A Study Of Pluronic-modified Cationic Dendrimers For Non-viral Gene Delivery Vectors

Efficient and safe gene delivery vector for gene transfer remains to be a major challenge to successful gene therapy. Compared with viral vectors, nonviral gene transfer vectors are most attractive due to their fewer safety concerns and ease to prepare. As a result, the development of novel efficient nonviral gene transfer vectors has attracted much attention although the efficiency is not quite satisfactory. In the present study, we try to integrate the advantages of Pluronics, a kind of triblock copolymer and, Polypropylenimine （PPI）, a dendritic cationic polymer together to make a novel nonviral vector-mixed copolymer system （MCS） for efficient nonviral gene delivery.The contents of the study are the following: synthesis and characterization of Pluronic-PPI; preparation and the methods optimization for MCS; MCS was used for delivery vector of reporter gene pEGFP-N2 encoding green fluorescence protein and pGL3-promoter encoding luciferase with cell lines hunman lung adenocarcinoma SPC-A1, Chinese hamster ovary cell CHO, human embryonic kidney cell 293T and human breast cancer cell MCF7 and MCF7/ADR resistant to adriamycin （ADR）. The internalization pathways and transport mechanism were investigated using specific endocytic inhibitors and laser confocal scanning microscope. We evaluated the possibility of P123-PPI as delivery vector for chemical synthetic small interference RNA （siRNA）.Three kinds of Pluronics, P123, L61 and F127 were reacted with the third generation of PPI dendrimer （PPI 1864Da） and the Pluronic-modified PPIs were obtained. The mixed copolymer system was prepared with the method of electrostatic coacervation. The preparation medium and way of mixing were optimized. The optimized preparation was that the MCS was prepared in deionized water and vortexed for 20 seconds. Following these conditions, the resulted MCS had an average diameter of 100 nanometers. The stability of MCS composed of P123-PPI/P123/DNA was explored on the indicator of particle size. When the N/P ratio is 20, complexes can exist without size changing for 48 hours at room temperature and thereafter, complexes aggregates with time elapsing, and particle size increased. The characteristics of MCS were investigated, including the abilities condensing and packaging plasmid DNA, anti-DNase I digestion , anti-dissociation against heparin and serum. The results suggested that MCS can completely condense plasmid DNA and inhibit its electrophoresis when N/P ratio is more than 2; MCS can protect DNA against 0.4U Dnase I /μg DNA digestion and serum dissociation when N/P is more than 10, even when the concentration of serum was as high as 50% （V/V）. Whether the MCS can resist dissociation against heparin sodium or not was dependent on the concentration of heparin. When the concentration of heparin was lower than 0.075 mg/mL, it can not dissociate MCS; whereas the concentration was higher than 0.125 mg/ml, it can dissociate MCS completely. The free P123 in MCS had no significant effect on its ability of anti-dissociation against heparin sodium.MCS was composed of Pluronic-modified PPI dendrimer, free Pluronics and the therapy gene. MCS was prepared with electrostatic coacervation and particle size and zeta potential were determined. Transfection efficiency of the MCS containing pEGFP-N2 on cells was measured with flow cytomertry （FCM）. With the percentage of positive cells expressing GFP as the evaluation index, the optimal composition andcorrespongding quantity in the MCS was screened. First, we compared the transfection efficiency of P123-2.5g-PPI and PPI and investigated the effect of free P123 addition on the transfection of PPI. The results indicated that the transfection ability of P123-2.5g-PPI was far stronger than that of PPI; The addition of free P123 increased the transfection of PPI significantly in the absence of 10% fetal bovine serm （FBS）, whereas decreased the transfection efficiency of P123-2.5g-PPI to some degree. Second, the transfection efficiency of different compositions of MCS on cell lines SPC-A1, CHO and 293T were evaluated with pEGFP-N2 as reporter gene in the absence and presence FBS. It was shown that the effect of free P123 was different for different cell lines in the absence of FBS. For SPC-A1 and CHO cells, the free P123 decreased the transfection efficiency of P123-2.5g-PPI, while for 293T cell line, the free P123 increased the transfection efficiency of P123-2.5g-PPI. In the presence of 10% FBS, the addition of free P123 enhanced the transfection efficiency of P123-2.5g-PPI for all the three kinds of cell lines and the greatest enhancing effect was achieved when the concentration of P123 was between 0.0045% and 0.0135%. Third, the transfection efficiency of MCS on SPC-A1 was quantified with plasmid pGL3-promoter encoding luciferase. The results are consistant with that obtained from pEGFP-N2 reporter gene. The effect of fetal bovine serum （FBS） and bovine serum albumin （BSA） on transfection was investigated. FBS and BSA can significant increase transfection efficiency in a certain concentration range and decrease transfecion when the concentration was too high （50%）. After being transfected with therapeutic pRNAT delivered by MCS, MCF7/Adr uptaked more R123 than the cells not transfected.With the optimized MCS that composes P123-2.5g-PPI, P123 and plasmid and model cell line SPC-A1, we investigated the internalization pathways and whether the certain pathway could result in effective transfection by using of specific endocytic inhibitors. Ways to inhibit clathrin mediated endocytosis （CME） include chloropromazine （CPZ）, hypertonic glucose solution （0.45M） and K⁺ depletion. For inhibition of caveolae mediated endocytois （CvME）, the inhibitors are filipin III and genistein. The inhibitors against macropinocytosis is 5-（N,N-Dimethyl）amiloride hydrochloride （DMA）. After inhibition of certain internalization pathway, the uptake of MCS and lucifetase expression was determined. The results showed that K⁺ depelation, hypertonic glucose and chloropromazine decreased the uptake by 20%, 45% and 60% seperately. Filipin III and genistein against CvME decreased the uptake by 50% and 40%. DMA had no significant effect on the uptake. The effects of inhibitors on transfection were quantified with pGL3-promoter as reporter gene. The results suggested that K⁺ depelation and chloropromazine decreased the transfection efficiency by 45% and 30% seperately. Filipin and genistein against CvME reduced the transfection efficiency by 30% and 56% seperately. DMA against macropinocytosis had no significant effect on the transfection efficiency. So, we can infer that MCS was internalized by the cells through both CME and CvME pathways and both of them lead to effective transfection. Macropinocytosis was not involved in the uptake of MCS. To further clarify the internalization routes, we labelled MCS, CME and CvME endocytic markers and colocalized their relative position in the cells afer internalization. The colocalization experiments proved that MCS are endocyzed by the cells with both CME and CvME pathways.Endosome-lysosome system, cell skeleton and motor proteins play very important roles in the transport of membrane vesicles. We investigated the effects of endosome-lysosome acidification inhibitors NH₄Cl and monensin, actin depolymerizing agent （Cytochalasin D）, microtubule-depolymerizing agent （nocodazole, NCZ）, microtubule-stablizing agent （paclitaxel, PTX）, dynein inhibitor sodium orthovanadate （SOV） and kinesin Eg5 inhibitor （monastrol） on the transfection efficiency by determination of luciferase expression. The results indicated that nocodazole decreased the transfection efficiency significantly whereas paclitaxel had little effect. Cytochalasin D decreased the transfection efficiency by about 50%. Sodium orthovanadate decreased the transfection efficiency by 35% and monastrol had no significant effect on the luciferase expression. These results demonstrated that endosome-lysosome system, cell skeleton and motor proteins had pronounced influnce on the transport of MCS.To further prove that endosome-lysosome system and microtubule were involved in the transport of MCS, their relative position was investigated with colocalization experiments. The results suggested that MCS was transported through endosome-lysosome and microtubule to nucleus after internalization.The dissertation also evaluated the possibility of P123-2.5g-PPI as delivery vector for chemical synthetic small interference RNA （siRNA）. The results demonstrated that P123-2.5g-PPI can effectively transfer siRNA into drug-sensitive and drug-resistant tumor cells in the absence of serum. The mRNA level of p-gp was decreased significantly when the siRNA interfering p-gp was transfered to MCF7/ADR cells by P123-2.5g-PPI. Uptake of rhodamine was significantly enhanced when MCF7/Adr cells was treated with P123-PPI/siRNA complexes.