Development And Application Of Novel Non-Viral Gene Delivery Vectors Based On Hydroxypropyl Cyclodextrin Cross-Linked By Low Molecular Polyethylenimine

Gene therapy is that, the occurrence, development and progress of disease is interfereed in by deliverying human normal or therapeutic gene into target cells in order to substitute to defect or correct the fault in genic structure or fuction or to kill the pathological cells or to increase the ability of cleaning the pathological cells. The study of gene thearapy is widely carried on, and some of it applied in clinical trial stage. There are already over 1200 gene therapy of approval clinical trials in the world, around 70% of it applied in treatment of malignant tumor.There are four important parts of gene therapy in practice: One is to look for effective therapeutic gene, two is to develop efficient and safe gene delivery vectors which can delivery gene into target cells and to be expressed, three is to select the effective routes to delivery, four is how to make therapeutic gene expressed in target celles specially, produced a marked effect. Among them efficient and safe gene delivery vectors is the main bottleneck of application in gene therapy. The gene delivery vectors applied in gene therapy are mostly Adenovirus and Retrovirus account to 50%. The non-virus vectors applied are liposome, is total amount of 8%.Viral vectors are used widely for gene delivery system in cancer gene therapy for clinical application because of its highly efficient gene delivery. But there are some disadvantage, they are immunogenic(repeat administration is dangerous), insertional mutagenesis in host genome, limited capacity of delivery DNA, limited scale of production. The first patient died from immunic response after Adenovirus voctors applied for gene therapy. There is insertion mutation in host genome because of viral vectors. DNA of virus integrated in host genome, changed the tumor-suppressing gene or activated the oncogene, cause the cells malignant transformation. So the application of virus vectors should be more safety evaluation.Non-viral vectors have some safety advantages over viral approaches,including low immunogenicity and loss capacity for insertional mutagenesis, as well as their low cost and ease of production. The application of non-viral vectors to human has, however, been held back by the poor efficiency of their delivery gene to cells. Optimal non-viral gene delivery vectors should be degradable, low cytotoxicity, high efficiency of delivery to cells, capable of targeting the cells and tissue.For optimal non-viral vectors, Polyfection with cationic polymers of different structures was shown to enhance the uptake and the expression of DNA. Poly(ethylene imine) (PEI) has emerged as a good candidate in the cationic polymers. PEI (polyethylenimine) is cationic polymer as non-viral gene delivery vectors used widely in research. PEI is linear or branch polymer. There is high positive charge on its surface, so PEI can condense DNA with negative charge. There are lot of protonate nitrogen atom, endosome and lysosome of PEI and DNA complex swell and rupture (proton sponge effect), DNA release and go into nucleus then express. The molecular weight of PEI varies greatly. The molecular weight of PEI varies from 5000-25000 Da for gene delivery vectors. Transgene efficiency of high molecular weight PEI is high, but toxicity is also high. The toxicity of low molecular weight PEI is very low, but generally the transgene efficiency is very low. PEI can make erythrocyte aggregate and bind non-specificly with albumin in blood . Transgene efficiency is low applied in vivo. So modification of PEI is in need . This study selected low molecular PEI as a part of non-viral gene delivery vectors. Here new idea is a new way to develop non-viral gene delivery vectors, a kind of nontoxic polyhydroxy chemical and another kind of low toxic polyanmino chemical are coupled with a linker, some copolymers formed with a degradable esteratic linkage. Copolymers are degradable and low toxic gene delivery vectors. Then they will be determined for advantage of vectors of high efficiency of their delivery to cells. Effective vectors are choosed to be coupled to targeting oligopeptide, improved its speciality targeting specific cells and tissue. Finally, new targeting, low toxic, degradable, highly efficient gene delivery vectors are prefered. They are expected for application in cancer gene therapy.The study includes two parts:1. High water soluble and nontoxic polyhydroxy hydroxypropyl cyclodextrin (HP α-CD(2-hydroxypropyl)- α-cyclodextrin) HP β-CD(2-hydroxypropyl)-β-cyclodextrin) or HP γ-CD(2-hydroxypropyl)-γ-cyclodextrin) were selected as backbone, low molecular polyethylenimine (branch 600 Da or linear 423 Da) were selected as side chain. Two chemical are coupled with CDI (1,1'-Carbonyldiimidazole). The copolymers formed with a degradable esteratic linkage. They were determined for advantage of vectors of high efficiency of their delivery DNA into cells.2. Targeting oligopeptide was coupled to the copolymer, the oligopeptide could bind specially with the extracellular part of Her-2 receptor. The targeting vector will bind specially with Her-2 receptor positive tumor cells, improving the speciality. The speciality of targeting pepetide was determined in vitro and in vivo. The vector was capable of targeting tumor cells with Her-2 receptor.Study Part I : Development of novel non-viral gene delivery vectors based on hydroxypropyl cyclodextrin cross-linked by low molecular polyethylenimine.Objective: Development of novel degradable, low toxic and highly efficient non-viral gene delivery vectors.Methods: High water soluble and nontoxic polyhydroxy hydroxypropyl cyclodextrin (HP α-CD(2-hydroxypropyl)- α-cyclodextrin) HP β-CD ((2-hydroxypropyl)-β-cyclodextrin) or HP γ-CD(2-hydroxypropyl)-γ-cyclodextrin) were selected as backbone, low molecular polyethylenimine (branch 600 Da or linear 423 Da)were selected as side chain. Two chemical were coupled with CDI. Copolymers were characterized with some charactertion methods such as ~1H-NMR, FTIR and TGA. Particle size and zeta potential of copolymers was determined. The capacity of retarding DNA was determined by gel retard. Copolymer's cytotoxicity was determined by MTT method. Copolymers and plasmid DNA of pEGFP or pCAG of luciferase complex were used to transfect different cell lines, the green fluorescence of cells was observed and luciferase activity was determined by assay of transfection efficiency. Transfection efficiency of copolymers in serum and serum free medium was determined by assay of transfection efficiency too. The degradability of copolymer wasdetermined.Results: Copolymers named HP α-CD-PEI 600, HP β-CD-PEI 600, HP γ-CD-PEI 600 HP β-CD-PEI 423, HP γ-CD-PEI 423 were characterized. The copolymers were novel compond. The copolymers formed with a degradable esteratic linkage beween CD and PEI. The composition of copolymers was analysised by integral ratio in ~1H-NMR. There is 3.3 PEI 600 in every CD of HP γ-CD-PEI 600. The ratio of coupling of branch PEI 600 is higher than linear PEI 423 significantly. The N/P ratio of the minimum particle size of HPα-CD-PEI 600 and plasmid DNA complex is 400, particle size is 325 nm. The N/P ratio of that of HP β-CD-PEI 600 is 300, particle size is 195 nm. The N/P ratio of that of HP γ-CD-PEI 600 is 40, particle size is 174 nm. When the N/P ratio is higher than 200, zeta potential of HP α-CD-PEI 600 and DNA complex is over +10 mv but not over +20mv. When the N/P ratio is higher than 150, zeta potential of HP β-CD-PEI 600 and DNA complex is about +20 mv. When the N/P ratio is higher than 40, zeta potential of HP γ-CD-PEI 600 and DNA complex is about +20 mv. The minimum particle size of HP β-CD-PEI 423 or HP γ-CD-PEI 423 and plasmid DNA complex was about 389 nm or 468 nm, When the N/P ratio was higher than 200, zeta potential of them was over +10 mv. When the N/P ratio was 12, HP α-CD-PEI 600 could retard DNA completely. That of HP β-CD-PEI 600 is 8 and that of HP γ-CD-PEI 600 is 4. The toxicity of HP β-CD-PEI 600 was lowest, and next was HP α-CD-PEI 600, The toxicity of HP γ-CD-PEI 600 was higher than both of them. The toxicity of three copolymers were much lower than PEI 25 kDa. The N/P ratio of optimal transfection efficiency of HP β-CD-PEI 600 was 300 and that of HP γ-CD-PEI 600 is 40 or 60 applied in different cell lines. The optimal transfection efficiency of HP β-CD-PEI 600 and HP γ-CD-PEI 600 was comparable to PEI 25 kD. The transfection efficiency of HP α-CD-PEI 600 was low. The transfection efficiency of PEI 25 kDa decreased obviously in serum compared with that in serum free medium. But that of HP α-CD-PEI 600, HP β-CD-PEI 600, HP γ-CD-PEI 600 almost did not decrease. HP γ-CD-PEI 600 was degradable in near physiological condition.Discussion: Copolymers were HP α-CD-PEI 600, HP β-CD-PEI 600, HP γ-CD-PEI 600, HP β-CD-PEI 423, HP γ-CD-PEI 423. The capacity of condesingDNA of HP β-CD-PEI 600, HP γ-CD-PEI 600 was high. The transfection efficiency of HP β-CD-PEI 600, HP γ-CD-PEI 600 was high. The transfection efficiency of HP γ-CD-PEI 600 was stable in serum and in serum free medium. HP γ-CD-PEI 600 was selected as vector to be modified in part two for its high transfection efficiency, low dosage and stabe transfection efficiency in serum and in serum free medium.Study Part II: Speciality of vector coupled to targeting Her-2 receptor oligopeptideObjective: Modification of HP γ-CD-PEI 600 for targeting Her-2 receptor positive breast or ovary tumor cells.Methods: Oligopeptide identified from sreeening of phage display selection was coupled to HP γ-CD-PEI 600 with the linker SPDP. The sequence of oligopeptide is MARAKE which bound specially with the extracellular part of Her-2 receptor. Coupled vector was determined for the linked oligopeptide by ~1H-NMR. Coupled vector and plasmid DNA of pEGFP or pCAG of luciferase complex was used to transfect different cell lines of Her-2 positive cells (SKOV-3c, SK-BR-3, T 47D cells) and Her-2 negative cells(MCF-7 cells ), the rate of positve green fluorescence cells was determined by FACS, and luciferase activity was determined by assay of transfection efficiency compared between coupled vector and vector.Coupled vectors with plasmid containing gene of IFN-α were injeceted into tumor of athymic mice bearing tumor by subcutaneously inoculated with SKOV-3c cells. The inhibition of subcutaneous tumor growth and the survival time was observed.Results: The green fluorescence positve rate of positve cells increased by 87 percent in SKOV-3c cells and 58 percent in SK-BR-3 cells.The relative light units increased by 63 percent in SKOV-3c cells and 77 percent in SK-BR-3 cells after tranfecting the two cell lines with coupled vectors or not coupled vectors with plasmid. It did not increase in MCF-7 cells(Her-2 receptor negative cells). The transfection efficiency of coupled vector did not increase after the blocking the Her-2 receptor with free oligopeptide in Her-2 receptor positive cells. Tumor growth was most significantly inhabited and the survival time of mice was longerafter the therapy with new vector coupled to oligopeptide compared with mice received therapy of PEI 25 k Da or new vector not coupled to oligopeptide.Discussion: The vector was easily modified by coupling other functional groups. The transfection efficiency of coupled vector increased compared with that of not coupled vector. Its speciality targeting Her-2 positive tumor cells was boosted.ConclusionThe development of new non-viral gene delivery vectors, suitable material of vector is essential in first. Then modification of the vector are carried on for its specific bingding with target cells and for its endocytosis and release of DNA. High water soluble and nontoxic polyhydroxy hydroxypropyl cyclodextrin was selected as backbone, low molecular polyamino polyehylenimine was selected as side chain. Two chemical are coupled with CDI. The copolymers formed with a degradable esteratic linkage. The copolymers were excellent candidate for gene delivery vectors. They were low toxic, degradable and efficient in delivery DNA into cells. The ratio of coupled PEI 600 to cyclodextin determined the the capacity of condensing DNA and transfection efficiency of copolymers. It was feasible to modify the copolymers by coupling to suitable target group so as to bingding specially with taeget cells an tissue. After being coupled to oligopeptide targeting Her-2 receptor, its speciality targeting Her-2 positive tumor cells was boosted.Cyclodextrin are capable of deliverying drug for its hydrophobic cavity. So the novel vectors will be used to delivery DNA and drug. Its potential effect will be better.The new way to design novel vectors is feasible in the study, we think the fruther optimization of them will do better than now.