Preparation Of PLGA Nanoparticles Encapsulating AZD9291 Modified With Chitooligosaccharides

Osimertinib(AZD9291)is the third generation of epidermal growth factor receptor tyrosine kinase inhibition(EGFR-TKI),which is clinically used to cure advanced non-small cell lung cancer(NSCLC)patients with selective T790M mutations.Although AZD9291 has strong targeting and good efficacy,it is expensive for patients to take it,and easy to cause drug resistance and side effects.It is generally believed that the nano drug delivery system can enhance the efficacy and reduce the dosage of drugs.Therefore,this study aims to design and construct a multi-functional,controllable preparation and release AZD9291 nanoparticles based on poly(lactic-co-glycolic acid)(PLGA)and chitooligosaccharides(COS),so as to improve the efficacy of AZD9291 and provide technical support for its further use.Firstly,AZD9291 was encapsulated by emulsifier-solvent evaporation using PLGA with a molecular weight of 17,000 Da,and COS was modified on the surface of the nanoparticles to obtain AZD-PLGA-COS NPs.The preparation process of nanoparticles by emulsion solvent evaporation was determined as follows:the concentration of PLGA was 10 mg/mL,the ultrasonic intensity was 97.5 W,the concentration of COS was 5 mg/mL,and the volume ratio of the internal water phase to the external water phase was 1:8.The particle size of AZD-PLGA-COS NPs was 176.6±0.4nm,the PDI was 0.186± 0.022,the Zeta potential was 18.65 ± 0.38 mV,and the encapsulation efficency was 95.22 ± 0.49%.The particle size of AZD-PLGA-COS NPs decreased,the Zeta potential increased and the encapsulation efficency increased after modification.In the drug release experiment in vitro,AZD-PLGA-COS NPs gradually released AZD9291 with the extension of release time,which had a certain degree of sustained release.Secondly,the biosafety of nanoparticles was evaluated by cell compatibility and blood compatibility.The cell safety of blank PLGA NPs and blank PLGA-COS NPs were evaluated by MTT cytotoxicity test.Within the concentration range of nanoparticles,the effect on the survival rate of L929 and H1975 cells was insignificant,indicating that the nanoparticles had almost no cytotoxicity.Through quantitative analysis,the hemolysis rate of both blank PLGA NPs and blank PLGA-COS NPs was less than 1%,with no hemolysis and good blood compatibility.The calcium recovery time of blank PLGA-COS NPs was closer to that of blank plasma,which indicated that the modification of COS could improve the anticoagulation ability of nanoparticles and improve the blood compatibility of nanoparticles.Finally,nanoparticles containing Cy3 fluorescent dyes were prepared and the results of cell uptake were observed by fluorescence microscopy.In H1975 cells,the mean fluorescence intensity of Cy3@PLGA-COS NPs was stronger than that of Cy3@PLGA NPs,indicating that H1975 cells were more inclined to absorb Cy3@PLGA-COS NPs.H1975 cell survival test showed that after-AZD9291,AZD-PLGA NPs,AZD-PLGA-COS NPs treatment for 72 h,the IC50 of H1975 cells were 88.68 ± 3.79 nM,75.15 ± 2.21 nM and 47.98±0.99 nM,respectively.The IC50 of AZD-PLGA-COS NPs group decreased significantly.This result was also confirmed in the cell apoptosis test:the late apoptosis rate after AZD-PLGA-COS NPs treatment was 38.09±1.40%,significantly higher than that of AZD9291 group(23.54±0.82%,p<0.001)and AZD-PLGA NPs(30.41±0.92%,p<0.01).Therefore,AZD-PLGA-COS NPs could promote cell apoptosis by promoting cell uptake,so as to enhance drug efficacy and reduce drug dosage.Western blot results showed that the nanoparticles induced apoptosis of H1975 cells by regulating the expression of endogenous apoptosis-related proteins.The results of H1975 cell scratch test showed that AZD9291,AZD-PLGA NPs and AZD-PLGA-COS NPs had certain inhibitory effects on the migration of H1975 cells,and the effect of AZD-PLGA-COS NPs modified by COS was better than that of AZD-PLGA NPs.Simultaneously,COS was found to inhibit the expression of PD-L1 at mRNA and protein levels in H1975 cells.Meanwhile,PD-L1 expression promoted by AZD9291 could be significantly down-regulated.AZD-PLGA-COS NPs could also inhibit the expression of PD-L1 at mRNA and protein levels.Therefore,COS played the role of immunomodulator while serving as carrier material.The above experimental results indicated that AZD-PLGA-COS NPs is expected to become a dual-effect platform of targeted drugs combined with immunotherapy.