Study On The Brain-targeted Gene Delivery Vector Modified With Angiopep-2

With the development of molecular biology and the advances of science andtechnology, gene therapy has becoming a new method of treatment for various centralnervous system diseases. How to penetrate the blood brain barrier (BBB) and then deliverythe therapeutic gene to the brain phathological site, has becoming an urgent problem. Anideal brain targeting gene delivery vector need meet these requirements, such as goodsecurity, high transfection efficiency, effective permeability of the BBB, no interferenceunder the in vivo environment, and so on.In this study, liposome was added into PEI/DNA complexes, in order to prepare atype of ternary complex containing liposome, PEI and DNA. Then PEG grafted withAngiopep-2peptide was modified to the ternary complexes, which was a brain targetingpeptide and make the complexes possessed brain targeting function.Firstly we studied the related factors of preparing PEI/DNA complexes, whichcontained the solvent PH, ionic strength, temperature, the ratio of composition, and so on.Through the investigation of the physical and chemical properties, such as the particle sizeand the surface zeta potential, we found that PEI/DNA complexes with smaller size couldbe obtained in the following preparation conditions: room temperature, the solvent PHbetween3to5, the ionic strength less than0.02mol/L, and the ratio between PEI and DNAin the range of4to10. PEI/DNA complexes were prepared in three solvents, whichconfirmed with the above-mentioned conditions. The cytotoxicity of complexes wasdetected by MTT analysis. The luciferase expression of the pGL-3plasmid in Hela cellwas evaluated. Finally, the optimized preparation conditions of PEI/DNA were determined.The N/P ratio was8, and the solvent was deionized water. On this basis, the liposome was added to the PEI/DNA complexes, in order to reducethe cytotoxicity of PEI. The liposome modified with positively charged lipid and PEG wasevaluated, that could affect the characteristics of ternary complexes. The results showedthat the ternary complexes pCaLip/PEI/DNA containing cationic liposome modified withPEG, has the size less than200nm, the suitable zeta potential of20mV, high transfectionefficiency, and good security. Moreover, the transfection efficiency had none affection atthe presence of10%serum.Then, the brain targeted peptide Angiopep-2was covalently grafted with the PEG–DSPE. The correct connection was detected by NMR. The brain targeting ternarycomplexes Ang-pCaLip/PEI/DNA were prepared, that had the size of180nm, and the zetapotential of15mv. The results of in vitro transfection experiment showed that thetransfection efficiency of Ang-pCaLip/PEI/DNA in the positive cell line hCMEC/D3andC6were significantly improved (p<0.05), compared with none targeting modificationcomplexes. However, the difference of transfection efficiency in negative Hela cell was notfound. The human brain microvascular endothelial cell line hCMEC/D3was used toestablish the blood brain barrier cell model, and then the permeability of the ternarycomplexes was evaluated used the in vitro BBB cell model. The results showed thatAng-pCaLip/PEI/DNA could effectively facilitate the plasmid to penetrate the BBB cellmodel and induce the apoptosis of C6cells by Trail plasmid.In summary, we successfully prepared a ternary composite gene delivery vector ofliposome/PEI/DNA modified with Angiopep-2. The in vitro experiments showed that thisvector had better security and higher transfection efficiency, and it could penetrate theBBB cell model effectively. So the brain targeting ternary complex maybe a potentialvector used in brain targeted gene delivery.