Synthesis And Properties Of PLGA Carrier Based On Tumor Microenvironment Response

Malignant tumors are the main diseases affecting human health,and current chemotherapy is still an important method for clinical treatment of tumors.In the course of chemotherapy,90%of cancer patients will develop drug resistance,which becomes a key obstacle affecting the efficacy of chemotherapy.With the wide application of nanotechnology in the field of biomedicine,the research of drug delivery system is developing in the direction of high efficiency,intelligence and targeting.Now it is represented by the slow release controlled nano drug delivery system.The mode of administration has shown good prospects.Anti-cancer drug delivery based on environmentally responsive carriers has received wide attention due to its potential to improve drug efficacy,reduce unwanted side effects,and bypass cell-mediated drug resistance,synthesizing different responsive groups The same polymer can be used to obtain multiple responsive polymers,which can react differently according to the stimulation of different environments in the environment,so as to release more drugs accurately.Poly（lactic-co-glycolic acid,PLGA）is a synthetic lipophilic polymer that is an FDA-approved biodegradable pharmaceutical excipient due to its good biocompatibility.It is used as a drug carrier for sexual and biodegradability.However,there are still some limitations in practical applications:for example,lack of specific binding to the body cells,the drug can not reach a specific target organ;the burst effect is obvious,resulting in low water-encapsulation rate and unstable drug release.Therefore,the corresponding modification of PLGA to overcome its shortcomings is the hot spot of PLGA as a drug carrier research.In this paper,PLGA-COOH was modified to reduce the disulfide bond and the oxidized response thioether bond by chemical coupling polymerization to construct a PLGA vector capable of recognizing the redox environment in the specific environment of cancer cells,and to study its performance.The results are as follows:1.Using carbodiimide condensation method（EDC/NHS）coupling chemical catalysis of PLGA-COOH to obtain PLGA activated ester,which is amidated with cystamine（Cystamine）to obtain PLGA-Cys.A disulfide-bonded PLGA vector having reduction responsiveness was prepared.The small molecule bis（4-nitropheny）diethyl sulfide（Des）having a thioether bond is synthesized by 2,2-thiodiethanol and 4-nitrophenylchloroformate.The prepared PLGA-Cys and bis（4-nitropheny）diethyl sulfide（Des）together with cystamine were synthesized into a polymer polymer PLGA-b-p（Des-alt-Cys）having a dual response effect.Infrared spectroscopy and nuclear magnetic resonance spectroscopy were used to detect the corresponding characteristic peaks of PLGA-NHS,PLGA-Cys,bis（4-nitropheny）diethyl sulfide,PLGA-b-p（Des-alt-Cys）.At the same time,the molecular weight of PLGA-b-p（Des-alt-Cys）was verified by gel permeation chromatography,and 13 disulfide bonds and thioether bonds（Des-alt-Cys）were linked by PLGA..2.The redox performance of the hydrophobic dye-containing Nile Red（NR）PLGA-b-p（Des-alt-Cys）nanoparticles PLGA-b-p（Des-alt-Cys）-NPs was studied.Fluorescence spectrophotometer was used to detect 10 mM,40 mM DTT,0.5 μM,100 μM H2O2 without dithiothreitol（DTT）or hydrogen peroxide（H2O2）.The fluorescence intensity of Nile red in different time periods（0.5 h,1 h,3 h,6 h,8 h,10 h,24 h,48 h,72 h,96 h,120 h）.The experimental results show that Nile red has obvious release behavior with time under the treatment of 10 mM,40 mM DTT,0.5 μM and 100 μM H2O2,and the fluorescence intensity is very weak at 120 h.It indicates that the PLGA-b-p（Des-alt-Cys）vector can recognize the redox environment and complete drug release.The experimental results show that the synthesis of the polymer provides an experimental basis for the development of a novel anti-tumor sustained-release preparation.3.Nile red（NR）PLGA-b-p（Des-alt-Cys）nanoparticles were co-cultured with mouse colon cancer cells（CT26 cell line）for 1 h,4 h,8 h,and 18 h.The fluorescent strength of the encapsulated Nile Red polymer carrier nanoparticles at different time intervals was different for mouse colon cancer cells.The fluorescence intensity was 18 h>8 h>4 h>1 h.Nile Red（NR）PLGA-b-p（Des-alt-Cys）Nanoparticles PLGA-b-p（Des-alt-Cys）-NPs（4 mg/mL）and PLGA Nanoparticles PLGA-NPs（4 mg/mL）Co-culture with mouse colon cancer cells（CT26 cell line）for 4 h and 18 h,fluorescence confocal microscopy showed that PLGA-b-p（Des-alt-Cys）compared with PLGA-NPs（4 mg/mL）)-NPs（4 mg/mL）Nanoparticles were concentrated in the cell membrane at 4 h and 18 h,showing a large number of spots,indicating that the polymer can recognize the cancer cell environment and achieve active targeting.However,PLGA-NPs（4 mg/mL）showed only a few spots around the cell membrane under the microscope at 18 h,and the fluorescence intensity was very low,indicating that the original drug PLGA-NPs did not have the characteristics of active targeting,fluorescence.The intensity is PLGA-b-p-NPs>PLGA-NPs.4.The PTX-PLGA-b-p（Des-alt-Cys）-NPs encapsulating paclitaxel was prepared by modified emulsification and volatilization method.The particle size distribution was 254.1±20.5 nm,the zeta potential was₂3.5±2.5 mV,and the drug loading was 11.75 ±0.04%,the encapsulation efficiency is 84.6±0.02%.The results of scanning electron microscopy showed that the obtained PTX-PLGA-b-p-NPs were more uniform and rounded.The peak of anticancer drug paclitaxel was not detected by differential scanning calorimetry（DSC）and X-ray diffraction（XRD）,and the paclitaxel was determined to be amorphous.The presence in the interior of the polymeric carrier indicates that the entrapped paclitaxel nanoparticles have been successfully prepared.5.PLGA-b-p（Des-alt-Cys）-NPs lyophilized powder was obtained by using mannitol as a lyoprotectant.After reconstitution,the particle size stability was measured by laser particle size analyzer（DLS）within 24 h.The results show that the particle size is stable within 24 h without DTT or H2O2 redox agent,but there are still different degrees of aggregation.Under 40 mM DTT or 100 mM H2O2 treatment,the particle size change became smaller at about 3 h,probably due to the cleavage of disulfide bonds and thioether bonds,which caused the particles to become smaller.After 7 h,the particles aggregated to cause hydration.The particle size becomes larger,and the experimental results show that the synthesis of the polymer provides an experimental basis for the development of a novel anti-tumor sustained-release preparation.6.The in vitro release test of PTX-PLGA-b-p NPs was carried out under the release of 40 mM DTT and 100 μM H2O2 without the addition of dithiothreitol（DTT）or hydrogen peroxide（H2O2）,PTX-PLGA-b-p NPs have sustained and stable release characteristics,compared with PTX-PLGA-NPs in the absence of DTT or H2O2,the release of PTX-PLGA-b-p（Des-alt-Cys）-NPs,at 40 mM Under the condition of DTT and 100 μM H2O2,PTX-PLGA-b-p（Des-alt-Cys）-NPs showed rapid release and more drug release,indicating that PLGA-b-p（Des-alt-Cys）can recognize redox The environment is capable of ensuring consistent and stable release characteristics under redox conditions.