Oligonucleotide-carrying Brain-targeted Biomimetic Nano Delivery System Anti-parkinson’s Disease Study

Background and objective:Parkinson’s disease(PD)has the second highest prevalence among neurodegenerative diseases,and it is estimated that by 2030,at least 8million people worldwide will suffer from PD,representing a huge burden on the health care of modern society and the health of the elderly.The pathogenesis of PD involves multiple molecular mechanisms,among which alpha-synuclein(α-Syn)protein misfolding and diffusing in the brain is widely recognized as the main causative agent of PD.Antisense oligonucleotide(ASO)drugs are currently an important technical tool in the field of gene therapy.However,ASOs are difficult to cross the bloodbrain barrier,have weak targeting properties,and have a very short halflife in the blood in the free state.The heteroduplex oligonucleotide(HDO),which is designed based on the single-stranded ASO structure and carries small molecules of biological origin,is more easily taken up by cells,has a longer duration of action,and has a more significant effect on the regulation of target gene expression levels.Currently,most of the ASO drugs on the market are administered by invasive means,which is a complicated and inconvenient treatment process with poor safety.Nano drug delivery systems are widely used in the field of drug targeting therapy,in which artificial materials are easy to produce,stable and reliable,but have low biocompatibility;bioactive components such as cell membranes are highly biocompatible and have a good ability to cross the blood-brain barrier,but have low yield and high heterogeneity,and the combination of the two can effectively complement each other.Based on the mentioned background,the Chol-HDO,a DNA heterologous oligonucleotide double-stranded modified by cholesterol,was designed for the first time and showed higher inhibition efficiency than ASO.Then,DSPE-PEG2000-Levodopa was constructed to modify the cell membrane-encapsulated PLGA nanoparticles.The nanoparticles were used to carry Chol-HDO for drug delivery in the hope of achieving efficient penetration of the blood-brain barrier and targeted delivery to dopaminergic neuronal cells in the brain,reducing α-Synuclein protein levels in the target regions of the brain,decreasing Lewy body deposition,improving motor function in model animals,and reversing the progression of PD symptoms in experimental subjects.This research provides ideas and directions for the development of nucleic acid-based PD therapeutic drugs and corresponding delivery vehicles.Methods:The ASO sequences were mixed 1:1 with corresponding co-DNA or co-RNA concentrations and annealed to obtain double-stranded Chol-HDO.Gene repression efficiency was compared by q PCR after administration of ASO,Toc-HDO,and Chol-HDO on cells,respectively.Nanoparticles were prepared by the nanoprecipitation method.They were characterized by NTA,TEM,and Western-Blot.The cell membranes of vascular endothelial cells b End.3 cells were extracted and sonicated by adding DSPE-PEG2000-Levodopa to obtain hybridized cell membranes.HDO@NP solution was mixed with the normal cell membrane and hybridized cell membrane and sonicated again to obtain membraneencapsulated nanoparticles HDO@MNP and targeted dopaminergic neuron nanoparticles HDO@LMNP.Uptake and lysosomal escape of nanoparticles in b End.3 cells were investigated.To evaluate the difference in genetic inhibition efficiency and in vitro release behavior of nanoparticles and to examine the in vitro trans-BBB efficiency and effect on cell viability.A53T mice were used to establish a PD model,and the therapeutic effects were investigated by behavioral assays,α-Syn protein levels and m RNA detection in striatal/nigral regions,IHC staining of TH in corpus striatal/ substantia nigra regions,HE staining,and serum biochemical assays to evaluate nanoparticle safety after drug administration.Results:Chol-HDO showed the best effect on α-syn m RNA inhibition among all groups of drugs,with an optimal action concentration of 200 n M in in vitro experiments.HDO@NP,which had an average particle size of 172 nm and an electrical potential of +29 mv,had a circular vesicle-like structure under TEM,HDO@MNP had a particle size of 180.3 nm and an electrical potential of +2 mv,and HDO@LMNP had an average particle size of approximately 181 nm and an electrical potential of-2 mv.A distinct cell membrane layer structure was observed at the periphery of HDO@MNP and HDO@LMNP by TEM.The encapsulation rate of HDO@NP was81.77 ± 3.38.The encapsulation rate of HDO@MNP was 92.76±2.65 and92.20±3.04 for HDO@LMNP.HDO@MNP and HDO@LMNP not only could better escape from lysosomal phagocytosis but also were more easily enriched to mitochondria.The PD mouse model administered with HDO@LMNP showed better behavioral performance than the model control,free HDO,HDO@NP and HDO@MNP groups.Mice in the HDO@LMNP group had the highest number of TH+ neurons in the substantia nigra(SN)and corpus striatum region with the lowest α-Syn protein and m RNA levels.HE staining and serum biochemical results showed that Chol-HDO and biomimetic nanoparticles had good biosafety.Conclusion:Chol-HDO has a higher cellular uptake rate and higher efficiency of m RNA inhibition than ASO.The biomimetic nanoparticle HDO@LMNP has a good safety profile,can efficiently penetrate the blood-brain barrier to target the brain,and has a more significant therapeutic effect on PD mouse models than free Chol-HDO and other nanoparticles,and is a promising delivery system for delivering nucleic acid drugs across the BBB to target dopaminergic neurons for the treatment of PD.