Preparation Of Galangin-loaded Erythrocyte Membrane Nanoparticles And Evaluation Of Blood-brain Barrier Permeability

Central nervous system diseases such as Alzheimer’s disease,Parkinson’s disease,and glioblastoma have a high global prevalence and a long pathological cycle,and most drugs cannot cross the blood-brain barrier to reach the lesion due to its obstructive effect,which makes clinical drugs inadequate for their therapeutic effect.Therefore,it is crucial to investigate a strategy that can enhance the blood-brain barrier passage rate of drugs.Galangin has attracted much attention due to its various pharmacological effects such as antioxidant,anti-inflammatory,anti-tumor,and anti-Alzheimer’s disease,but its poor water solubility and low blood-brain barrier permeability have resulted in low brain entry of galangin.The bionic nano-delivery system is an innovative brain delivery strategy with properties such as increased drug circulation time,lesion site targeted accumulation and immune evasion.Based on this,a bionic nano drug delivery system aimed at enhancing the blood-brain barrier passage rate for the treatment of brain diseases was designed and constructed in this project.The specific research of the project is as follows.（1）PLGA nanoparticles loaded with galangin（GL-NPs）were constructed using a modified emulsification solvent volatilization method by optimizing the preparation process with encapsulation rate and drug loading capacity as the measurement criteria through orthogonal tests.The prepared GL-NPs were spherical in appearance with uniform size and good nanoparticle dispersion,with a particle size of 159.8±1.4 nm,zeta potential of-3.76±0.49 m V,encapsulation rate of 71.65±0.67%and drug loading capacity of 4.87±0.05%.（2）In order to enhance the long circulation time as well as the blood-brain barrier passage rate of GL-NPs,erythrocyte membranes were wrapped on the nanoparticle surface using a liposome extruder to obtain erythrocyte membrane-wrapped galangin-loaded nanoparticles（RBCm-GL-NPs）.The prepared RBCm-GL-NPs showed a clear membrane-shell spherical structure with a particle size of 179.4±2.1 nm,zeta potential of-3.53±0.76 m V,encapsulation rate of 69.75±1.12%,and drug loading capacity of 4.75±0.08%.Stability investigation experiments demonstrated that RBCm-GL-NPs could also remain stable in vivo with good stability.In vitro release experiments showed that RBCm-GL-NPs have obvious slow release effect,which can prolong the drug action time and reduce the number of dosing.（3）To further investigate the brain-targeting effects of RBCm-GL-NPs,three types of brain cells,PC12 cells,h CMEC/D3 cells and U-118MG cells,were selected for the experiments.Firstly,the cytotoxicity was examined by MTT method;secondly,the proliferation effect of nanoparticles on PC12 cells damaged by Aβ_25-35 induction was investigated;then the cellular uptake of nanoparticles was measured qualitatively and quantitatively using fluorescence microscopy and flow cytometry;finally,an in vitro blood-brain barrier model was established using U-118MG cells and h CMEC/D3 cells co-culture to investigate the permeability of nanoparticles.The results of in vitro experiments showed that the nanoparticles at different concentrations were not significantly cytotoxic to the three cells;both GL-NPs and RBCm-GL-NPs were more protective than galangin to PC12 cells,and the protective effect of RBCm-GL-NPs would be slightly stronger than GL-NPs;the results of cellular uptake experiments showed that the RBCm-coated NPs would significantly increased the uptake of drugs by cells;and the results of cellular co-culture blood-brain barrier model showed that the blood-brain barrier passage rate of RBCm-NPs increased gradually over time compared with that of NPs,and by 12 h,the blood-brain barrier passage rate of RBCm-NPs reached 9.25%,while that of NPs was only 5.30%,which fully demonstrated that the use of red blood cell membrane to wrap nanoparticles can facilitate the passage of nanoparticles through the BBB.In conclusion,the bionanoparticles containing galangin were successfully constructed with small particle size,good stability,slow release and reproducible preparation process.In vitro cellular experiments showed that the wrapping of erythrocyte membrane significantly enhanced the uptake of the drug and improved the blood-brain barrier passage rate.This project has enhanced the brain passage of the natural product galangin,increased the possibility of galangin for the treatment of central nervous diseases,and provided new ideas for the development of safe and effective nano-delivery systems for brain diseases.