Evaluation About The Employment Of Cationic Polymerβ-CD-PAMAM As Gene Carrier Of Neuroblastoma Tumor Cells

Multiple sclerosis is an autoimmune disease of the central nervous systeminflammatory demyelinating disease, characterized by the demyelination caused bymyelin-specific T cell-mediated autoimmune attack, the local T cell and macrophageinfiltration, axonal damage and loss of nerve function.MS permanent neurological deficits are associated with the loss of neurons andthe injury of axonal. Except that MS inflammatory mechanism can lead todemyelination and axonal injury, the immune cells also have neuroprotectiveproperties, which are partly mediated by the release of neurotrophic factors (brainderived neurophic factor, BDNF).Some study indicated that when BDNF was added to the neural precursor cells invitro, the effect was mainly promoting their differentiation into neurons. As BDNF isa natural protein molecules which is difficult to pass the blood-brain barrier, it ramainhas a problem how to sent it to the damage point of the central nervous system.Genetransfection is an efficacy treatment nowadays that can provide a long-term efficacy.The key steps of this technology are to transduct the gene into the cells by vector. And,this technique allows modification of the vector, so as to go through the blood-brainbarrier.Gene transduction processes required for efficient, high security carrier. Currently gene transfer vector used in gene therapy research is divided into two majorcategories of viral vectors and nonviral vectors.The commonly used viral vectors, although via genetic engineering, removingpathogenic while retaining the hige gene transfection efficiency, but the difficulty inpreparation, target gene insertion length restriction, there are potential immunereaction and biological safety, greatly limiting their in vivo experiment application.Therefore, people have been committed to finding new gene transduction conductorNon-viral vector is synthesized with positively charged cationic gene vectors,and the advantages of synthetic vectors are safe and good adaptability, easy totransform. The novel cationic polymers are currently hot research field of biologicalmaterial, its advantages include:Cationic polymer is one kind of non-viral carriers. They are mostlynon-immunogenic，less expensive to produce,and relatively safer. The sulface bondingforce and chemical activity can also be controlled. However, the relationship betweentransfection efficiency and the polymer structure is stilled been studied. The cationicpolymer carrier combines DNA with electrostatic interation and then enters cellsthrough cellular internalization. The cationic polymer namedβ-CD-PAMAM used inthis study is synthesized by the Chemical and Chemical engineering institute ofSunYat-sen University. So we synthesized the star-shape cationic polymerβ-CD-PAMAM, researchedthe load capacity of plasmid DNA and detected its forming nanometer complexparticle size, Zeta-potentials, morphology and structure; At the same time in vitro wetested its cytotoxicity and transfection efficiency, and further studied the effect ofserum on transfection efficiency.The whole study was composed of two parts:1. Synthesis and characterization of β-CD-PAMAM/DNA2. Synthesis and characterization of a novel nonviral vectorβ-CD-PAMAM forgene delivery in vitro.Chapter1Synthesis and characterization of the complex formed ofstar-shape cationic polymerβ-CD-PAMAM and DNAObjectiveThe complex formed of star-shape cationic polymerβ-CD-PAMAM and DNAwas synthesized and characterized, and further researched the load capacity ofplasmid DNA.MethodsAt a certain N/P ratio (N/P ratio refers to the NH4~+of the cationic polymerdivided by the PO3-molar proportion of the DNA). When the cationic polymeruniformly mixed to form a complexes, the particle size and z-potentials of thenanocomposite in aqueous system were measured at25oC using a ZetaPALS. Themorphology of the β-CD-PAMAM/pDNA complexes at the N/P ratio of10wasobserved by transmission electron microscopy. The agarose gel electrophoresis wasused to detect the ability of loading plasmid DNA by β-CD-PAMAM.Results1. Particle size and zeta potential of nano-materials complexThe particle size of the complexes decreased with the increase of N/P ratio from1to30and then remained in the100–200nm range with further increase of N/P ratio to30; all complexes were positively charged and the z-potential increased with theincrease of N/P ratio from1to30, and theβ-CD-PAMAM/DNA binding stability.2. Shape and structure of nano-materials complexWhenβ-CD-PAMAM/pEGFP-N3formed at N/P ratio of10, the size of thecomplex investigated by electron transmission microscopy does not exceed200nm, ithas spherical, compact structure, good for endocytosis in gene transfection.3. Agarose gel electrophoresisWhen N/P ratio is10, β-CD-PAMAM and DNA is fully combined, agarose gelelectrophoresis showed that plasmid DNA were blocked in loading holes. So whenN/P=10, β-CD-PAMAM can be used as gene transfer vectors.ConclusionsSynthesis of star-shaped cationic polymerβ-CD-PAMAM can be used for invitro transfection in order to understand the β-CD-PAMAM gene transfectionefficiency and cytotoxicity to lay a foundation in theory and practice, we detectedtheir cytotoxic and transfection efficiency in vitro.Chapter2Synthesis and characterization of a novel nonviralvectorβ-CD-PAMAM for gene delivery in vitroObjectiveTo prepare for further in vivo gene therapy, we synthesized nanometer materialsβ-CD-PAMAM andβ-CD-PAMAM/pEGFP-N3complexes and tested their toxicityand transfection efficiency in cell lines.MethodsPAMAM dendrimer type as positive control group, we cultured humanembryonic kidney cells (Hek293cells) and neuroblastoma tumor cells (SH-SY5Ycells) in vitro, and prepared containing EGFP gene expression vector pEGFP-N3,using the MTT colorimetric test (MTT) research of nanometer materials β-CD-PAMAM andβ-CD-PAMAM/PEGFP-N3complex cells toxic effect; then respectively applied β-CD-PAMAM and PAMAM two kinds of carrier pEGFP-N3transfected into serum-free culture and serum-free culture of human embryonickidney cells (Hek293cells) and neuroblastoma tumor cells (SH-SY5Y cells) aftertransfection,24h were detected by flow cytometry in transfection efficiency, confocallaser scanning microscope and fluorescence microscope observation of effect oftransfection.Results1. Cell toxicity testIn both cells, when N/P <10, all nano-materials carriers and their complexesshowed low toxicity,cell viability in more than80%(P <0.05); when N/P>20, viabilityof cells carring β-CD-PAMAM and β-CD-PAMAM/pEGFP-N3complex washigher than those carring PAMAMå'ŒPAMAM/pEGFP-N3complex (P<0.05).2. Cell transfection experimentsWhen β-CD-PAMAM/DNA complex combined at the N/P value of20,30and40,the transfection efficiency of SH-SY5Y cells was higher than PAMAM andpEGFP-N3combined at N/P value of20. When N/P was20, the highest transfectionefficiency was found, reach up to20%(P<0.05).When the complex was combined at the same N/P, transfection efficiency wasmildly higher in groups with DMEM medium containing10%FBS than those withoutFBS. However， there were no statistically difference(P>0.05). Laser scanningconfocal microscope and inverted fluorescence microscope displayed an intuitivetransfection results above.ConclusionIndicate the cytotoxicity and transfection efficiency of β-CD-PAMAM,confirmed that β-CD-of PAMAM can be used as a carrier for cell gene transfer ofHEK293and SH-SY5Y. It can further be used in the combined gene therapy ofmultiple sclerosis in EAE model.