Synthesis Of PEI Derivatives And Their Application In Tumor Gene Delivery

Recent years, cancer has become a major killer of threatening human life and health. InChina, the annual number of deaths due to malignant tumors reached2.2million, representingthe first cause of death in our residents. Thus, cancer therapy has become a worldwideproblem to be solved, which has an extremely important strategic significance. Today,common treatments in clinical cancer include chemotherapy, radiotherapy and surgicaltreatment. However, these treatment strategies are harmful to normal tissues and organs,which bring patients great sufferings. What’s more, long-term use of chemotherapy willproduce the phenomenon of multidrug resistance (Multidrug resistance, MDR), reducing theeffect of chemotherapy drugs. Compared with conventional treatment, gene therapy isfundamentally expected to treat the disease, which has become the focus of the internationalacademic research. Gene therapy is considered to be a revolution in the field of pharmacy andmedicine, which is one of the most important milestones of today’s biomedical development.Gene therapy is bound to have a profound influence and impact on the traditionalpharmaceutical industry. It is expected to become the "darling" in the clinical future treatmentof cancer.Gene therapy includes three main areas: gene medicine, gene diagnosis and gene transfer.With the successful implementation of human genome project (human genome project, HGP),gene diagnosis and production of gene medicine is no longer a barrier to gene therapy.However, safe and effective drug delivery and playing a therapeutic effect are difficult, whichhas become one of the bottlenecks to get good results in gene therapy. Effectiveness andsafety of gene delivery mainly perform in the following areas: safety of the carrier, targetingand immune response of the gene delivery system. Gene delivery methods can be commonlydivided into physical, chemical and biological methods. Chemical and biological methods arebased on the gene vectors carrying drugs. The carrier’s position is extremely important.Successful gene therapy depends on the efficiency of gene transfer vectors. Vector species and development have become critical issues in the development of gene therapy. Biologically,gene vectors can be divided into plasmid vectors and viral vectors according to the sources.While, chemical researchers are eager to see plasmid as a kind of DNA, and they divided thecarrier into viral vectors and non-viral vectors. Currently, the most widely used vectors inclinical are still viral vectors. In part of them, transfection efficiency can be more than90%.However, there are such as immunogenicity deficiencies in viral vectors, which are difficult toovercome. In contrast, non-viral vectors are becoming more and more concern because oftheir many advantages. Polyethyleneimine (PEI) molecule has become the fastest growingmolecule in the field of non-viral gene vectors and a class of most research value and appliedpotential gene transfer vector.For transfection efficiency, the range of ideal PEI molecular weight is about11.9×103~70.0×103. Among which, the weight average molecular of25K hyperbranched PEI (PEI25K)has the highest transfection efficiency, and thus, PEI25K has become the "gold standard" tomeasure other non-viral gene vectors. Unique "proton sponge" effect of PEI are good toachieving endosomal intracellular escape. However, as a non-viral gene vector, PEI25K stillhas many defects, such as the relatively low efficiency of gene transfection, cell toxicity andnon-degradable polymers. Thus, it is particularly necessary to improve its structure.In this section, after EDC/NHS catalysis, we first couple N-acetyl-leucine with differentnumber of hydrophobic with amino group of PEI25K. And study the influence of the numberof N-acetyl–leucine on PEI25K as a material of gene delivery. Next, based on previousresearch work of others, we screen the relatively most appropriate derivative and proveN-isopropyl acrylamide is successfully modified on PEI using H NMR. After deliveringexogenous p53gene to prostate cancer PC-3cell lines, we explore its related role andcompare with the results of wild-type p53in Hela cell lines, to discuss whether p53geneplays a different role among different types of tumor cells. And a series of experimentalchemistry in vitro and experiment of cells of the derivative material structure, cytotoxicity,ability to bind nucleic acids, gene transfection efficiency, and a series of related genes andevaluation of transfection of p53are studied. Part I: Construction and evaluation of N-acetyl-leucine modified PEI25k gene vector1. We firstly obtain three N-Ac-Leu modified PEI derivatives using EDC/NHS catalyticsynthesis, which are N-Ac-Leu-PEI (60:1), N-Ac-Leu-PEI (80:1), N-Ac-Leu-PEI (100:1),respectively. Next, we verify their polymer structures using1H NMR.2. Three N-Ac-Leu-PEI polymers can well combine with the plasmid pEGFP indicated bygel electrophoresis. However, compared to unmodified PEI25K, their binding capacitiesare all reduced. Surface potentials of the three N-Ac-Leu-PEI are at+30mV based on thesurface potential measurement, which can exactly verify that the ability of N-Ac-Leu-PEIbinding with the plasmid is lower. The measurement results show that the particle size ofN-Ac-Leu-PEI polymer particle size in the range of180~220nm.3. Compared with PEI25K, hemolytic effect of N-Ac-Leu-PEI gene carriers obviouslydecreases and they hold good blood compatibility. Compared with PEI25K, values ofN-Ac-Leu-PEI adsorbed proteins are significantly lower in protein adsorptionexperiments and the value will slowly decrease with increasing protein adsorption time.These results prove that the introduction of N-Ac-Leu could greatly improve thebiocompatibility of PEI, which is expected to be performed in vivo.4. MTT results show that the cytotoxicity of N-Ac-Leu-PEI in HeLa cells is significantlylower than PEI25K.5. Three N-Ac-Leu-PEI polymers can effectively mediate gene transfection, and theirtransfection efficiency is higher than PEI25K. N-Ac-Leu-PEI (100:1) will in the highesttransfection efficiency under conditions of a mass ratio of3.0.Part II: Preparation and Properties of PEI derivatives gene vector1. Five kinds of PEI derivatives are obtained by refining feed ratio. We preliminary screen acarrier by Gel retardation assay and cytotoxicity test. Next, the structure of the selectedmaterial was characterized and confirmed by H NMR.2. Vectors, plasmids and plasmid composite particle size and surface charge are detected bythe potential of nano particle size.3. Carrier’s cytotoxicity is performed by MTT assay, which proving that modified vector hold fewer toxic compared with PEI.4. The carrier can achieve complete protection of DNA under the quality of the plasmidvector is not less than0.8in the DNase I enzyme degradation experiment. In comparison,after modification, the ability of protection in PEN is slightly declined compared to inPEI.5. Qualitative and quantitative investigation of a carrier of the gene transfer properties areperformed respectively by green fluorescent protein plasmid transfection experiments andPGL-3plasmid transfection experiments. Such data show that the introduction of theN-isopropyl acrylamide on PEI can reduce the surface charge of the polymer andintroduce a certain amount of hydrophobic groups. However, the more introductions ofhydrophobic groups will reduce the efficiency of gene vector transfection. In qualitativetransfection experiments, the best transfection emerges in plasmid mass ratio of2. Inquantitative transfection experiments, PEI demonstrated very good transfection efficiency.Although PEN is less than PEI, the transfection efficiency of PEN is almost the samewith other commercial transfection reagent.6. The mRNA and protein expression of p53gene are proved by RT-PCR and Western blotfrom, respectively. Next, we prove that expression of p53protein performs a normalfunction by checking the expression of relative proteins. The activity of caspase familyproteins and mitochondrial membrane potential changes can increase the credibility of theformer result.7. Cell cycle arrest and apoptosis are performed by flow cytometry, which found that thereis no cycle arrest phenomenon in Hela cell lines. The existence of cell apoptosis is provedby DAPI staining.8. In all, although the transfection efficiency is reduced to some extent after modification,the toxicity of obtained derivative is low, and its gene transfection efficiency iscomparable to commercial transfection reagent. What’s more, in the delivery experimentof the p53gene resulting in apoptosis of cancer cells, excellent transfection performanceis shown, and subsequent apoptosis-related experiments also proved exogenous p53genecan indeed be replaced or the compensation of endogenous missing or invalid p53gene.