On Construction Of Ultra-pH-sensitive,Synergetic And Polymeric Prodrug Delivery System And Its In Vitro And In Vivo Antitumor Activity

Chemotherapy is one of the most important methods in the clinical treatment of cancer.However,traditional chemotherapeutic drugs are not effective due to poor tumor selectivity,and are often accompanied by serious toxic and side effects.In recent years,with the continuous development and in-depth research of nano-drug delivery system,polymer prodrugs with good biocompatibility have become a hot research topic in tumor treatment.Compared with traditional physical encapsulated nano-drug carriers,chemically bonded polymer prodrugs can significantly improve drug loading capacity,avoid drug diffusion in blood circulation,significantly reduce toxicity and side effects,improve bioavailability of drugs,and regulate drug release behavior in tumor cells accurately.However,at present,polymer prodrugs are still not used in clinic.The reason is that there is physiological barrier in tumor site.It is impossible to obtain ideal drug concentration in tumor site only through high permeability and retention(EPR)effect of tumor.After being ingested by tumor cells,high polymer prodrugs could not release drugs quickly,which affected the ability of killing tumor cells.In view of these,there is an urgent need to develop a new type of polymer prodrug nano-delivery system,which can significantly improve the targeting enrichment of drugs in the tumor site and the ability of rapid release of drugs in cellsTumor tissue cells proliferate abnormal and provide energy through glycolytic,this abnormal metabolism of lactic acid resulting in a gradual decrease in the tumor tissue from blood vessels to the inside of the tumor tissue pH.The condition of low pH,relative hypoxia and abnormal expression of specific protein in tissue can be used to design intelligent polymer precursor drug controlled release system.Because acidic environment is a kind of characteristic of tumor tissue,and there is the gradient difference of pH:pH is 7.4 in blood vessel;tumor tissue(extracellular)decreases gradually from outside to inside,the range of pH is 6.5-7.0;and pH is 4.0-6.0 in intracellular organelles(endosomes and lysosomes)of tumor cells.Therefore,the change of pH gradient in tumor tissue can be used to design multi-functional controlled release system of polymer prodrugs.In our research group,a novel ultra-pH-sensitive drug carrier was prepared based on the poly(ortho ester).It is possible to construct a pH-sensitive drug delivery system by further structural regulation and molecular optimization.In addition,the combination chemotherapy can significantly improve the efficacy of chemotherapyIn this paper,the pH-sensitive polymer prodrug delivery system was prepared by graft reaction between poly(ortho ester)(POEA-g-NH2)and 5-fluorouracil derivatives.Then the ultra-pH-sensitive polymer prodrug delivery system was constructed by embedding hydrophobic chemotherapeutic drug Doxorubicin(DOX)Finally,the chemotherapeutic effect was evaluated by a series of antitumor experiments in vivo and in vitro.The specific contents of the study are as follows(1)Firstly,5-fluorouracil derivatives(5-fluorouracil-l-acetic acid,5-FUA)were synthesized by the reaction of 5-fluorouracil with chloroacetic acid in the presence of potassium hydroxide.Then 5-FUA was grafted onto the poly(ortho ester)(POEA-g-NH2)by amide reaction in different proportions.Finally,the final products(P-FUA20,P-FUA50 and P-FUA80)were obtained by dialysis and lyophilization The molecular structure,drug loading and graft rate of the polymer prodrugs were determined by Nuclear Magnetic Resonance(NMR)and ultraviolet spectrophotometry,respectively.The 5-FUA loading contents of the three polymer prodrugs were 6.14%,6.14%,and 6.14%,respectively,and the 5-fluorouracil graft ratio were 19.58%,49.38%and 79.25%,respectively.(2)Amphiphilic polymer prodrugs could form blank prodrug micelles(P-FU20,P-FU50 and P-FU80)and DOX-loaded prodrug micelles(P-FU20-DOX,P-FU50-DOX and P-FU80-DOX)by self-assembly in aqueous solution.The particle size and Zeta potential of the micelles were measured by Dynamic Light Diffractometer(DLS).The average particle size of the blank micelles(P-FU20,P-FU50,and P-FU80)were 158.2 nm,148.5 nm and 168.2 nm,respectively,and Zeta potential of the blank micelles were-20.6mV,-18.1mV and-15.3mV,respectively.The average particle sizes of the DOX-loaded prodrug micelles(P-FU20-DOX,P-FU50-DOX and P-FU80-DOX)were 211.5 nm,235.0 nm and 223.6 nm,respectively.The drug loading content were 6.4%,7.5%and 6.7%,respectively.And the drug loading efficiency were 53.1%,55.3%and 49.1%,respectively.The critical micelle concentration of(CMC)was measured by fluorescence spectrophotometer The CMC of P-FU20,P-FU50,and P-FU80 micelles were 9.0 × 10-4 mg/mL,8.0 ×10-4 mg/mL and 1.5 × 10-4 mg/mL,respectively.Nuclear Magnetic Resonance(NMR)and DLS were used to detect the hydrolysis rate and particle size change of micelles under different pH conditions.At the same time,the drug release in different pH environment was detected by enzyme labeling instrument and ultraviolet spectrophotometry respectively.The experimental results were as follows:Under the condition of pH-7.4,there was no degradation and no significant change in size of each prodrug micelles.The maximum release amount of drugs in each micelles were less than 16%(DOX)and 8%(5-FUA)within 24 hours.Under the condition of pH-6.5,the ortho ester was degraded in 12 hours,and the particle size increased continuously in 24 hours,and the maximum particle size was more than 900 nm;The maxijmum drug release within 24 hours was less than 60%(DOX)and 40%(5-FUA),respectively.Under the condition of pH-5.5,the ortho ester was degraded rapidly.After 6 hours,P-FU20 and P-FU50 were degraded completely,while P-FU80 micelles were hydrolyzed up to 94%,and the size of micelles increased rapidly.After 12 hours,it reached about 800 nm,and after 24 hours,the micelles disintegrated.The drug release amount of DOX and 5-FUA in each drug-loaded micelle reached 80 to 90%(3)H22 cells and HepG2 cells were used to study the relationship between blank prodrug micelles and DOX-loaded prodrug micelles in vitro.The MTT method was used to detect the cytotoxicity of blank/DOX-loaded micelles.The results showed that both the physically embedded and chemically bonded drugs in the micelles could produce cytotoxicity equivalent to the free drugs in the micelles,and the toxicity of the two drugs in the micelles was similar to that of the free drugs.Laser Confocal Microscopy was used to analyze the uptake of DOX-loaded micelles by planar cells and the ability to penetrate into HepG2 multicellular spheres(HepG2 MTCS).The results showed that the micelles of DOX-loaded micelles could be ingested and released by cells.And has better tissue penetration ability in HepG2 MTCS.Flow Cytometry and Image J software were used for quantitative analysis of uptake of DOX-loaded micelles and permeability of DOX-loaded micelles in HepG2 MTCS.The results were consistent with those of qualitative experiments.The inhibitory effect of the micelles on the growth of HepG2 MTCS was observed under the Fluorescent Inverted Microscope.The results showed that the synergetic prodrug micelle groups had a better inhibitory effect on the growth.(4)H22 tumor-bearing mice were used to investigate the drug distribution and tumor inhibition ability.The results of drug distribution test showed that the synergetic micelles could effectively prolong the circulation time of drugs in the blood and improve the enrichment ability of drugs in the tumor.Compared with the free drug group,the maximum concentration of DOX and 5-FUA in the tumor in the groups of P-FU20-DOX,P-FU50-DOX and P-FU80-DOX increased by two-fold,and could maintain the relatively high drug concentration effectively.The results of antitumor experiment showed that the synergistic micelles had better antitumor activity.At the same time,the combination of prodrug micelles can effectively reduce the adverse effects of drugs on the heart and enhance the destruction of tumor tissue by the analysis of body weight and tissue sections in miceIn conclusion,the ultra-pH-sensitive polymer micelles have the following characteristics:1)the particle sizes are suitable and uniform,the lower CMC is beneficial to the long circulation of particle blood,and the particle size is stable under physiological pH;2)after reaching the tumor tissue,the rapid response of extracellular pH,resulted in the increase of micellar size and the enhancement of drug retention.3)after being ingested by tumor cells,under the action of lower intracellular pH and enzyme,the micellar size was further enlarged and then disintegrated,the drug was released rapidly,and the ability of killing tumor cells was enhanced.Through further functional modification and structural optimization,the ultra-pH-sensitive polymer system based on poly(ortho ester),5-fluorouracil and DOX is expected to be used in clinical research.