Construction And Characterization Of Tumor- Targeted Erythrocyte Membrane-encapsulated PLGA Nanoparticles

In this study,a drug carrier,folate-targeted erythrocyte membrane-encapsulated PLGA nanoparticle?FA-RBCm-PLGA?was prepared by nanoprecipitation and liposome extrusion,and loaded with paclitaxel?PTX?and the third-generation P-glycoprotein?P-gp?inhibition,Tariquidar?TQR?.The novel nano drug delivery system?FA-RBCm-PLGA-PTX-TQR?was conducted with both tumor targeting and anti-tumor multidrug resistance.The drug-loaded system utilized the safety of natural red blood cell membrane,the tumor targeting of folic acid ligand and the anti-tumor multidrug resistance of TQR,which was expected to reduce the side effects and improve the anti-tumor effect of PTX,so that cancer could be remedied more effectively.The preparation procedure,physicochemical properties,in vivo pharmacokinetics and in vitro antitumor activity of the drug-loaded system were evaluated.The research details were as follows.The PLGA-PTX nanoparticles were prepared by nanoprecipitation method.The orthogonal design was carried out to determine the optimal preparation process on the drug loading ratio,oil-water volume ratio and ultrasonic intensity with the indexes of encapsulation and drug loading efficiency.The results showed that PLGA-PTX nanoparticles had the best encapsulation and drug loading efficiency when the drug loading ratio was 1:5,the oil-water volume ratio was 1:5,and the ultrasonic probe was 500 W for 10min.The RBCm was extracted by hypotonic lysis method,and FA-RBCm-PLGA-PTX nanoparticles were prepared by lipid extrusion.The particle size,potential,morphology and in vitro release of PLGA-PTX and FA-RBCm-PLGA-PTX nanoparticles were characterized and compared.The membrane protein properties and storage stability of FA-RBCm-PLGA-PTX were also investigated.The results indicated that the encapsulation efficiency,particle size,and potential of PLGA-PTX were 76.1%,138.7 nm,-4.45 mV,respectively,While FA-RBCm-PLGA-PTX were 67.6%,159.8 nm,-10.98mV,and both were spherical particles with uniform particle size under transmission electron microscopy.Compared with PTX solution,both PLGA-PTX and FA-RBCm-PLGA-PTX released slowly,but FA-RBCm-PLGA-PTX had the most obvious sustained release characteristics.The membrane protein content of FA-RBCm-PLGA-PTX was not significantly reduced compared with natural red blood cells.The FA-RBCm-PLGA-PTX system had good stability and was no significant increase in particle size at 4°C for 21 days.FA-RBCm-PLGA-PTX was injected into the tail vein of rats to investigate pharmacokinetic behavior in vivo.The results showed that the AUC of the FA-RBCm-PLGA-PTX was 6.025 mg/L·h,MRT was 7.359 h,C_max was 3.328 mg/L,t_1/2 was 5.837 h.Compared with PTX solution,FA-RBCm-PLGA-PTX had a better long circulation.FA-RBCm-PLGA-PTX was injected into the tail vein of mice to investigate tissue distribution in vivo.The results indicated that FA-RBCm-PLGA-PTX increased the distribution of PTX in liver,spleen and kidney,and the drug content was significantly higher than that of PTX injection at 12 h,suggesting that FA-RBCm-PLGA-PTX can prolong the circulation of the drug.FA-RBCm-PLGA-PTX-TQR was prepared by the previous nanoprecipitation method and liposome extrusion method.The mass ratio of TQR and PTX was 10:1,and other preparation conditions were unchanged.The anti-tumor activity of FA-RBCm-PLGA-PTX-TQR was detected by MTT assay.The uptake behavior of nanoparticles by MCF-7/ADR cells was examined by fluorescence microscopy and flow cytometry from both qualitative and quantitative aspects.Uptake mechanism was also explored.The results showed that the IC₅₀ values of FA-RBCm-PLGA-PTX-TQR,FA-RBCm-PLGA-PTX and RBCm-PLGA-PTX for MCF-7/ADR cells were17.88?g/mL,39.52?g/mL and 130.0?g/mL,suggesting that tumor targeting of folic acid could increase cytotoxicity.The presence of TQR in the nanoparticles made it have anti-tumor multidrug resistance effect,and the reversal factor was 2.21 times.Folic acid inhibition experiments indicated that FA-RBCm-PLGA-PTX-TQR could be taken up by MCF-7/ADR cells through folate ligand receptor specific binding.The investigation of uptake mechanism revealed that FA-RBCm-PLGA-PTX-TQR could also entry into cells by clathrin and caveolin-mediated endocytosis,and this process was affected by temperature,intracellular ATP levels,pH and cell membrane fluidity.In summary,the dual-loaded FA-RBCm-PLGA-PTX-TQR nanoparticles have a stable preparation process and good safety,which can extend the circulation time of PTX in rats,improve the distribution in tissues.The nanoparticles also have tumor-targeting effect and anti-tumor multidrug resistance properties,which can enhance the therapeutic effect and reduce the side effects of PTX.It is a nano drug delivery system with excellent performance and good application prospects.