The Study Of Brain-targeted Delivery Of Rdp Cell-penetrating Peptide-mediated DNA And Its Transmembrane Transport Mechanism

The blood-brain barrier (BBB) is the main reason for a slowdown of the development of Central nervous system (CNS) drugs, it is difficult to take effect for numerous drugs with therapeutic potential. Therefore, the key of treatment for brain diseases is to find out a method of overcoming BBB or to make drugs penetrate in the brain to reach an effective concentration. Traditional methods of treating brain diseases such as stereotactic surgery, injection drugs into brain and direct delivery of gene carriers are high invasive, but they have disadvantages such as low efficient of expression, in a small range of diffusion and a short maintaining time, which restricted their widely use.Now a new technology of viral or non-viral vectors which can carry macromolecular active agents into the brain is proposed. Viral vectors have significant side effects and difficult to carry out targeted modifcation, whereas non-viral vectors have have advantages like biodegradabilities and biocompatibilities, which endue them a good prospect in the treatment of CNS diseases.Many researches have justified that as non-viral vectors, peptides can carry oligonucleotides, siRNA or DNA to pass through the BBB, so that to achieve the brain-targeted transport. Our research is on whether a novel deriviative peptide RDP (RVG-derived peptide)origin from rabies virus can mediate DNA in progress of brain-targeted delivery, as well as its transmembrane transport mechanism.In the study of DNA brain-targeted delivery mediated by RDP, RDP was connected to plasmids with LacZ and EGFP reporter gene in non-covalent way, and by agarose gel shift assay and circular dichroism spectrum, we determined the best ratio and interaction of RDP and DNA complexes. In vivo experiments, mice were divided into experimental group (Injecting optimal mixing ratio of RDP and DNA complexes in caudal vein), the control group (Injecting DNA in caudal vein) and the blank (Injecting saline in caudal vein), and the mice were killed on fixed time to get their brain, liver, kidney and other tissues. Then make frozen sections to confirm the expression of the target protein in various tissues by X-gal staining and fluorescence. The results showed that target protein can be detected in the brain, kidney and spleen in RDP/pDNA group, and the protein level is highest in the brain. However, the targeted protein was only detected in the kidney and spleen in control group. Therefore it can be assure that as a suitable target for gene therapy of brain carrier, RDP have great potential in the future application of non-viral vectors.In vitro study of transmembrane mechanism, we found that among HeLa, CHO, and SH-SY5Y cells, RDP only showed specificity to SH-SY5Y neuronal cells. Penetration experiments were conducted at4degrees and37degrees, RDP and RDP-EGFP can get into cells at37degrees but not at4degrees, which indicate RDP get into cells are energy dependence. When endocytosis inhibitor, colchicine, was added into the cells, a significant inhibitory action was found, thus we judged that the way of RDP and RDP-EGFP get into the cells are endocytosis, but not directly penetration. Next, we use competitive inhibition of RDP and RDP-EGFP labeled with Rho and the block of neurotransmitters for RDP get into the brain to prove that the mediated receptor of endocytosis is like to be receptors that have a similar structure as GABA, but not the nicotinic acetylcholine receptor.