Construction And Evaluation Of FA Modified Nano-erythrocyte Targeting Drug Delivery System

In this study,a hypotonic lysis method and nano-extrusion process were used to prepare the nano-erythrocyte membrane drug carrier which co-modified with FA and PEG,and a novel nano drug delivery system?FA/PEG-DOX-Nano-RBCs?with dual roles for long circulation and tumor targeting were constructed by loading doxorubicin hydrochloride.The drug-loaded system not only makes use of the natural biocompatibility of erythrocyte membrane and the long circulation of the PEG,but also has specific tumor targeting which is expected to improve the antitumor efficacy and can greatly reduce the side effects of DOX,which has great clinical significance.In this study,the preparation process,physicochemical properties,in vivo drug release behavior,anti-tumor efficacy and safety of the carrier system were evaluated.The details are as follows.The erythrocyte membrane was extracted by hypotonic lysis method,and the bifunctional modified FA/PEG-DOX-Nano-RBCs nanoparticles were prepared by nano-extrusion process.The selection of drug loading temperature,loading sequence,DOX concentration,the volume ration between DOX and RBC,the loading and resealing time were investigated with the index of DOX encapsulation efficiency.The result indicated that when the temperature was 4?,the low permeability solution was25%×PBS,the drug loading time was 30 min,the healing time was 90 min,the concentration of DOX was 4.0 mg/mL,DOX and erythrocyte membrane volume ratio was 4:1,FA/PEG-DOX-Nano-RBCs have the best encapsulation efficiency.The morphology of FA/PEG-DOX-Nano-RBCs was observed by transmission electron microscopy,and the particle size,zeta potential,surface protein content and entrapment efficiency were characterized.The in-vitro release behavior was evaluated by the dialysis method,at the same time,the stability of FA/PEG-DOX-Nano-RBCs was also investigated.The results shows that FA/PEG-DOX-Nano-RBCs were spherical particles with uniform particle size of 160.2 nm,the zeta potential was-12 mV,the encapsulation efficiency was 29%,and the protein content has no significant difference compared with erythrocyte membrane.The FA/PEG-DOX-Nano-RBCs system has obvious sustained-release characteristics and can be continuously released for 24 h compared with DOX solution.And the drug delivery system has good stability,no significant increase in particle size and decrease in the encapsulation rate within 30 days of storage at 4°C.The FA/PEG-DOX-Nano-RBCs were injected through tail vein,and aimmed to investigate its pharmacokinetics and tissue distribution in vivo compared with DOX injection.The pharmacokinetics results showed that the C_max of the drug-loaded nanoparticle group was 4.34 mg/L,t_1/2 was24.74 h,MRT was 22.46 h,and AUC was 41.11 mg/L·h,indicating that its body has a better long circulation.The tissue distribution results showed that compared with DOX injection,the nanoparticle experimental group significantly reduced the distribution of DOX in the heart and increased the distribution of the drug in the liver,spleen and lung,and can effectively lasted to 24 h,which suggesting that the drug-loaded nanoparticle experimental group can effectively reduce the cardiotoxicity which caused by DOX injection and prolong the residence time of the drug.MTT assay was used to detect the antitumor activity of FA/PEG-DOX-Nano-RBCs group and DOX-Nano-RBCs group in vitro.The cellular uptake behavior of the drug was qualitatively and quantitatively investigated by laser confocal and flow cytometry techniques.The pathways and influencing factors for the transport the particles by cells were studied by a variety of conditions and inhibitory agents.The results showed that FA/PEG-DOX-Nano-RBCs could cause a large number of apoptosis in Hela cells at lower concentrations,and the IC₅₀ was only 2.52?g/mL.Laser confocal results showed that FA/PEG-DOX-Nano-RBCs could be taken up by cells.The transport process is an active transport process,which is affected by temperature,pH,and osmotic pressure,at the same time,it is regulated by clathrin and caveolin-mediated endocytosis.The results showed that the process was regulated not only by temperature,ATP,environmental pH and osmotic pressure but also by clathrin and caveolin.The Balb/c mouse tumor model of H22 hepatocarcinoma cells was established,and the FA/PEG-DOX-Nano-RBCs group and the DOX injection group were injected through the tail vein.The tumor volume was measured in different experimental groups during the treatment period,and the changes of serum TNF-?and IL-6 levels were detected by ELISA and Western-Blot.At the same time,the in vivo targeting characteristics were observed by in vivo imaging technology,and the H&E staining analysis was performed on the heart and tumor tissues.The results showed that compared with DOX injection,FA/PEG-DOX-Nano-RBCs can effectively inhibit the growth of tumors in mice,reduce the inflammatory response of DOX injection,which can significantly reduce the toxicity of DOX on cardiac tissue and irritation caused by intravenous injection.In summary,the dual-function modified FA/PEG-DOX-Nano-RBCs delivery system has stable preparation process and reproducibility,and can significantly prolong in-vivo circulation time of DOX and improve tissue distribution.At the same time,it has a tumor-targeting effect,can enhance the therapeutic effect and reduce the toxic and side effects of DOX.It is an excellent nano delivery delivery system.