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In recent years, non-viral gene vectors have attracted great attention for efficient gene delivery due to the advantages, including low toxicity, low immunogenicity and simple preparation. Polyethylenimine (PEI) is one of the typical non-viral gene carriers that have been widely utilized for gene delivery owing to its superior capabilities in gene compression and buffering capacity. This article discusses the processes of gene delivery and the barriers of PEI-based carrier during the gene delivery, such as low biocompatibility, cytotoxicity, lack of specific targeting and insufficient gene release, etc. Therefore, we summarize the multiple approaches for the modifications of PEI in terms of improved biocompatibility, degradability, specific targeting and buffering capacity. Furthermore, we also review on the recent impressive progresses of smart stimuli-responsive PEI carriers, including endogenous stimuli (pH, reactive oxygen species, glutathione, biomolecular, etc), exogenous stimuli (light, temperature, magnetic field, etc) and dual-responsive strategies, which might provide guidance for the development of more efficient and safer non-viral gene vectors.
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Objective To synthesize a novel prostate cancer targeting gene vector PAMAM-PEG-C2min and improve gene transfection efficiency targeting on prostate cancer. Methods The aptamer (C2min) and polyamide-amine (PAMAM) were ligated by polyethylene glycol (PEG). The structure of the synthesized PAMAM-PEG-C2min was identified by NMR. The biological characteristics of the nanoparticles were examined by the uptake experiments and gene transfection experiments (the loaded gene was siR-M) with the prostate cancer cells (PC3 and LNCaP). Besides, the in vivo targeting was investigated using in vivo image system. The in vivo targeting results indicated that PAMAM-PEG-C2min can achieve the simultaneous targeting of two prostate cancer tissues. Results The PAMAM-PEG-C2min synthesis was confirmed by NMR. Cell uptake experiments showed that the cell uptake efficiency of PAMAM-PEG-C2min was concentration dependent. In vitro experiments showed that the PC3 and LNCaP cells transfection efficiency and targeting of PAMAM-PEG modified with C2min were significantly improved compared with the PEG modified PAMAM. Conclusion PAMAM-PEG-C2min is a potential targeted drug delivery vehicle. It provides a new technology platform for comprehensive and specific targeting treatment of prostate cancer.
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The study was designed to synthesize a novel dendritic copolymer composed of polyamidoamine dendrimer G0 as the inner core and poly(L-glutamic acid) grafted low molecular weight polyethylenimine (PGLP) as surrounding arms for gene delivery vector. The molecular structure of PGLP was confirmed by 1H NMR (proton nuclear magnetic resonance spectroscopy). The DNA combination capability of PGLP was examined by gel retardation electrophoresis. The particle sizes and zeta potentials of PGLP/pDNA complexes were determined by dynamic light scattering (DLS). The cytotoxicity of PGLP was evaluated by Cell Counting Kit-8 (CCK-8) and hemolysis assays, which was approved by Research Ethics Committee of the First Affiliated Hospital of Nanchang University. The in vitro transfection efficiency of PGLP was measured by a flow cytometry. The results of physicochemical properties suggested that PGLP could self-assemble with DNA to form complexes with average particle sizes of about 105-200 nm and zeta potentials of about +10 - +28 mV, which could protect DNA from serum degradation. The results of biological properties suggested that PGLP showed more higher transfection efficiencies but lower cytotoxicity than PEI 25K or Lipofectamine 2000 in various cell lines (HEK 293T, HeLa, BEL 7402, RASMC). Importantly, it was found that PGLP/pDNA complexes at w/w = 8 showed more strong serum-resistant capacity than PEI 25K/pDNA complexes. Therefore, PGLP is a promising candidate vector for gene delivery.
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The key of gene therapy is to deliver the functional gene to the target tissue in the body. The safe and efficient gene carrier is particularly important in the targeted delivery. Multifunctional envelope-type nano device (MEND), based on concept "Programmed packaging", is a new type of gene carrier system, with high encapsulation efficiency, favourable stability, high transfection efficiency, easy preparation, etc. MEND is designed to control intracellular trafficking as well as the tissue distribution of encapsulated compounds such as nucleic acids/proteins/peptides, permitting them to function at the appropriate location. In this paper, research progresses in MEND are reviewed in accordance with three types of payloads:the small interfering RNA (siRNA), DNA and proteins/peptides in recent years.
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Objective To explore the feasibility and efficacy of an MRI-visible,targeted,nano-vector which is synthesized by attaching a targeting ligand,the GD2 single chain antibody (scAb GD2),to the distal ends of PEG-g-PEI-SPION as a carrier for gene delivery into human bone marrow mesenchymal stem cells (hBMSCs) and in vitro cellular MR imaging.Methods scAbGD2-PEG-g-PEI-SPION was synthesized as previously reported.Gel electrophoresis was performed to assess the pDNA condensation ability of scAbGD2-PEG-g-PEI-SPION.The particle size and Zeta potential of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes were observed by dynamic light scattering.Cytotoxicity of scAbGD2-PEG-g-PEI-SPI-ON was evaluated by CCK-8 assay using hBMSCs.Gene transfection efficiency of scAbGD2-PEG-g-PEI-SPION in hBMSCs was quantified by flow cytometry,PEG-g-PEI-SPION,scAbGD2-PEG-g-PEI-SPION,scAbGD2-PEG-g-PEI-SPION+ free AbGD2 and scAbIgG2a-PEG-g-PEI-SPION group was established.The cellular internalization of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes was observed by confocal laser scanning microscopy and Prussian blue staining.MRI of scAbGD2-PEG-g-PEI-SPION was performed by cellular MRI scanning in vitro.Results scAbGD2-PEG-g-PEI-SPION condensed pDNA to form stable nanocomplexes of 80-100 nm in diameter and showed low cytotoxicity to hBMSCs.At the same N/P ratio,the transfection efficiency of scAbGD2-PEG-g-PEI-SPION group was significantly higher than those of other groups (P＜0.001).At the optimal N/P ratio of 20,scAbGD2-PEG-g-PEI-SPION/pDNA obtained the highest transfection efficiency of (59.60 ± 4.50) ％ in hBMSCs.Furthermore,hBMSCs labeled with scAbGD2-PEG-g-PEI-SPION showed sensitive low signal intensity on MRI T2/T2 *-weighted images in vitro.Conclusion scAbGD2-PEG-g-PEI-SPION is an efficient MRL visible targeted nano vector for gene delivery into hBMSCs.
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Objective To construct a Luciferace reporter vector containing the 3'untranslated region (3'UTR) of NFAT5 and measure the correlation between NFAT5 and miR-155.Methods The miR-155 targeting NFAT5 3'UTR was predicted by Target Scan,Mir Base and Pic Tar.NFAT5 and mutant NFAT5 sequence(NFAT5-mu) were then designed and synthesized,and they were cloned into pMIR-REPORTTM Luciferace reporter vector.Human embryonic kidney-293AD (HEK-293AD) cells of the 4th passage were divided into 4 groups according to the random number table.cells in plasimd +miR-155 mimics groups were transfected with pMIR-NFAT5 recombinant plasimid,pRL-Tk plasmid and miR-155 mimics;cells in plasimd + miR-155 mutated groups were transfected with pMIR-NFAT5-mu recombinant plasimid,pRL-Tk plasmid and miR-155 mimics;cells in plasimd + miR-155 control groups were transfected with pMIR-NFAT5 recombinant plasimid,pRL-Tk plasmid and miR-155 Negative control;cells in plasimd +miR-155 inhibitor were transfected with pMIR-NFAT5 recombinant plasimid,pRL-Tk plasmid and miR-155 inhibitor;and were respectively transfected into together by liposome.After culture for 24 h,the luciferase activity was detected by dual luciferase reporter assay system.Results TargetScan,Miranda and PicTar shared the results that NFAT5 has the complementary binding sites with 3'UTR of miR-155.And luciferase reporter vectorwas constructed.Therefore the result of sequencing and double digesting of recombined plasmid were completely correct.Dual-luciferase reporter assay showed that miR-155 possesses a target effect on 3'UTR of NFAT5.Compared to the pMIR-NFAT5 + miR-control group,the luciferase activity of the pMIR-NFAT5 + miR-1 5 5 mimics group was decreased,with statistically significant difference(P＜0.01),while there was no significant difference at other time points(P＞0.05).Conclusion The pMIR-NFAT5 recombinant plasmid and pMIR-NFAT5 recombinant mutated plasmid were confirmed with successful construction.and it was found that miR-155 can target NFAT5 mRNA 3'-UTR.The results provide the experiment data for further disclosing the mechanism of inhalation injury on the level of gene expression.
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Objective To explore the feasibility and efficacy of an MRI-visible,targeted,nano-vector which is synthesized by attaching a targeting ligand,the GD2 single chain antibody (scAb GD2),to the distal ends of PEG-g-PEI-SPION as a carrier for gene delivery into human bone marrow mesenchymal stem cells (hBMSCs) and in vitro cellular MR imaging.Methods scAbGD2-PEG-g-PEI-SPION was synthesized as previously reported.Gel electrophoresis was performed to assess the pDNA condensation ability of scAbGD2-PEG-g-PEI-SPION.The particle size and Zeta potential of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes were observed by dynamic light scattering.Cytotoxicity of scAbGD2-PEG-g-PEI-SPI-ON was evaluated by CCK-8 assay using hBMSCs.Gene transfection efficiency of scAbGD2-PEG-g-PEI-SPION in hBMSCs was quantified by flow cytometry,PEG-g-PEI-SPION,scAbGD2-PEG-g-PEI-SPION,scAbGD2-PEG-g-PEI-SPION+ free AbGD2 and scAbIgG2a-PEG-g-PEI-SPION group was established.The cellular internalization of scAbGD2-PEG-g-PEI-SPION/pDNA nanocomplexes was observed by confocal laser scanning microscopy and Prussian blue staining.MRI of scAbGD2-PEG-g-PEI-SPION was performed by cellular MRI scanning in vitro.Results scAbGD2-PEG-g-PEI-SPION condensed pDNA to form stable nanocomplexes of 80-100 nm in diameter and showed low cytotoxicity to hBMSCs.At the same N/P ratio,the transfection efficiency of scAbGD2-PEG-g-PEI-SPION group was significantly higher than those of other groups (P＜0.001).At the optimal N/P ratio of 20,scAbGD2-PEG-g-PEI-SPION/pDNA obtained the highest transfection efficiency of (59.60 ± 4.50) ％ in hBMSCs.Furthermore,hBMSCs labeled with scAbGD2-PEG-g-PEI-SPION showed sensitive low signal intensity on MRI T2/T2 *-weighted images in vitro.Conclusion scAbGD2-PEG-g-PEI-SPION is an efficient MRL visible targeted nano vector for gene delivery into hBMSCs.
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Biodegradable polyamines have long been studied as potential recombinant viral gene vectors. Spermine (SPE) is an endogenous tetra-amine with excellent biocompatibility yet poor gene condensation capacity. We have previously synthesized a polyspermine based on SPE and poly(ethylene glycol) (PEG) diacrylate (SPE-alt-PEG) for enhanced transfection performance, but the synthesized SPE-alt-PEG still lacked specificity towards cancer cells. In this study, folic acid (FA) was incorporated into SPE-alt-PEG to fabricate a targeted gene delivery vector (FA-SPE-PEG) via an acylation reaction. FA-SPE-PEG exhibited mild cytotoxicity in both cancer cells and normal cells. FA-SPE-PEG possessed higher transfection efficiency than PEI 25 K and Lipofectamine(®) 2000 in two tested cancer cell lines at functional weight ratios, and its superiority over untargeted SPE-alt-PEG was prominent in cells with overexpressed folate receptors (FRs). Moreover, in vivo delivery of green fluorescent protein (GFP) with FA-SPE-PEG resulted in highest fluorescent signal intensity of all investigated groups. FA-SPE-PEG showed remarkably enhanced specificity towards cancer cells both in vivo and in vitro due to the interaction between FA and FRs. Taken together, FA-SPE-PEG was demonstrated to be a prospective targeted gene delivery vector with high transfection capacity and excellent biocompatibility.
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@#This study was aimed to prepare sheddable PEG modified miRNA-complexing nanoparticles and investigate in vitro cellular uptake effect and in vivo distribution profile. The sheddable PEG material was synthesized through condensation. The sheddable PEG modified miRNA-complexing nanoparticles were successfully prepared by electrostatic interaction between gene vector and miRNA, and then ibuprofen was added to deshield PEG layer. The in vitro cellular uptake effect and in vivo distribution profile of nanoparticles were investigated on 4T1 model cells. As a result, the particle size of nanoparticles was 107. 7 nm and Zeta potential was 15. 8 mV. Compared to unsheddable PEG group, the cellular uptake effect by 4T1 tumor cells as well as the concentration on tumor regions was significantly improved in the sheddable PEG group. Results showed that this systen has a great potential application in the field of tumor treatment.
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This study aims to investigate the function of two SNPs (rs8904C > T and rs696G > A) in 3' untranslated region (3'UTR) of NFKBIA gene by constructing luciferase reporter gene. A patient's genomic DNA with rs8904 CC and rs696 GA genotype was used as the PCR template. Full-length 3'UTR of NFKBIA gene was amplified by different primers. After sequencing validation, these fragments were inserted to the luciferase reporter vector, pGL3-promoter to construct recombinant plasmids containing four kinds of haplotypes, pGL3-rs8904C/rs696G, pGL3-rs8904C/rs696A, pGL3-rs8904T/rs696G and pGL3-rs8904T/rs696A. Then these plasmids were transfected into LS174T cells and the luciferase activity was detected. Compared with pGL3- vector transfected cells (negative control), the luciferase activity of the four kinds of recombinant plasmids was significantly decreased (P A, the luciferase activity of the recombinant plasmids containing A allele (pGL3-rs8904C/rs696A and pGL3-rs8904T/rs696A) was about 45.1% (P P T, there were no significant differences in the luciferase activity between the recombinant plasmids containing T allele and those with C allele. Together, the luciferase reporter gene vectors containing SNPs in NFKBIA gene 3'UTR were constructed successfully and rs696G > A could decrease the luciferase activity while rs8904C > T didn't have much effect on the luciferase activity.
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Cationic lipid nanoparticles possessing favorable biocompatibility and controllable particle size has at-tracted increasing attention in the field of non-viral vectors for delivering nucleic acids in vitro and in vivo.1,2-Dilinoleyloxy-3-dimethylaminopropane(DLinDMA)and its derivatives,one of the most important cationic lipid materials,have been widely studied as the mainly composition of cationic lipid nanoparticels.This paper summa-rized the structure,characteristics and clinical application of DLinDMA nanoparticles.
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OBJECTIVE: To identify the possibility of branched polyethyleneimine (BPEI), polyamidoamine dendrimers (PAMAM) and linear polyethylenimine (LPEI) as gene vector for miRNA-15a and miRNA-16-1 gene therapy in prostate cancer. METHODS: The diameter and Zeta potential of complex were measured by Zetasizer Nano-ZS9, and its stability was also observed. The efficiency of transfection in vitro was detected by flow cytometer and the positively transfected cells were detected by fluorescence microscope. The inhibition of complex on LNCaP cell was determined by CCK8 assay. The expression of protein BCL-2, CCDN1 and Wnt3a were detected by Western blot method. RESULTS: BPEI, PAMAM and LPEI with miRNA formed nano-sized particles. The transfection rate of BPEI/miRNA-CY3 was significantly higher than that of PAMAM/miRNA-CY3 and LPEI/miRNA-CY3 at N/P ratio (P < 0.01). The protein level of BCL-2, CCDN1 and Wnt3a in BPEI, PAMAM and LPEI were all lower than the control group in LNCaP cells. CONCLUSION: With good stability and transfection rate, BPEI/miRNA, PAMAM/miRNA and LPEI/miRNA can be a promising nano-vector of miRNA transfer system. The complex can inhibit the expression of BCL-2, CCDN1 and Wnt3a gene specifically and efficiently, which may be used for prostate cancer treament.
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Objective To construct a gene delivery carrier with aptamer-polyethylene glycol-dendrimer-polyamidoamine (APT-PEG-PAMAM) ,forming nanoparticles to specifically target prostate cancer cell lines ,carrying prostate cancer cell pro-liferative suppressor microRNA :miRNA-34a .We investigated the transfection efficiency of this gene delivery system as well as functionally studied its inhibitory effect on prostate cancer (PCa) cell proliferation .Methods The construction of APT-PEG-PAMAM gene carrier was identified and confirmed by nuclear magnetic resonance (NMR) .The nano-complex sizes and zeta potential of APT-PEG-PAMAM gene carrier complexes were measured by zeta sizer .The efficiency of gene transfection of APT-PEG-PAMAM /miRNA nano-complexes were investigated by measuring the expression miRNA-34a in prostate cancer cells (PC3 and LNCaP);the PCa specific cell proliferation inhibition of APT-PEG-PAMAM / miRNA-34a nano-complexes were investigated by measuring CCK-8 cell proliferation inhibition experiments by comparing with APT-PEG-PAMAM and APT-PEG-PAMAM /miRNA-34a nano-complexes .Results NMR results demonstrated that APT-PEG-PAMAM /miRNA-34a nano-complexes were successfully synthesized by structural identification .Qualitative and quantitative transfection efficien-cy experiments data show that the cellular uptake of vectors were concentration-dependent ,after the APT further modified it significantly and increased the LNCaP cell transfection efficiency and specificity of PCa cells targeting ability .CCK8 cell prolif-eration assay data indicated that APT-PEG-PAMAM/miRNA-34a has the anti-PCa cells effect .Conclusion APT-PEG-PAM-AM/miRNA-34a may prove to see its efficacy for near future in pre-clinical and clinical study on the treatment of PCa .
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OBJECTIVE: To synthesize nonviral gene carrier poly(L-glutamic acid)-graft-polypropylenimine(MP-g-PPI) and investigate the capability of the polymers to condense DNA and the stability of MP-g-PPI DNA complexes.
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OBJECTIVE: To synthesize a water-soluble cationic polymer of quatemized chitosan modified by folic-acid for active targeting and to study its role as a gene vector. METHODS: Folic-acid was connected to quatemized chitosan by bi-functional PEG, CSTM-g-PEG-FA/pDNA nanoparticles were prepared by self-assembly, and the gene transfection efficiency mediated by the nanoparticles into 293T cells in vitro was observed. RESULTS: When weight ratio of CSTM-g-PEG-FA/pDNA was 5 or greater than 5, pDNA was completely wrapped. And the transfection efficiency of CSTM-g-PEG-FA/pDNA nanoparticles was significantly higher than CSTM/pDNA nanoparticles. CONCLUSION: Cationic polymer CSTM-g-PEG-FA can electrostatically bind and condense anionic DNA into nanometersized particles and has the function of active targeting.
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ObjectiveThe aim of the present study was to investigate the incorporation of plasmid DNA (pDNA) onto a coronary stent by chemo-immuno-conjugation for achieving site-specific gene delivery.MethodsA gene eluting stent was fabricated by reacting with polyallylamine bisphosphonate (PAA-BP) to introduce amine reactive groups on the surface.Then an anti-DNA antibody was chemically coupled and pDNA was immunologically tethered on the stent surface.Radioactive-labeled antibody was used to evaluate binding capacity and stability.ResultsThe presence of amine groups on the modified stent surface was confirmed by XPS and AFM analysis.The isotope label assay indicated that the amount of antibody chemically linked on the stents was 15-fold higher than that of the control stent and its retention time was also significantly longer.ConclusionThe results suggested that a large amount of reactive amine groups were introduced on the PAA-BP modified 316L coronary stent surface.This study provide a potential metal surface modification method that could facilitate coupling and tethering of biological molecules such as anti-DNA antibody and plasmid DNA (pDNA) to achieve sustained and highly localized gene delivery for substrate-mediated gene transfection.
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OBJECTIVE: Aim to construct a novel polymer gene delivery system based on a new kind of dendrimer-polyamidoamine (PAMAM). With the modification of polyethylene glycol (PEG) and YPSMA-1 monoclonal antibody (mAb), PAMAM-PEG-mAb was successfully synthesized as a novel gene vector targeting to the prostatic cancer. METHODS: NMR was used to characterize PAMAM-PEG-mAb. The cellular uptake and prostatic cancer (PCa) distribution experiments were employed to explore its biological characteristics and PCa cell (PC3 and LNCaP) targeting ability. RESULTS: NMR results demonstrated the successful synthesis of PAMAM-PEG-mAb. The cellular uptake of vectors was concentration-dependent. The gene expression in vitro indicated that the modification of mAb could increase the gene expression efficiency and PCa targeting ability of PAMAM vectors to LNCaP ( PSMA overexpressing prostate cancer cells). CONCLUSION: PAMAM-PEG-mAb is a potential gene delivery vector targeting to PCa. Copyright 2012 by the Chinese Pharmaceutical Association.
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Objective To investigate the impact of chitosan and alkylated chitosan DNA nanoparticles on the function of human naive CD4+T cells.Methods The secretion of cytokines (IL-4 and TNF-γ) was observed after the co-incubation of human naive CD4+T cells with nanoparticles 12 h,24 h and 48 h,respectively.ResultsNone of the nanoparticles induced the production of cytokines ( IL-4 and TNF-γ ).Conclusion Chitosan and alkylated chitosan DNA nanoparticles will not induce the differentiation of human naive CD4+ T cells into T1 or T2 and may be considered as a safe gene carrier.
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Objective:To explore the relationship between TK gene expression regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells.Method:The reformed reconstructed enhanced vector, pGL3-basic-EGFP-TK-hTRETp-CMV enhancer, and hTERT mono-promoter vector, pGL3-basic-EGFP-TK-hTRETp(as controls), were transfected into telomerase(+) nasopharyngeal carcinoma 5-8F cell lines,telomerase(+) human breast cancer MCF-7 cell lines and telomerase(-) normal vascular endothelium cell lines respectively. TK gene green fluorescent protein was observed by fluorescence microscope. The expression of TK gene mRNA was measured by the real-time fluorescent quantified PCR and the telomerase activity was determined by the method of TRAP argentation in maligment tumour cells pre- and post-transfected by enhanced vector . Meanwhile the relationship beteewn TK and telomerase was analyzed.Result:①A strong TK gene fluorescent show and TK mRNA expression were displayed after the enhanced suicide gene vector was transfected into nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which were more stronger than those of mono-promoter group,pGL3-basic-EGFP-TK-hTRETp,and ECV cells transfected by enhanced suicide gene vector. Meanwhile,real-time fluorescent quantified PCR showed that the A value of enhanced vector group was higher than that of controls. ②Telomerase activity after transfection of enhanced vector and GCV was lower than those before by the method of TRAP argentation in nasopharyngeal carcinoma cell lines,but no change in normal control cells after transfection of enhanced vector and GCV.③ After adding GCV, the obvious inhibitory effect of tumour cells growth induced by pGL3-basic-EGFP-TK-hTRETp-CMV enhancer were observed in nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which was higher than those of mono-promoter, pGL3-basic-EGFP-TK-hTRETp,pGL3-basic-EGFP3 and blank controls, but without inhibitory effect in ECV cells transfected by enhanced vector. Conclusion:TK gene expression is regulated by hTERT promoter and CMV enhancer, and then the telomerase activity is reduced and the cancer cells are specifically killed.But it is unclear how the telomerase are down-regulated by TK gene.
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Cyclodextrin(CD) is gradually applied in the nonviral gene vector system,due to its biocompatibility and flexibility of tailing via structural modification,polymerization or supramolecular combination.The ideas and research progress of the CD,its low molecular derivatives,CD polymers and CD supramolecular combination in the field of norviral gene vectros were reviewed,and their "structure-safety-transfection efficiency" relationships were discussed.