Construction Of Folic Acid Modified Amphiphilic Paclitaxel Micellar Drug Delivery System And Its Targeted Drug Delivery For Cervical Cancer

Background:Malignant tumors are recognized as one of the most serious diseases that are harmful to human health.With the changes in human living environment and living habits,the overall growth of tumor patients and the trend of rejuvenation of disease have caused the clinical demand for anti-tumor drugs to rise sharply.However,most chemotherapeutics have treatment problems such as low treatment efficiency and large side effects.Paclitaxel（PTX）has unique anti-cancer activity against breast cancer,advanced ovarian cancer,cervical cancer,acute leukemia,etc.,and has been widely used in clinical treatment.However,paclitaxel has low solubility,which limits its clinical application.At present,paclitaxel injection widely used in clinical practice adds polyoxyethylene castor oil as a solubilizer to improve its solubility.However,with the addition of the solubilizer,the probability of severe allergic reactions increases.Therefore,it is still a big challenge for pharmaceutical researchers to develop a PTX targeted drug delivery system with lower toxicity and better tolerance to replace existing dosage forms.Objective:Paclitaxel was selected as the target drug,and amphiphilic chitosan material modified with folic acid was used as the carrier to construct a new type of paclitaxel drug-loaded micelles with high efficiency and low toxicity that target the folate receptor and have p H sensitivity,which is the clinical application of paclitaxel Bring new ideas.Method:1.Synthesis and characterization of folic acid-cholesterol-chitosan polymer.Folicacid（FA）,cholesterol（CH）,chitosan（CS）are synthesized by aminoacylation reaction and decarboxylation reaction to synthesize folic acid modified amphiphilic cholesterol chitosan（FACC）polymer,infrared spectrometer（FT-IR）and nuclear magnetic resonance（¹H NMR）to characterize its structure,fluorescence spectroscopy to detect the critical micelle concentration（CMC）value of polymer FACC.2.Preparation and quality evaluation of paclitaxel drug micelles.Paclitaxel drug-encapsulated micelles（PTX-FACC）were prepared by dialysis,and the encapsulation efficiency and drug loading of micelles were detected by HPLC.The particle size and morphology of the micelles were detected by laser particle size analyzer and transmission electron microscope,and the PTX-FACC micelles were detected by HPLC in a PBS solution of p H 5.0,p H 7.4 and p H 9.0（containing 1%Tween-80）;take paclitaxel injection（released in p H 7.4 PBS solution）as a control to investigate the stability and release effect of PTX-FACC micelles in different p H media.3.In vitro targeting study of FACC polymer.With paclitaxel solution（PTX）as the control group,the cytotoxicity of PTX-FACC micelles on Hela（high folate receptor expression）cells and A549（folate receptor low expression）cells was measured and calculated by the thiazole blue colorimetric（MTT）method.Using immunofluorescence,FACC polymer was labeled with fluorescein isothiocyanate（FITC）,rhodamine（TRITC）,and Hoechst 33342,and the FACC polymer was observed in Hela cells by laser scanning confocal microscope.And the distribution of A549 cells.4.In vivo targeting study of FACC polymer.A tumor-bearing nude mouse model carrying Hela tumor cells was constructed,and the drug-loaded micelles were fluorescently labeled with DIR,and the distribution of FACC polymer in mice was studied by in vivo imaging technology.Results:1.The characteristic peak of the FT-IR spectrum at 1652-1940 cm^-1shows the formation of CH-CS polymer.The characteristic peak at1637cm^-1 indicates that folic acid is successfully coupled to the chitosan backbone and FACC polymer is formed.The ¹H NMR spectrum showed new peaks at 2.5 ppm,5.95 ppm and 6.65 ppm,further confirming the synthesis of FACC polymer.The CMC value of FACC is 64.13μg/m L,indicating that FACC can form very stable micelles at low concentrations.2.The size of the PTX-FACC micelle prepared by the optimal formulation is253±0.56nm,and its encapsulation efficiency and drug loading are 65.1±0.23%and9.1±0.16%.The test results of drug release rate can reach 92%under acidic conditions（p H=5.0）,which is higher than physiological conditions（p H=7.4）and alkaline conditions（p H=9.0）.This p H-dependent release behavior indicates that the release rate of PTX-FACC micelles in the weakly acidic tumor microenvironment is higher,the toxicity to normal cells is lower,and it is targeted.Under the condition of p H 7.4,PTX-FACC micelles can slowly release PTX within 96 hours,and the release rate is much lower than the control group,indicating that FACC micelles have a good sustained release effect.3.According to the results of MTT detection,when the FACC micelle concentration reached 500μg/m L,the cell survival rate was still above 80%,indicating that FACC micelles had no obvious cytotoxicity to Hela cells and A549 cells.The cytotoxicity of PTX-FACC micelles at different concentrations was higher than that of the PTX control group,especially for Hela cells.Fluorescence microscopy showed that FACC has a strong fluorescence signal in Hela cells,and a very weak fluorescence signal in A549 cells,proving that FACC micelles are targeted to Hela cells.4.In vivo imaging tests showed that compared with the DIR solution of the control group,FACC micelles had stronger fluorescence intensity and longer duration on tumor sites in tumor-bearing mice,proving that FACC micelles had a targeting effect on cervical cancer.Conclusion:The FACC polymer micelles constructed in this paper have suitable particle size,uniform morphology,high encapsulation efficiency for paclitaxel,strong drug-loading ability,sustained release,and strong targeting of cervical cancer cells.FACC polymer is a good drug carrier for targeted delivery of PTX,which provides a reference for the development of new formulations of PTX.