Preparation And Antitumor Activity Of Novel 2-methoxyestradiol Orally Micro-nano Complexes

2-Methoxyestradiol(2-ME)is a kind of steroid hormone antitumor drugs.The compound has strong killing activity on tumor cells,and does not affect normal cells,so it has received widespread attention from researchers.Although the oral preparation has the advantages of convenient taking and good patient compliance,the 2-ME is extremely difficult to dissolve in water,the short half-life after oral administration,and the low bioavailability limit its clinical application.Therefore,how to increase the effective concentration of 2-ME at the tumor site and improve the bioavailability is an urgent problem to be solved.One of the main physiological characteristics of the gastrointestinal tract and tumor cells is the change in pH.Based on this,this subject designed and prepared a pH-responsive micro-nano oral drug delivery system,which consists of 2-ME-containing polylactic acid-polyhistidine(PLA-PLH)and yeast-derived glucan microcapsules(YGM).The YGM shell can protect the drug-loaded nanoparticles to efficiently cross the acid barrier and the enzyme barrier;the drug-loaded nanocore utilizes the pH responsiveness of PLA-PLH,not only can be released from the micron shell in the intestine,and efficiently cross the mucus barrier and intestinal epithelial barrier into the blood circulatory system,and after entering the tumor cells,can increase the effective concentration of 2-ME in the cell through lysosome escape,and finally improve 2-ME bioavailability and anti-tumor activity.The research content of this paper mainly includes the following aspects:1.Synthesis and in vitro characterization of YGM/PLA-PLH/2-ME.First,histidine is prepared by anhydride ring-opening polymerization to obtain PLH,and then PLH is condensed with PLA to obtain block copolymer PLA-PLH,and nanoparticles are prepared by solvent exchange.The structure and morphology of PLA-PLH were characterized by fourier infrared spectroscopy,nuclear magnetic resonance hydrogen spectroscopy,dynamic scattered light,and transmission electron microscope.The experimental results showed that PLA-PLH is spherical nanoparticles with average particle size and average potential of(224.93±13.05)nm and(5.42±1.01)mV,respectively.The pharmacological experiment of the drug-loaded nanoparticles PLA-PLH/2-ME showed that when the mass ratio of 2-ME to PLA-PLH was 0.6:1,the drug loading rate and encapsulation rate were(27.86±0.19)%and(64.38±0.50)%respectively.Using electrostatic interactions,PLA-PLH/2-ME nanoparticles are loaded into YGM to form a 2-ME oral micro-nano composite(YGM/PLA-PLH/2-ME).The simulated gastrointestinal environment was used to investigate the drug release characteristics of the drug delivery system.The experimental results showed that under simulated gastric juice conditions,2-ME and nanoparticle PLA-PLH/2-ME 2 h release were(17.31±0.50)%and(14.72±0.21)%,in simulated intestinal fluid,2-ME and nanoparticle PLA-PLH/2-ME released in one hour were(2.04±0.14)%and(42.26±1.52)%.The experimental results show that the YGM/PLA-PLH/2-ME drug delivery system can effectively reduce drug degradation.Moreover,PLA-PLH/2-ME can be quickly released from the microparticle drug delivery system.This provides a structural basis for drug-loaded nanoparticles to enter the blood circulation.2.In vitro anti-tumor activity study of YGM/PLA-PLH/2-ME.The drug-loaded nanoparticles PLA-PLH/2-ME will be released from the micro-nano drug delivery system YGM/PLA-PLH/2-ME in the intestine,and then reach the tumor through the blood circulation system,so,we focused on the anti-tumor activity of nanoparticles.Cell uptake experiments show that PLA-PLH nanocarriers can significantly increase the uptake of drugs by colon cancer cell CT26.Cell proliferation rate experiments show that PLA-PLH nanocarriers have no significant inhibitory effect on the proliferation rate of CT26 cells in the concentration range of(2-160)?g/mL,PLA-PLH/2-ME nanoparticles can significantly increase 2-ME Inhibition of CT26 proliferation.Cell cycle and cell apoptosis experiment results showed that PLA-PLH/2-ME group mainly blocks CT26 tumor cell cycle in G2/M phase,and which can promote tumor cell apoptosis.Cell scratches experiments showed that PLA-PLH/2-ME can enhance the inhibitory effect of 2-ME on CT26 cell migration and invasion.In addition,we also studied the anti-tumor activity of YGM/PLA-PLH/2-ME.The experimental results show that the micro-nano drug delivery system can effectively inhibit the migration of tumor cells and has good anti-tumor activity in vitro.3.In vivo pharmacokinetic study of YGM/PLA-PLH/2-ME.In this study,Sprague-Dawle(SD)rats were used as animal models,and the high-performance liquid chromatography method was used to determine the drug concentration of 2-ME in rat plasma.Compared with the 2-ME group,the micro-nano drug delivery system YGM/PLA-PLH/2-ME group had significantly increased peak time and peak concentration.Tmax was significantly extended from(1.00±0.00)h to(2.30±0.67)h,Cmax increased from(0.54±0.04)?g/mL to(0.95±0.15)?g/mL,and the bioavailability increased by about 8 times.The results showed that the drug delivery system YGM/PLA-PLH/2-ME can significantly improve the half-life and oral bioavailability of 2-ME oral administration.4.In vivo anti-tumor activity study of YGM/PLA-PLH/2-ME.AOM/DSS method was used to construct a mouse colon cancer model,and the tissue distribution,nanoparticle release in vivo and antitumor activity of YGM/PLA-PLH/2-ME were investigated.In vivo imaging results of mice showed that YGM/PLA-PLH/DIR can significantly increase the accumulation of fluorescent dye in colon tissue after oral administration.In vivo nanoparticle release experiments showed that PLA-PLH/2-ME nanoparticles can be released from microparticles after YGM/PLA-PLH/2-ME enters the intestine.Mouse body weight curve,organ index,blood routine,liver function and organ slice experiments showed that the drug delivery system has good biocompatibility and no obvious toxicity in mice.The results of pharmacodynamic experiments showed that the tumor suppression rate of YGM/PLA-PLH/2-ME drug delivery system could reach 70.89%,which was significantly higher than that of the nanoparticle PLA-PLH group and the free drug group 2-ME group,indicating that oral micro-nano The drug delivery system YGM/PLA-PLH/2-ME can enhance the oral antitumor activity of 2-ME.