Study On The Targeted Red Blood Cell Membrane Nano-Drug Delivery System And Its Preparation

Malignant tumors are one of the main lethal causes of human health,and traditional anti-tumor drugs have poor efficacy.In recent years,microparticle drug delivery systems,such as drug-loaded nanoparticles,liposomes,microspheres,and microcapsules have become hot spots in research.However,their stabilities are very poor,and they are easy to be captured and cleared by the human immune system after administration,and the bioavailability is low.As an endogenous cell,red blood cells have the advantages of high biosafety and low immunogenicity.It is a new type of drug delivery system with great application prospects.It has become a research hotspot in related fields,and the aim is great to improve the bioavailability of the drug and the therapeutic effect.In this paper,a biomimetic targeted drug delivery system with high biocompatibility and precise targeted delivery-folate erythrocyte membrane doxorubicin chitosan nanoparticles(FA-RBC-DOX-CS-NPs)was designed and prepared.With the materials of Folic acid(FA)and amino polyethylene glycol phospholipid(NH2-PEG-DSPE),folic acid-targeted phospholipids were prepared and targeted cell membranes were constructed.At the same time,doxorubicin hydrochloride(DOX)was selected as the model drug,and chitosan(CS)and sodium tripolyphosphate(TPP)was used to construct chitosan doxorubicin nanoparticles by ion cross-linking.In order to combine the advantages of both nanoparticles and cell membranes,folic acid targeted cell membranes and chitosan doxorubicin nanoparticles were made into targeted biofilm nanoparticles so as to improve the bioavailability and therapeutic effect of drugs.The main research contents and results of this paper are as follows:1.In the folic acid solution,added 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC)as the hydroxyl activation reagent and N-hydroxysuccinimide(NHS)as the cross-linking agent for activation.By reacting with NH2-PEG-DSPE through the formation of an amide bond,1H-NMR analysis was performed on the obtained compound to confirm its structure,which proved the successful synthesis of folic acid phospholipid(FA-PEG-DSPE).2.Red blood cells were obtained by centrifuging the whole blood of rat,and the intraluminal material was released by freezing and thawing to obtain a clean cell membrane.The FA-PEG-DSPE was incubated with the cell membrane to prepare the target erythrocyte membrane,and the resulting cell membrane was characterized by fluorescence imaging with FITC instead of FA,and again proved by FT-IR.3.Taking doxorubicin hydrochloride as a model drug,chitosan and TPP were used to prepare chitosan doxorubicin nanoparticles by ion cross-linking,and their physical and chemical properties such as particle size,Zeta potential,apparent morphology,and in vitro stability were evaluated.Through single factor inspection to obtain the best-optimized conditions and conduct a confirmatory test,the drug loading of DOX-CS-NPs was(15.09±1.36)%;the encapsulation rate was(60.99±0.92)%,the particle size of DOX-CS-NPs was(152.5±2.554)nm,PDI was(0.124±0.042),Zeta potential was(10.1±1.342)mV measured by dynamic light scattering particle size analyzer.Though the transmission electron microscopy(TEM),it was observed that the chitosan doxorubicin nanoparticles were nearly spherical,and the particle size was consistent with the results measured by the MalvernZetasizer particle size analyzer;in vitro stability studies showed that DOX-CS-NPs had no obvious particle size change within 10 days,and PDI was less than 0.2,which showing good stability4.The folic acid targeted cell membrane was broken into cell membrane vesicles by ultrasonic method,and then the DOX-CS-NPs were coated with cell membrane vesicles by liposome extruder to prepare FA-RBC-DOX-CS-NPs,then investigated the obtained products for in vitro properties such as particle size,Zeta potential,apparent morphology,and in vitro release.The particle size of FA-RBC-DOX-CS-NPs(254.2±2.651)nm,PDI(0.199±0.031)and Zeta potential(-10.1±0.213)mV were measured by dynamic light scattering particle size analyzer.TEM showed that the nanoparticles wrapped with the film showed a spherical morphology and were evenly distributed.The results of in vitro release showed that the release rate of FA-RBC-DOX-CS-NPs at pH=7.4 was lower than DOX and DOX-CS-NPs;the release rate at pH=6.5 was faster than that at normal,and slightly slower than the release rate of DOX and DOX-CS-NPs.5.By the MTT test,it evaluated the safety of folate erythrocyte membrane chitosan blank nanoparticles bio carriers(FA-RBC-CS-NPs)and FA-RBC-DOX-CS-NPs against MCF-7 and HUVEC in vitro drug efficacy of cells.The material toxicity experiment showed that the blank nanoparticles with cell membrane had good cell compatibility when the concentration was 0.01～1000μg/mL;Cytotoxicity experiments found that when DOX concentration was 0.1～5μg/mL,the cytotoxic effect of FA-RBC-DOX-CS-NPs on MCF-7 breast cancer cells was significantly stronger than DOX and DOX-CS-NPs,and the greater the concentration,the better the effect.The results of cell uptake experiments found that under the conditions of drug concentration 5 μg/mL,uptake time from 1 h to 4 h,the uptake of folic acid-targeted nanoparticles was slightly larger than that of free DOX,but the uptake of free DOX was much greater than that of(FA+FA-RBC-DOX-CS-NPs)intake.6.Establish MCF-7 breast cancer tumor model,observe the distribution of ICG in various tissues and organs of nude mice after the breast cancer nude mice tail vein injection of indocyanine green(ICG),ICG-CS-NPs and FA-RBC-ICG-CS-NPs through the live imaging system at 2 h,8 h,12 h and 24 h.The results showed that ICG in ICG group and ICG-CS-NPs group was distributed throughout the body,mainly accumulating in liver and kidney,while ICG in FA-RBC-ICG-CS-NPs group was mostly distributed in tumor.It was further proved that the FA-RBC-ICG-CS-NPs group had a stronger inhibitory effect on MCF-7 breast cancer solid tumors than the ICG group and ICG-CS-NPs group.In a word,a new drug delivery system of targeting folic acid-targeted erythrocyte membrane nanoparticles was constructed in this subject.Studies have shown that the system had good biocompatibility and targeting,which could significantly delay the release rate and improve biocompatibility.