Design And Characterization Of Polycation Liposomes As Novel Non-viral Gene Delivery Vectors

Gene therapy provides great opportunities for treating diseases from genetic disorders, infections and cancer.The development of efficient and safe gene transfer systems could be one of the most important factors for successful gene therapy.This study constructed a novel non-vrial gene delivery vector,polycation liposomes(PCLs),which combined the advantage of two widely used gene delivery systems,cationic polymers and cationic lipids.The character of PCLs,cytotoxicity,cellular uptake and transfection efficiency in vitro were studied.Then the mechanism of PCLs intemalization,kinetics of cellular uptake,intraceular distribution and the tumor cells inhibition by telomerase inhibitor transferred by PCLs were also investigated.Furthermore,we designed combined vectors,PCLs/protamine/DNA complexes,for increasing transfection efficiency,and then the tumor growth inhibition of PCLs-D/pCMV-IL-12 complexes and PCLs-D/protamine/pCMV-IL-12 complexes were studied.In this work,the compound PEI 800-Chol was synthesized using PEI with low molecular weight and Cholesteryl chloroformate.The NMR and IR results indicated that cholesterol was covalently conjugated to the backbone of PEI.PEI 800-Chol was a amphiphilic compound with hydrophilic part PEI 800 and hydrophobic Chol.The apparent CMC of PEI 800-Chol was 0.1012 mg/ml,determined with a fluorescence probe technique using pyrene,which was very useful for PEI 800-Chol to modified the surface of liposomes.Chol part anchor to bilayer of liposomes and PEI 800 part bring the liposomes positive charge.PCLs-S(SPL,Chol and PEI 800-Chol)and PCLs-D(DOPE and PEI 800-Chol)were prepared using film hydration method.The mean particle size of PCLs was 133 nm and the zeta potential was 50.1±2.6 mV which resulted to high DNA condensing ability. When the N/P ratio larger than 2,both PCLs-S and PCLs-D could bind most part DNA, while the former was prefer.Little difference was found between the cytotoxicity of PCLs-S and convention liposomes.The IC₅₀value of PCLs-S against HeLa cells was 603.19μg/ml which notablely higher than that of LipofectamineTM 2000(48μg/ml). Positive charge of PCLs-S could improve ODN internalization by HeLa cells and protect from degradation.Due to PEI anchord onto the surface of liposomes, considerable buffering capacity of PCLs-S was observed,which enhanced the endosomal escape ability of liposomes and increased the transfection efficiency.At N/P ratio 10,the highest transfection efficiency of PCLs-S/DNA complexes was obtained, having the equivalent transfection activity with Lipofectamine^TM2000.Interestingly,the transfection activity of PCLs-S was not influenced in the presence of serum,even improved GFP expression at highr N/P ratios.Hydrophilic PEI layer might be helpful for remaining stability of complexes in the presence of serum and increasing transfection efficiency.PCLs-D composed of DOPE and PEI 800-Chol has two endosomal escape abilitys,membrane destabilization and protonation.The GFP expression percentage of PCLs-D(68%)was significantly increased in comparison with that of PCLs-S and Lipofectamine^TM2000(～44%).The mechanism of PCLs internalization,kinetics of cellular uptake and intraceular distribution were investigated.The relationship between internalization pathway and final transfection efficiency might be exited,while different internalization mechanism among various cell lines.When A549 cells were pre-treated by chloropromazine(CPZ), which could specifically inhibit clathin-dependent uptake,the ODN uptake and transfection activity were decreased and fluorescence was concentrated on cell membrane comparing with that CPZ non-treatd determined by laser scanning confocal microscope.The result indicated that the dominance internalization of PCLs in A549 cells was clathin-dependent uptake,while in MCF-7 cells the mean of internalization was more multiple,combining with nonclathin-dependent and nonengery-dependent process.The kinetics of cellular uptake results shown that the cellular uptake of free ODN,PCLs-S/ODN complexes and PCLs-D/ODN complexes trend to saturation after 4 h incubation.PCLs-S and PCLs-D could increase uptake rate by 2.20-fold and 5.45-fold, respectively.Additionally,the eliminate rate of ODN could be decreased by PCLs-D, resulting to longer intracellular retain time.According to the study of PCLs character and transfection activity,tumor cells inhibition of ASODN against hTERT mediated by Cls and PCLs was compared.The results indicated that CLs could significantly increase the inhibition effect of ASODN and the IC₅₀value was decreased from 153.9μmol/L to 1.28μmol/L.Cls could also improve the intracellular stability of ASODN,achive continued tumor cell groth inhibition during 120 h.PCLs had the same enhancement effect to ASODN with the lower cytotoxicity,which might be more useful for in vivo study.Plasmid DNA nuclear entry was one of the committed steps for gene transfection. This study increased the nuclear targeting ability of PCLs-D by adding protamin and formed PCLs-D/protamine/DNA complexes.The combined vectors prepared using"Pre-mixed"and"Post-mixed"methods could both improve transfection efficiency obviously,85-fold and 12-fold higher than that of PCLs-D/DNA complexes;however, no transfection activity was found when protamine-DNA nanpartilces used.Though using the same method to prepare combined vectors,other cationic polymers without nuclear targeting such as chitosan and PEI 25k,didn't shown the same effect as protamine.These results suggested nuclear targeting ability was very important in non-viral vectors,while endosomal escape was precondition.Laser scanning confocal microscope confirmed ODN could be carried into nucleus by combined vectors.The protamine-DNA nanoparticles could well protect the integrity of plasmid DNA under ultrasonic condition and might retain transcriptional activity.A549 cells transferred by combined vectors kept high transgene expression during 72 h.At last,pCMV-IL-12 was used as therapic gene and the tumor growth inhibition of PCLs-D/pCMV-IL-12 complexes and PCLs-D/protamine/pCMV-IL-12 complexes were investigated.No antitumor effect was observed after intratumor injection of naked pCMV-IL-12 at different concentration and the IFN-γlevel in serum was low.When pCMV-IL-12 transferred by two vectors,tumor inhibition effect was appeared and IFN-γlevel increased too.Tumor inhibition rate of the group PCLs-D/protamine/pCMV-IL-12(5μg)reached to 65.8%,doubled than group PCLs-D/pCMV-IL-12(5μg).Intratumor injection of pCMV-IL-12 mediated by PCLs-D and combined vectors for antitumor treating in vivo could obtain high tumor inhibition rate as well as avoid the side effect of recombinate IL-12 protein direct incection.In conclusion,the results of our work provid new methods for reasonable design of non-viral gene vectors and some experimental proofs for further in vitro and in vivo application of non-viral gene delivery systems.