| BackgroundGene therapy is developed with the development of recombinant DNA technology. As a novel treatment strategies, it usually refers to deliver the normal gene or therapeutic potential of DNA/RNA into target cells to correct or replace defect genes, suppress the abonormal gene activity and expression, so as to achieve the interfence with the occurrence,development and progression of disease (1).One of the great challenges for gene therapy is to design a suitable gene transfer vector, which is critical for a good treatment efficiency of gene therapy. Gene therapy carrier systems usually include viral vectors and non-viral vectors. These two kinds of gene transfer system have been widely used in gene therapy in vivo and gene transfection in vitro.Although gene transfection and expression efficiency have been demonstrated for the viral vectors compared with non-viral vector systems,the viral vectors have obvious disadvantage such as potential security risks, complex preparation, no direct delivery of treatment medicine as well as more expensive. However,non-viral vector systems, not only delivery target genes,but also transfer the antibody, oligonucleotides, siRNA. Therefore, due to the low toxicity, low immunogenicity, flexible and controllable structure as well as convenient synthesis and low cost, non-viral vector systems is becoming promising therapeutic modality for the treatment of genetic and acquired disorders(2).Calcium phosphate, as earlier non-viarl vector for in vitro gene transfection, can not be applied well in basic research and clinical gene delivery due to its low transfection efficiency for many cells(1). Cationic liposome mediated gene transfection established by the mid 80’s, because of similar features to overcome the cells barrier with the viral vector and easy to penetrate the cell membrane as well as higher efficiency In vitro gene transfection, become a new non-viral vector system for gene transfection. However, it could be cleared by serum easily In vivo and accumulated in the lung tissue so that it could induce a strong inflammatory response and have higher cell toxicity.Therefore, the cationic liposome carrier has not been well applied.A new generation of cationic polymer carrier should satisfy the requirements for gene transfection and gene therapy such as easily condensing and packaging DNA or RNA, high delivery efficiency, low cell toxicity, relative stability and easy serum removal.In recent years, a variety of cationic polymers carrier such as polyethylene imine (PEI)(4), poly lysine [PLL poly (L-lysine) (8)and PAMAM (9) dendrimer is becoming as a new non-viral vector(10). However, the cationic polymer vector transfection efficiency is still relatively low, and the relationship between structure and function of these specific groups of cationic polymer and its functional effect is still not very clear. Therefore, the structure of cationic polymer have been modified to increase the gene and siRNA delivery efficiency In vivo through different forms of the modification and synthesis(11).Recently, a new kind of containing disulfide bond in the main chain of biodegradable poly (amino amine (PPA) was developed(12-18). Because of this new kind of polymer has good biodegradable, so it has a very low cytotoxicity (19). In the PPA cationic polymer containing ranched chain, because of different types of amino in the branched chain, it makes the branched chain cationic polymer can interact with the cell membrane, thus affecting cell absorption, escape and transfection process(20,21).Based on the structure characteristics of branched polyphosphoramides, we developed a new kind of cation polyphosphoramide (denoted as PPA) with amino moities in the main chain by click chemistry. This novel dendrimer cationic polymers could be used as a safe and efficient gene transfer vector.PurposeThe purpose of this study is to chemical synthesis a new kind of of branched polyphosphoramides as a gene carrier characteristic of low toxicity, and high transfection efficiency. It will be tested transfection efficiency as a carrier for gene and siRNA into target cells using a series of chemical and cell biological experiments and also toxic effects produced in the process of transfection. This study will provide the basis for the novel dendrimer cationic polymers as gene or siRNA carrier to the clinical treatment of diseases.MethodsIn this article, we developed a new kind of cation polyphosphoramide (denoted as PPA) with amino moities in the main chain by polycondensation of ethyl dichlorophosphate with N1-(2-aminoethyl)-N1-(1-methyl)-1,2-ethanediamine, and then evaluated the gene and siRNA delivery efficiency in A549 cells and HEK293 cells by fluorescent microscopy and flow cytometry assay. Cytoxicity of cell proliferation by CCK8 assay were determined the effect of cation polyphosphoramide on toxicity of different cells. Immunofluorescence staining and Western blot confirmed whether the gene and siRNA could be delivered effectively into target cells by cation polyphosphoramide.Results1. Chemical synthesis of polyphosphramide and NMR spectrometer measurementPolyphosphoramide were synthesized by N1-(2-aminoethyl)-N1-(1-methyl)-1,2-ethanediamine reacted with ethyl dichlorophosphate under N2 protection. The successful synthesis of polyphosphoramide was demonstrated by NMR spectra. The resonances at 4.51-4.72 ppm (a) and 1.09-1.31 ppm (b) are the characteristic signals of methylene protons (-POCH2CH3) of the phosphoester. Resonances at 2.62-3.08 ppm (c,d,e) are the characteristic signals of methylene protons (-NCH2CH2)N(CH3) CH2CH2N-) of the polyphosphoramide main chain. We also analyzed their structures by 31P NMR spectroscopy. The 31P NMR spectrum of polyphosphoramide (Figure 1B) gave a strong resonance at δ10.02 ppm, assigned to the phosphorus atom in polyphosphoramide block.2. DNA binding ability of polyphosphoramideTo evaluate the DNA binding ability of PPA, gel retardation assays were performed by electrophoresis of PPA/pDNA polyionic complexes using a 1.0% agarose gel containing ethidium bromide. The gel retardation assays showed that both lower content of PPA (0.5μg/μL) and higher content of PPA (1.0μg/μL) in the same content of pDNA (0.5 μg) could complex pDNA well at certain N/P values, the PPA/pDNA was completely retained in the samples at N/P 8 and higher ratio, indicating effective DNA condensation. In comparison, with higher content of pDNA (1.0μg) and an increase the N/P ratio, the complexes completely lost mobility when formed at a lower N/P value of 4, indicating a high complex capacity of full DNA chain with PPA.3. Particle size and zeta potential measurement for the polyphosphoramide and DNA complexes.The particle size and zeta potential of PPA/pDNA complexes have been shown to influence the cellular uptake, endocytosis pathway, nuclear entry, and transfection efficiency. PPA/pDNA polyionic complexes were prepared by simple and direct mixing of pDNA and PPA solutions at various weight ratios (N/P ratio) in 10 mM Tris-HC1 buffer, pH 7.4. The particle size and zeta potential of polyionic complexes weight ratios ranging from 1:1 to 16:1 were measured by Nano-ZS ZEN3600. The results showed that all the complexes were very small with sizes ranging from 100 to 166 nm. The particle sizes decreased slightly with increasing N/P ratio.Zeta potentials of the polyionic complexes, characterized by a zeta potential analyser with dynamic light-scattering capability, increased from 26.6mV to 38.4mV with the increasing NP ratio. The positive zeta potential of PICs made complexes easier to be uptaken by cells, attributed to the electrostatic interactions between negative characteristic.4. Cytotoxicity of polyphosphoramideWe evaluated the cytotoxicity of PPA at different concentration by CCK-8 assay in human lung adenocarcinoma A549 cells, human embryonic kidney HEK293 cells and human prostate cancer DU145 cells. Over the tested range of PPA concentration (10-1000lμg/μ L), no significant difference of cell viabilities in A549 cells, HEK293 cells and DU145 cells were observed from 10Lg/μ L to 1000μg/μ L compared to the DMSO control, In order to investigate whether the cell cytotoxicity of PPA was in time dependent manner, HEK293 cells were incubated with 100OLg/μ L PPA for 6h,12h,24h,48h,72h. Cell survival assay showed that there was no significant for the cell cyotoxicity between PPA and DMSO control at indicated time. These results suggested that PPA can be further used In vitro or In vivo gene carrier.5.Transfection efficiency of plasmids by polyphosphoramide and detection of the functional activity for the target geneWe investigated the in vitro transfection efficiency of PPA/plasmid complexes at different ratio in HEK293 cells lines using a pEGFP plasmid, encoding for enhanced GFP protein. A similar trend of transgene expression in HEK293 cells was observed between lower concentration (0.5 μg/μL) and higher dose (1.0 μ.g/μL) of PPA with the same pGFP(0.5 ug) in weight ratios at N/P ratio (3:1 or 5:1). In contrast, polyionic complexes containing 1.0μg/μL PPA with 1.0μg pGFP at N/P ratio(3:1) showed higher transfection efficiency than at N/P ratio(5:1). These results suggested that polyionic omplexes at lower ratio of PPA/DNA (3:1) exhibited higher transfection efficiency and fewer ration of PPA/DNA (5:1) may contribute to the lower transfection efficiency.We then investigated the in vitro transfection efficiency of PPA/plasmid polycomplexes in HEK293cells lines using a pEGFP plasmid, encoding for enhanced GFP protein.The transfection efficiency was visualized by observation of GFP-positive cells using inverted fluorescence microscope, the results showed that the transfection efficiency of PPA at N/P ratio(3:1) were comparable to the commercially available transfection reagents lipofectamine 2000 and Hilymax, showing similar fluorescence intensity for GFP protein in target cells (Figure 6A).Moreover, Flow cytometry was further performed to precisely quantify GFP-positive cells by in HEK293 cells, as shown in Figure 6B, more than 60% (64%)of cell were fluorescence GFP-postitive in PPA transfcetion group, showing comparable transfection to lipofecatmine 2000(62% of GFP-positive cells) and higher transfection than Hilymax(48% of GFP-positive cells), another commercial transfection reagent.Because the phosphorylation of PKD and IκB degradation cascade are the critical event that PKD specific agonist mediated NF-κB signalling passway, we then explored whether exogenous PKD2 delivered by PPA can have biological activity and activate downstream target IKB degradation of NF-KB signalling pathway in response to PKD specific agonist phorber ester PMA. Overexpression of GFP-PKD2 could be found in A549 cancer cells transfected with polyionic complexes containing 1.0μg/μL PPA and 1.0μg GFP-PKD2 at N/P ratio(3:1), indicating that exogenous GFP-PKD2 plasmid could be transfected efficiently and overexpressed in A549 cancer cells, while, overexpression of GFP-PKD2 could enhance the endogenous and exogenous phosphorylation of PKD at active loop(S744/748) and promote IkB degradation of NF-JB signalling passway in response to PMA(100nM) treatment for 30min. All these result suggested that our novel synthesized PPA could effectively deliver exogenous gene into target cells and showed functional role of protein expressed by interest of gene in specific signalling passway.Next, we evaluated whether more than one exogenous genes can also be effectively delivered into target cells by PPA transfection and whether related protein or kinases expressed by these target genes can exhibit biological function in cells, so we cotransfected NF-κB-1uc reporter gene, internal control Rellina luciferase plasmid (pGL4.74[hRluc/TK]) with pEGFP or GFP-PKD2 plasmid with PPA (1.0μg/MI) into HEK293 cells,24 h after transfection the cells were serum starved for 12h and stimulated with or without PMA (100 nM) for another 16 h. Dual-Luciferase reporter assay showed that PMA-induced DNA-binding activity of NF-κB to a consensus promoter sequence(2X NF-κB) increased remarkably in HEK293 cells transfected with GFP-PKD2 plasmid compared with pEGFP transfected HEK293 cells, indicating that PKD2 mediated PMA-induced NF-κB transcriptional activation in target cells. Collectively, these results suggested that our synthesized PPA could effectively deliver one and more exogenous genes into target cells and transfected genes by PPA also show physiological role in the target cells.6. Transfection efficiency of siRNA by polyphosphoramide and measurement of target protein nuclear translocationSynthetic siRNAs are chemically synthesized double-stranded RNAs with structures that mimic the cleavage products of the enzyme Dicer. Upon introduction into the cytoplasm, synthetic siRNAs are incorporated into the RNA interference (RNAi) machinery in the same way as endogenous small RNAs. The great therapeutic potential of siRNA is a result of its ability to silence nearly any targeted gene after introduction into cells. To investigate whether our synthetic novel PPA can deliver the siRNA and silence the target gene of siRNA in cells, siRNA control(siCTL) or siRNA of PKD2 (si-PKD2) were encapsulated with PPA at N/P ratio(3:1) to form a PPA-siRNA complex and then added to A549 cells or HEK293 cells, after 48h, the cell lysate were harvested. Western blotting showed that siRNA PKD2 transfection by PPA significantly reduced endogenous PKD2 expression in A549 cells or HEK293 cells, indicating that siRNA could be delivered by PPA effectively into target cells, and exhibited silencing the target gene in target cells.We then performed imrnunofluorescence staining and confocal microscopy to explore whether knockdown of PKD2 by PPA transfection can reduce TNF-a-induced NF-κB p65 nuclear distribution, which is the critical characteristic of NF-κB activation of gene transcription, in response to TNF-a treatment. TNF-a stimulation significantly increased NF-κB p65 nuclear distribution in siCTL-PPA transfected A549 cells, while siRNA knockdown of PKD2 by si-PKD2/PPA transfection remarkably reduced NF-κB p65 nuclear translocation induced by TNF-a treatment in A549 cells, suggesting that siRNA could also be encapsulated and delivered into target cells effectively, as well as exhibition of silencing function in physiological condition.In order to evaluate the siRNA silencing with PPA transfection in different cells, we also transfected with siCTL or siRNA of PKD2 into HEK293 cells, the transfected cells were treated with TNF-a for 30 min. Confocal microscopy showed that similar results of NF-κB p65 nuclear translocation can be found in HEK293 cells transfected by siRNA of PKD2 with or without TNF-a treatment. Taken together, these results indicated that our synthesized PPA not only can effectively delivery the plasmids into target cells, but also could form complex with siRNA and show knockdown efficiency as well as biological function of siRNA into the target cells.ConclusionIn this study, we have developed a novel cation polyphosphoramide (denoted as PPA) with amino moities in the main chain by polycondensation of ethyl dichlorophosphate with N1-(2-aminoethyl)-N1-(1-methyl)-1,2-ethanediamine. The PPA could efficiently retard mobility of DNA at lower N/P ratio, and exhibited a decreased size and increased zeta potential. The cytotoxicity assay and in vitro gene transfection further revealed that PPA were non-toxic to different cells and showed higher transfection efficiency at lower N/P ratio (3:1). Moreover, this kind of synthesized PPA can effectively deliver more than one exogenous genes into target cells and overexpression of these gene could show functional role in cells. More importantly, the novel PPA could also encapsulate the siRNA into different cells and exhibited silencing the target gene as well as the signaling pathway in A549 cancer cells and HEK293 cells. This study provides a new insight into the design of efficient and biocompatible gene and siRNA carriers using cation polyphosphoramide with amino moieties in the main chain as scaffolds. |
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