Research Of Double-tailed Fluorous Chains Modified Polymers As Vehicles For Gene And Protein Delivery

Gene and protein therapy has become a newly emerging approach for diseases. Compared with traditional therapy methods, there are a number of advantages such as high targetability and high efficacy, less side effects, so it has been the focus of research in the biomedical field today. Gene and protein are difficult to enter cells by themselves, which makes it essential to transport them into cells by vehicles. As reported, Cationic polymers are most studied vectors. However, these polymers are usually critictized by low transfection efficacy and high cytotoxicity, so various ligands are attached to polymers, including lipids, saccharides, amino acids, peptides and proteins. Although these modifications improve performances of polymers for gene and protein delivery, they still could not resolve such problem as cytotoxicity fundamentally in transfection process. Therefore it seems more urgent to explore gene and protein vectors with high efficacy and low cytotoxicity.Fluorination is a novel modification that could improve gene transfection performance of cationic polymers. Fluorinated polymers could increase gene transfection efficacy at low nitrogen to phosphorus (N/P) ratios which could reduce the surface charge density of complexes. They exhibit minimal cytotoxicity. However, fluorinated polymers performing efficient transfection must be grafted with a high density of fluoroalkyl chains, which would lead to a series of problems. For example, intensive surface grafting would cause serious steric hindrance which influences the binding between polymers and nucleic acid and limits the further functional modification on the polymers surface. In addition, a high density of fluoroalkyl chains leads to severe cytotoxicity. In terms of these problems, here this paper used a double-tailed fluorous compound 2-Chloro-4,6-bis[3-(perfluorohexyl)propyloxy]-1,3,5-triazine (CBT) to modify polymers in order to achieve efficient DNA, siRNA, protein transfection under low grafting ratios. We studied the viability of these modified polymers and got the following result:Different generations of PAMAM dendrimers (G1, G2, G5 PAMAM) and branched PEI with a molecular weight of 1800 Da (bPEI1.8K) were modified with different ratios of CBT. The average grafting numbers of products were calculated by well-established ninhydrin assay and fluorine element analysis. The modified polymers were named GI-CBT1.9, G2-CBT1.5,G5-CBT1.3 and bPEI1.8K-CBT1.3 (the subscripts represent the average grafting numbers). The results show thatl-2 CBT chains-modified polymers could bind DNA tightly and transfect enhanced green fluorescent protein (EGFP) and Luciferase efficiently. Among these synthesized products, G1-CBT1.9 transfects nearly 70% HEK293 cells, such highest efficacy approaches that of commercial transfection reagent Lipofectamine 2000 and is significantly superior to SuperFect and PolyFect. In addition, there is no observed cytotoxicity under optimal transfection conditions.CBT-modified polymers not only could deliver DNA efficiently, but also show high siRNA delivery efficacy. For example, all of CBT-modified polymers show high Luciferase silencing efficacy with low off-target effects on HeLa and MDA-MB-231 cells. G1-CBT1.9 shows comparable gene silencing efficacy to Lipofectamine 2000 and lower cytotoxicity. Transfection efficacy of G1-CBT1.9is also much more than non-modified G1 PAMAM in vitro and in vivo. The results indicate G1-CBT1.9 has become a potential candidate for gene and protein delivery.Recently there are a large number of reports about DNA and siRNA delivery vehicles, but only a few studies of protein vectors could be seen. Our researches show G1-CBT1.9,G2-CBT1.5 and bPEI1.8K-CBT1.3 all could transport BSA-FITC enter HeLa cell. Among the three modified polymers, G1-CBT 1.9 also shows the highest protein delivery efficacy, which is consistent with our previous results of gene delivery research. Besides of BSA-FITC, G1-CBT1.9 Could efficiently deliver β-Gal with minimal cytotoxicity as well.In a word, here we propose a novel modification method in order to improve performances of polymers for gene and protein delivery. Compared with traditional fluoroalkyl chains modification, polymers modified with extremely a few double-tailed fluoroalkyl chains grafting ratios could achieve high transfection efficacy and low cytotoxicity. Such a simple strategy has a broad commercial application prospect. The research further completes the fluorinated polymers delivery system and promotes the application of fluorinated polymers in protein delivery.