| Chitosan-g-stearic acid (CS-SA) micelle which was effectively taken up into cells even cell nucleus has been demonstrated as an effective gene carrier in vitro and in vivo. Because of its weak ion-buffer capabilities, gene loaded CS-SA was restricted in lysosome. In this study, spermine was conjucated to CS-SA (Spermine modified chitosan-g-stearic acid, SP-CS-SA) to construt the new SP-CS-SA/pDNA delivery system The study was designed to promote the gene escape from the endo-lysosome and evaluate the effect of spermine on gene transfection efficiency and treatment of gene p53 and also explore its mechanism.Chitosan with the molecular weight of 18.1 kDa was prepared by enzymatic degration. Chitosan (CS)-g-stearic acid (SA) copolymer (CS-SA) was synthesized via the reaction of carboxyl groups of SA with primary amino groups of CS catalyzed by 1-ethyl-3-(3-dimethylamin-opropyl) carbodiimide (EDC). Spermine modified chitosan-g-stearic acid (SP-CS-SA) was synthesized in the presence of sodium periodate. The chemical compositions were comfirmed by NMR and Elemental Analysis (EA). Stearic acid graft ratio was 3.76% determined by 2,4, 6-trinitroben2ene sulfonic acid (TNBS) method. Breaking percentage of the ring on chitosan backbone was 12.42%. Spermine graft ratio was 7.59%. The critical micelle concentration (CMC) for CS-SA and SP-CS-SA determined by by pyrene fluorescence method in aqueous medium was 116.3 ± 16.3 μg/mL and 108.2 ± 3.8 μg/mL. The particle sizes by number were 133.6 ± 23.2 nm and 128.4 ± 19.8 nm, zeta potential were 22.8 ± 3.2 mV and 18.2 ±1.8 mV measured by dynamic light scattering. Transmission electron microscopy showed that both micelles have uniform distribution, irregular spherical, smaller particle size than measured before. Ion-buffer capability was greatly enhanced after modification by spermine and cytotoxicities of CS-SA and SP-CS-SA were lower than Lipofectamine TM2000 and PEI 25K.CS-SA/pDNA and SP-CS-SA/pDNA gene delivery system were prepared by incubation together. pDNA was condensed tightly with uniform diameter and protected from DNase. The transfection efficiency of SP-CS-SA/pEGFP-C1 on EC-1 and Bel-7402 cells were 44.6 ± 4.9% and 33.5 ± 2.3% seperatively higher than that of CS-SA/pEGFP-C1,***p < 0.001. Co-localization intracells showed that SP-CS-SA/pDNA delivery system could promote the escape of DNA from endo-lysosome, and little cytotoxicity was observed under the transfection dosage.The therapeutic effect of CS-SA/p53-EGFP-N1 and SP-CS-SA/p53-EGFP-Nl were evaluated on Bel-7402 cells. The results showed that percentage of the apoptotic cell for SP-CS-SA/p53-EGFP-N1 was 37.3 ± 1.1% higher than that of CS-SA/p53-EGFP-N1 (10.1 ± 3.0%), **P< 0.01. The percentage was close to that of PEI 25K/p53-EGFP-N1, but lower than Lipofectamine TM 2000/p53-EGFP-N1,*P< 0.05. The expression of SP-CS-SA/p53-EGFP-N1 was enhanced by SP, leading to G1 phase retardation and cells apoptosis. |
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