A Preliminary Study Of Cationic Laminarin As A Gene Carrier

Laminarin is also known as laminaria or alginate starch, it is aheteropolysaccharide widely present in the alginate, and adjacent monosaccharide isconnected by β-1.3glycoside bond. It has an advantage of non-toxic, biocompatible,natural widespread and inexpensive. Natural laminarin displays electronegativity, butit does not has the ability of DNA transfection, so we should made it cationic. PEI is acommonly used cationic polymer in gene transfection, the higher molecular weightthe higher transfection efficiency but greater toxicity. Low molecular weightpolyethylene imine （600Da） has lower cell toxicity and low transfection efficiency. Inthis paper, we use a low molecular weight polyethylene imine grafte on laminarinthen we make a research on the feasibility of Lam-PEI as a non-viral gene vector, itsmain contents are as follows:1. Synthesis and characterization of Lam-PEI. A novel non-virus gene delivery vector（Lam-PEI） was successfully synthesized by grafting low-molecular branched PEI（600Da） onto the backbone of laminarin with N,N’-carbonyldiimidazole as a spacer.GA and zeta potential of Lam-PEI determined. FTIR shows there is a characteristicpeaks of ester carbonyl in1700cm-1, and the zeta potential of Lam-PEI is about+37mv. The results demonstrated that Lam-PEI had been successfully synthesized.2. The interaction between gene vector and nucleic acids in vitro. Gel electrophoresisretardation assay demonstrated that Lam-PEI can wrap DNA and RNA withelectrostatic interaction excellently. The Lam-PEI showed relative low cytotoxicityagainst SKN, MCF-7, SMMC-7721and A549, even at high concentration （up to48μg/mL）. In vitro transfection experiment displayed that Lam-PEI could deliverypEGFP-C1and FAM-siRNA into MCF-7cell and the transfection efficiency ofLam-PEI/siRNA is almost100%.3. The anti-tumor activity in vitro of Lam-PEI/siRNA-II. Three Bmi-1siRNAsequences were chosen according to GeneBank of Bmi-1mRNA, SequenceNM₀05180. In order to screen the targeting Bmi-1sequence, we use RT-PCRdetecting the RNAi effect of three different siRNA. The results showed that siRNA-IIfragments reduced Bmi-1mRNA expression obviously. Then we investigate the anti-tumor activity of Lam-PEI/siRNA-II in vitro. The cell viability was determinedby MTT method and the Bmi-1mRNA expression was determined by RT-PCR. Theresults indicated that inhibition rate was34.95±1.97%after48h; the inhibition rate ofBmi-1mRNA is64.31±3.67%.4. Examination of the antitumor effects of Lam-PEI/siRNA-II in vivo. Usingtumor-bearing nude mice as model, by HE staining result comparing changes in theorganization of the different groups of mice; the tumor growth curve was showed thatLam-PEI/siRNA can significantly inhibit the tumor volume. The results of RT-PCRindicated the gene vectors can efficiently inhibit the expression of Bmi-1mRNA intumor and the inhibition rate of Bmi-1mRNA is65.81±4.59%.5. Then tumor targeting gene vector Lam-PEI-FA was synthesized by introducingfolic acid onto Lam-PEI for targeting FR positive cells. We preliminary investigatethe loading capability and inhibition rate of Lam-PEI-FA/siRNA-II. The cell viabilitywas determined by MTT method and the Bmi-1mRNA expression was determined byRT-PCR. The results indicated that Inhibition rate was33.78±0.76%after48h; thetransfection efficiency of Lam-PEI-FA/siRNA is99.7%.