| Gene therapy is the selective treatment method, which transport exogenous gene into receptor cells to repair or replacement of defective, damaged gene. It is of great significance for the development of new drugs or clinical differentiation treatment of genetic diseases, cancer, cardiovascular disease and immunodeficiency disease. It is well known that the prerequisite factor for gene therapy is gene transfer which is delivery of gene into tissue cells for expression. Nowadays, gene vector is divided into two categories: viral vectors and non-viral vectors. Although using viral vector can obtain high transfection efficiency and stable gene expression, it has disadvantages of non-orientation and immunogenicity which limit its clinical use due to safety concern. Compared to viral vectors, non-viral cationic polymer vectors attract more attention since it owns excellent properties of controllable size, specific three-dimensional structure, safety and changeable multifunctional surface group etc.Polyamidoamine (PAMAM) applied as biological materials is one of the most important cationic polymers in non viral gene delivery vector family. PAMAM used ethylenediamine as core was synthesized by repeatedly Michael addition reaction. PAMAM has been widely studied as gene carrier system because of its regular structure, suitable molecular weight, nanometer size, internal cavity, controllable physiochemical properties and high density of surface functional group. Cell transfection efficiency of pDNA delivered by PAMAM is generation dependent, but high generation PAMAM also brings severe cytotoxicity. PAMAM/pDNA polyplex has no serum-resistance ability, which greatly limited the practical application of PAMAM as gene vectors in vivo. How to improve the gene transfection efficiency mediated by PAMAM and maintaining cell viability, and produce serum-tolerance ability is the urgent problem for researchers.In this study, we used α-CD as inner core and grafted with low molecular weight PAMAM (PAMAM-G1, PAMAM-G2) to develop a new star shape functional cationic polymer as gene delivery system. The physicochemical properties, cytotoxicity, cell transfection in vitro and cellular uptake route were investigated to evaluate the possibility and efficiency of this new polymer system as gene delivery vector, the details are as follows:(1) First of all, CDI was used as linking reagent to activate the hydroxyl group of α-CD, and various generation PAMAM were grafted onto the α-CD outer surface to prepare a new kind of star shape cationic polymer system (α-CD-PAMAM conjugates, named CD-PG1and CD-PG2respectively).(2)1H-NMR and FT-IR were used to identify the structure of α-CD-PAMAM conjugates. The grafting ratio, molecular weight was calculated based on1H-NMR, and space conformation of polymers was also simulated. FT-IR results showed that in1718cm-1, the-OH peak of α-CD was disappeared and in3400, a new-COO-peak appeared, which proved that PAMAM was successfully grafted onto α-CD surface; there are average of6.4PAMAM-G1or5.6PAMAM-G2molecules on α-CD. The molecular weight of CD-PG1and CD-PG2were1.02×104and1.89×104respectively.(3) Standard MTT test was used to evaluate the cytotoxicity of CD-PG1and CD-PG2in different cell lines, and compared to PAMAM-G1, PAMAM-G2and α-CD as negative control and PEI-25K as positive control. The results showed that the cytotoxicity of CD-PG1and CD-PG2is remarkably lower than that of PEI-25K in Hela, HEK-293A, BEL-7402and COS-7cells. The existence of serum could improve the cell viability when incubated with cationic polymers. IC50data indicated that the tolerance of various cell lines to cationic polymers were HEK-293A> BEL-7402> COS-7> Hela respectively. Haemolysis test was also used to evaluate the safety after iv administration for CD-PG1and CD-PG2cationic polymers. Results showed that PEI-25K could induce sever haemolysis, but it was not happened in CD-PG1and CD-PG2test groups.(4) We evaluated the physicochemical properties of CD-PGl/pDNA and CD-PG2/pDNA polyplexes. Agarose gel retardation assay was used to investigate the DNA binding, condensing ability and protecting effect of cationic polymers from DNase â… . Mean particle size, particle size distribution and surface charge of polyplex was studied by DLS method, and the effect of serum on particle size stability was investigated as well. The results indicated that CD-PG1and CD-PG2could effectively bind with pDNA when N/P≥6, and could protect pDNA degradation form DNase I in low N/P ratio. The mean particle size of these two kinds of polyplexes was smaller than150nm with narrow size distribution. In serum-containing medium or with various incubation time, the particle size was stable. Surface charge of both cationic polymer/pDNA polyplexes were above20mv.(5) The transfection efficiency of CD-PG1/pDNA and CD-PG2/pDNA polyplex were evaluated in different cell lines and compared to that using PEI-25K and Lipofectamine2000as positive controls tested in serum-free and serum-containing medium condition. The best N/P ratios of CD-PG1and CD-PG2for transfection were80and30in HEK-293A cells. The transfection efficiency of two polyplexes were63.7%å'Œ71.2%in serum-free test conditon and77.1å'Œ79.4%in serum-containing test condition, which are higher than that of PEI-25K group (14.3%) and Lipofectamine2000group (2.9%). Transfection efficiency trend in HEK-293A, BEL-7402and COS-7cells were similar which proved that these cationic polymers had excellent serum tolerance ability and could maintain the transfection level in serum-containing environment.(6) We used confocal fluorescence microscopy to observe the intracellular trafficking of pDNA in different incubation time and to study the entry kinetics of cationic polymer/pDNA polyplex by flow cytometry analysis. The results showed that cellular uptake of polyplex increased in nonlinear characteristics. When the cells were transfected for4h, many fluorescent labeled polyplex spots were found in cell nuclei which indicated that this cationic polymer system could efficiently mediate gene delivery.(7) In this study we investigate the effect of a-CD on gene transfection mediated by PAMAM in order to confirm whether the cell transgene activities influenced by a-CD are concentration dependent and generally applicable in all test cell lines according to the results of cell transfection efficiency pretreated with various a-CD concentration. The results indicated that after a-CD preincubation, the transfection efficiency of PAMAM-G2/pDNA polyplex improved significantly compared with PAMAM-G2group (p=0.001, n=3). When a-CD concentration was or above0.5mg/mL, transfection efficiency of test samples were much higher than control group (p=0.018,p=0.002,p=0.000,n=3). Results of other test cell lines were similar to that of HEK-293A cells, but the results of cell transfection in serum-containing test condition presented no statistical difference, which means that a-CD could not improve serum tolerance for PAMAM/pDNA polyplex.(8) Using relative transfection efficiency as an index to verify the influence of different factors on the polyplex cellular uptake. The results indicated that transfection efficiency of polyplex uptake by cell was much lower (16.1%) in low temperature (10℃) test condition than that in normal test temperature (37℃). When metabolic inhibitor sodium azide was added to pretreat with cells, the transfection efficiency also decreased to69.5%, which proved that the uptake of polyplex was an active process. The hypertonic sucrose solution could inhibit the fluid-phase pinocytosis, and leaded to the active uptake of polyplex particle decreasing to37.9%. We used chlorpromazine as clathrin-mediated endocytosis (CME) inhibitor and genistein as caveolae-mediated endocytosis (CvME) inhibitor to study the polyplex uptake route. The results proved that cationic polymer/pDNA polyplex could transport into cells through both routes. Adding NH4Cl could change the intracellular pH and decrease the pH value in endosome. It restricted the polyplex endosomal escape which leaded to lower level pDNA release and expressed in cell nucleus. The effect of a-CD on polyplex uptake was calculated using two inhibitors. Result showed statistical difference in chlorpromazine group (p=0.034, n=3), which suggested that a-CD may regulate the CvME route to increase the polyplex cellular uptake to upgrade the transfection efficiency.(9) We used lactate dehydrogenase as the assay index to investigate the LDH leakage induced by various concentration of PAMAM-G2, and study the nano scale holes characteristics and influence factors including incubation temperature, PAMAM-G2concentration and a-CD concentration etc. Results showed that the formation of nanoholes on cell membrane was related to PAMAM-G2concentration. Cells incubated with higher concentration PAMAM-G2could release more LDH. Low incubation temperature could limit the cell membrane mobility and as a result decreased the LDH leakage. Cells were pretreated with different concentration α-CD solution and replaced with PAMAM-G2for culture. When α-CD concentration was lower than0.5mg/mL, the leakage of LDH showed no significantly difference (p=0.522, p=0.832, p=0.059, n=3). When α-CD concentration was above0.5mg/mL, the leakage of LDH showed significantly difference (p=0.000, n=3), which indicated that high α-CD concentration could induce the nano scale holes formation. Compared with control group, unlike α-CD group (p=0.696, n=3), the LDH leakage of α-CD group showed statistical difference (p=0.000, n=3) suggesting that this effect may occur through CvME mediation or solubilization of phospholipids on cell membrane by α-CD. |
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