Studies On Synthesis And Performance Of Gene Transfer System Mediated By Supramolecular Polyrotaxanes Derivatives

Cancer is one of the disease that greatly threatened human's health, people who pursuit in medicine is endeavoring to find a specific medicine to cure cancer. Gene therapy in the many ways becomes a 21st century hotspot. Gene therapy is an approach for human disease treatment by inserting therapeutic gene into cells in order to produce desired protein with specific functions and inhibits expression of abnormal genes. The clinical success of gene therapy depends on the development of suitable efficient and targeted gene transfer system. Gene delivery systems are classified into viral and non-viral system. Non-viral vector win us high favor for its several advantages including low immunogenicity and power of carrying high molecular weight of DNA.Poly(ethyleneimine) (PEI) who had emerged as a good candidate in the cationic Polymers is cationic polymer as non-viral gene delivery vectors used widely in research. It is repored that PEI is linear or branch polymer. There is high positive charge on its surface, so PEI can condense DNA with negative charge. PEI makes the lysosome rupture and releases the DNA swallowed into cytoplasm and nucleus for gene expression through famous protons sponge effect. The molecular weight of PEI varies from 5000-25000Da for gene delivery vectors. Higher is transgene efficieney of high molecular weight of PEI, but higher is also toxicity, or vice versa:the toxicity of low molecular weight of PEI is very low, for the transgene efficiency is very low. PEI can make erythrocyte aggregate and bind non-specificly with albumin in blood. Transgene efficieney was low when it was applied in vivo.Therefore, PEI need to be modified in order to reduce its cyctotoxicity. Usually, polyethylene glycol (PEG), cyclodextrin (CD), and chitosan (CHI) et al. in the Polymer biodegradable material were applied into modified PEI, respectively and together, such as copolymers and polyrotaxanes, what' s more, targeted groups is grafted to the modified PEI. Then, the carrying efficiency from non-virus vectors will be enhanced.Objective:To investigate the transgene efficiency, toxicity and properties of different molecular weight of PEI-polyroaxane (FPP), a series of FPP polymers which included PEI(0.6 KDa,10 KDa,and 25 KDa, respectively) and folate terminatd polyroaxane (RP) were synthesized according to different molar ratio between different molecular weight of PEI and PR for clinic applicational delivery system.Methods and contents:three parts.One part:Synthesis and characterization of PEI-PR polymers1, Preparation of Polymers:a, Preparation of Psuedo-PR; b, Preparation of folate-NHS; c, Preparation of PR; d, Preparation of FPP.2, Characterization of polymers:The FPP composition was estimated by measuring nuclear magnetic resonance (NMR), infrared spectrum (IR), ultraviolet spectrum (UV).3, Buffer capacity was calculated by acid-base titration andβ=dC(HCl)/dpH.4, The binding ability of condense DNA and against RNase I degrate in FPPs were investigated by agarose gel electrophoresis. Morphology of FPPs/DNA complexed was observed under scanning electron microscope.5, Study of Complex toxicity:a, Influence of different concertration of FPP (0.6 KDa,10 KDa,25 KDa)/DNA,BPP/DNA and PEI-25 KDa/DNA complexes on morphology of cultured Hela and 16 HBE cells; b, Influence of different N/P ratios of FPP (0.6 KDa,10 KDa,25 KDa) /DNA, BPP/DNA, and PEI-25 KDa/DNA complexes on morphology of cultured Hela and 16 HBE cells.Two part:Preparation and biophysical properties of FPP/DNA complexes1, Using alkaline lysis method, through different kind of processes include preparation of Escherichia coli cells, re-proliferation of pAAV-EGFP or pLuc which first insert to coli cells, and extraction of plasmid pEGFP which purity and size was identified by agarose gel.2, The Zeta potential and particle size of FPPs/DNA and PEI/DNA complexes were measured by Laser Particle Size Determination.Three part:In vivo and vitro study of biological properties in complexes1, Study of transgene efficience in vitro:a, Influence of N/P ratios forming complexes on transfection efficiency of FPP(0.6K)/pLuc, FPP(10K)/pLuc, FPP(25K)/pLuc, BPP/pLuc, and PEI-25K/pLuc for KB cells by Luciferase; b, Influence of serum or without serm on transfection efficiency of FPP(0.6K)/pAAV-EGFP for KB cells; c, Influence of KB cells and A549 cells on transfection efficiency of FPP(0.6K/pAAV-EGFP by FCM; d, Influence of 293T,KB,Lovo and A549 on transfection efficiency of all complexes by FCM.2, Study of transgene efficience in vivo:a, Preparation of animal model; b, Luciferase assay:PBS, Naked DNA, PEI-25K/pLuc, BPP/pLuc, FPP(0.6KDa)/pLuc, and Lipotanmine/pLuc groups were randomly divided on bearing-tumour mice. They are killed at 24h and 48h after intratumoral injection of the complexes for luciferase assay of tumor tissue homogenates. c, The effect of anti-tumour in polymers/p53 complexes treatment were determined by tumour weight assay and gene expression of caspase-9 and bax. d, HE of heart and kidney of mice in FPP(0.6KDa), FPP(10KDa),FPP(25KDa) and Naked DNA groups after treatment 25d.ResultsOne part:Synthesis and characterization of PEI-PR (FPP) polymers1, characterization of polymers:(1), NMR:a,13C-CP/MAS NMR spectra of pseudo-PR:the spectrum shows resolved C-1 and C-4 resonances particular C-1 and C-4 adjacent to a conformationally strained glycosidic linkage are observed at 80 ppm and 98 ppm, respectively, b,1H-NMR spectra of PR:Signals corresponding to PEG were observed at 3.7 ppm ([-CH2CH2O-]n), and the C1 proton and C2-C6 protons of a-CD were identified at 4.9 ppm and 3.0-4.2 ppm, respectively. FA signals in polymers ranged from 6.5 to 8.7 ppm. c,1H-NMR spectra of FPP: Except above signals, signals from PEI ethylene protons (-CH2CH2NH-) in FPP were observed at 2.4-3.0 ppm. (2), IR:Amide bonds form in 1650 cm'and Structure of benzene in 1605cm-1 and 701.4 cm-1 indicated PR were successfully synthesised. Strong absorption in 1705 cm-1,1031.42 cm-1 for ester formation between amino of PEI and O6H of a-CD reaction indicated FPP successfully were synthesised. (3), UV: Compared FA, UV absorption in FPP shifted from 250nm and 280nm to 255nm and 289nm, respectively.Percent grating PEI on FPP(0.6 K), FPP(10 K) and FPP(25 K) were respectively 86.67%,36.66%, and 15.33%; Mw on FPP(0.6 K), FPP(10 K) and FPP(25 K) were respectively 85920 Da,453520 Da, and 473520 Da.PEG:a-CD:PEI(0.6K,10K,25K)=1:20:(102,43.9,18.40).2, Buffer capacity in different molecular weight of PEI enhanced with the increases of molecular weight, but buffer capacity in FPP decrease with the increase of molecular weight.3, Gel retardation results showed that FPP (0.6 KDa,10 and 25 KDa KDa) between N/P ratio 2 and 4 has well combined with DNA, and its result was a bit of poorer than that in PEI-25 KDa.4, Relative cell viability of Polymers on Hela and 16 HBE cells:Compared with PEI-25KDa, toxicity of FPP(0.6 KDa,10 KDa and 25 KDa) significantly was reduced. LD50 was (Hela,1285ug/ml,1256 ug/ml,1150 ug/ml,1315 ug/ml; 16 HBE,1295ug/ml, 1266 ug/ml,1210 ug/ml,1365 ug/ml).Two part:Preparation and biophysical properties of FPPs/DNA complexes1, Purity and size of pAAV-EGFP or pLuc prepared by alkaline lysis and extracted by reagent was fit the experiment request. The results were A260/A280 (pAAV-EGFP) =1.90±0.055; A260/A280 (pLuc)=1.84±0.021, plasmid molecular weight of pAAV-EGFP= 5.3kb and pLuc=4.7kb; concentration of pAAV-EGFP=278.83±1.11μg/ml and pLuc=269.86±1.03 ug/ml, respectively.2, When N/P ratio reached 10, particle sizes of different molecular weights FPPs/DNA complex were under 140nm; When N/P ratio was up to 20, zeta potential varied from 20mv to 30 mv.Three part:In vivo and vitro study of biological properties in complexes1, Study of transgene efficience in vitro:Extent of transgene efficiency of FPP (0.6 KDa, 10 and 25 KDa KDa) was the higher than those in BPP and PEI-25K When N/P=20, and extent of FPP (0.6 KDa, 10 and 25 KDa KDa) in N/P=20 was the higher than those in other N/P ratio. The best carrying conditions for complex was respectively 2ug/well DNA; the forming complexes for 30min; complex on the cells for 4h; a significant increase transgene efficiency in serum conditions (*p<0.05 and *p<0.05); transgene efficiency of FPP (0.6K)/DNA in serum was respectively the 8.5 times and 10 times that in serum-free; compared with Opti-MEM (blank), transgene efficiency of FBS (10-15%) and ALB (0.1-0.2%) were expanded by 2.5-3.5 times; no obvious difference transgene efficiency amonge different concentrations of ALD and FBS.2, Study of transgene efficience in vivo:a, Successful bearing-tumors mice model, b, Liuciferase assay showed that transgene efficiency of FPP (0.6 k) (N/P=20) was respectively 6.1 and 5.3 times those of PEI-25K (N/P=10) and BPP in 24h (*p <0.05 and*p<0.05), and was almost 136 times the amount of naked DNA. c, An antineoplastic active evaluation in vivo:(a), compared with BPP/p53, PEI-25 KDa/p53, naked p53, PBS groups, tumor heavy of FPP (0.6 K)/p53 group significantly decreased (*p<0.05,*p<0.05,**p<0.05, and**p<0.05, respectively); (b), Compared with normal control group, PEI-25 KDa (N/P=10)/p53 group, BPP/p53 group, and FPP(o.6K)(N/P=20)/p53 group, expression of caspase-9 and bax in KB model group significantly reduced (*p<0.05,*p<0.05,*p<0.05, and*p<0.05), and a significant difference between FPP/p53 and BPP/p53, it is no difference for HE of heart and kidney of mice among naked DNA, FPP (0.6K), FPP (10k), and FPP (25K) groups, indicating of low cyctotoxicity of FPP(o.6K), FPP(10K), and FPP(25K).Conclusion:1, Successful building a new non-virus carrier (PEI-polyroaxane, FPP) supramolecular polymer2, Good cushioning capacity; Ideal size and potential; Better performance of DAN compression3, In vitro and in vivo, compared with PEI -25K, FPPs have low toxicity and high transgene efficience.4, FPPs/DNA complex have high transgene efficience on the cancer cells with rich folate receptors.