Enhanced Endosomal Escape Of The Brain Targeting System Based On Cationic Polyaspartamide For BACE1Gene Silencing

The brain is one of the most important organs, and many diseases are associated withthe brain. The central nervous system disorders such as Parkinson’s disease, Alzheimer’sdisease, brain tumors and so on, have been the serious hazards today. And the number ofpatients is increasing year by year. With the development of molecular biology and genetechnology, gene therapy has get more and more attention in the treatment of centralnervous diseases, due to the high molecular targeting and high specificity.Due to the presence of blood-brain-barrier (BBB), it is difficult to deliver drugs to thebrain, including genes. Therefore, preparation of efficient macromolecule drug carrier is someanful. Currently, among the existing non-viral gene vectors, cationic liposomes andcationic polymers are the most commonly used.Cationic polymers have goodbiocompatible, non-immunogenic and structure is easy to modifiy. Polyplex, which iscomposed of polyplexs and plasmid DNA (pDNA),can be adhered to the surface of cells.In our study, we constructed cationic polymer PEG-P [Asp (TEP)]-chole as a genevector, and selected RVG29which derived from rabies virus glycoprotein as primary braintargeting molecules.Studies show that RVG may combine with acetylcholine receptors andγ-aminobutyric acid receptor of BBB,and by the way of receptor-mediated endocytosis, toachieve brain transporter. In addition, studies have found that Tet-1is composed of12amino acids of small peptides, can specifically bind to nerve cells. WhenTet1-PEG-PEI/DNA complex was injected into the lateral via an injection, the braintransfection efficiency of Tet1-PEG-PEI/DNA was significantly higher than unmodifiedPEG-PEI/DNA complexes. Therefore, we chose Tet-1as a secondary target ligand in thebrain delivery to increase the transport of the carrier to brain.In the first part researches are focused on the synthesis of the polymer. First of all, wesynthesized the L-aspartic acid-N-carboxy anhydride (BLA-NCA) as the monomer of thesynthesis of block copolymer PEG-b-PBLA. Thereafter, the polyaspartic acid wasmodified bytetraethylene pentamine. Finally, the polymer cholesteric waslinked toAce-PEG-P [Asp (TEP)] and the desired product Ace-PEG-P [Asp (TEP)]-chole wasobtained.The target ligand was linked to the Ace-PEG-P [Asp (TEP)] through the reaction of acetal and thiol group. The above products were subjected to structural analysis by1HNMR and the results showed that all the produts were we desired.In the second part,different N/P ratios of the polyplex were prepared, and the size andzeta potential was measured by dynamic light scattering.The results show that, with thehigher of N/P, the size of polyplex is smaller and the zeta potial is higher,and the size isabout100nm and zeta potential is+20mv at N/P ratio16and gel retardation experimentsshow that pDNA can be encapsulated completely.In the third part, we studied the cell viability and the cytotoxicity in vivo.The experimentof cell viability shows that cell cytotoxicity is much lower than lipofectamin2000and thecell viability is more than85%at N/P ratio less than16, while the cell survival rate ofapplying Lipofectamin-2000at80%or less. By CD68immunohistochemistry study, weknow that there are certain toxicity in vivo,but the positive cells disappear quickly.In the fourth part of this article, we have focused on the evaluation of polyplextargeted to the brain.The qualitation and quantitation of in vitro transfection of Neruo-2acells both suggest that the RVG29/Tet1-polyplex has the highest tansfection. The assay ofCLSM observation and evaluation of endosomal escape shows that dual modified polyplexhas higher celler uptake and can escape from endosomes. The quantitation of cell uptakedifferent modified polyplexes by flow cytometry, the results show that thecellar uptakerateof RVG29-polyplex is19.3%higher than polyplex. The GFP and pGL3wasencapsulated in cationic polymer, and was added to Neuro-2a cells.After48hours, weanalysis that the transfection efficiency of RVG/Tet1-polyplex group is3.6times higherthan polyplex group. The results of transport studies of polyplex shows that thepenetration rate of dual ligands is the highest.The qualitative study of distribution ofdifferent polyplexes distribution in mouse.The in vivo imaging results display thefluorescence of RVG29/Tet1-polyplex is the most intense in brainand.The last part of the article, we studied the effect of the transfection of BACE1plamid pGPU6/GFP/Neo which loaded the siRNA sence of BACE1.First of all, wedetected the level of BACE1in Neuro cells after incubation with different polyplex after48hours. We know that polyplex can reduce the level of BACE1protein and the proteinlevel in polyplex-Tet1/RVG29group was reduced the most. Then, we chose the transgenicmice as AD model. Through Morris water maze behavioral performance, we know thatthe polyplex can improve spatial memory ability in mice, especially the mice ofRVG29/Tet1-polyplex group has significate difference than saline group. In conclusion, we can conclude that the dual ligands linked polyplex has a good braintransport characteristic based on the results above and it is an efficient non-viral gene braindelivery system. At the same time,we will do more experiments in vitro and in vivo tomake further demonstration.