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A combination of cationic liposome/green fluorescence protein (GFP)-p53 complexes and a chemotherapeutic drug,cisplatin, was evaluated for therapeutic activity in human carcinoma cells. Cationic liposome/GFP-p53 complexes andcationic liposome/PEI (polyethylenimine) 0.8k/GFP-p53 complexes were synthesized and evaluated in HeLa and in A549cells for uptake and cytotoxicity, alone and in combination with cisplatin. The particle size of cationic liposome/GFP-p53complexes and cationic liposome/PEI 0.8K/GFP-p53 complexes was 318 ± 18 to 754 ± 108 nm, and zeta potentials were-15.7±2.8–+27±08 mV. The GFP expression on the delivery by cationic liposome/pEGFP (enhanced green fluorescenceprotein) complexes and cationic liposome/PEI 0.8K/pEGFP complexes was demonstrated. Treatment of the cells with eithercationic liposome/GFP-p53 complexes or cationic liposome/PEI 0.8K/GFP-p53 complexes inhibited the cell growth. Onpost treatment with cisplatin, the growth inhibition of the complexes was further increased in HeLa cells and significantlyincreased in A549 cells on the lipid-to-DNA ratio. This study concluded that the sensitivity to the cancer cells to cisplatinwas dependent on the cell line and the ratio of cationic liposome and GFP-p53. GFP-p53 expression delivered by cationicliposome/GFP-p53 complexes would be useful to increase the effect of cisplatin on the treatment of cancer cells.
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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 investigate the biological function of SPA-PEI conjugate(staphylococcal protein A-polyethyleneimine cross-linker),which is one key component for construction of a novel antibody-targeted DNA delivery system.Methods The binding capacity of SPA-PEI conjugate with multiple sources of IgG was determined by enzyme-linked immunosorbent assay and neutralization inhibition assay.The binding capacity of SPA-PEI conjugate with DNA fragment was determined by DNA gel retardation assay,and its DNA condensing ability was measured by Ethidium bromide exclusion assay.Results SPA-PEI conjugate could bind well to many species-derived IgGs.SPA-PEI conjugate had no significant effect on the binding properties of SPA.SPAPEI conjugate could neutralize negative charges of the plasmid DNA or DzTi.Its DNA condensing ability was nearly same to that of free PEI,which suggested a excellent DNA condensing ability of the SPA-PEI conjugate.Conclusion SPA-PEI cross-linkers prepared by this project group maintained the biological activity of SPA and PEI.SPA-PEI cross-linkers could be used for the construction of a novel antibody-targeted non-virus DNA delivery system.
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Objective To select polyethylenimine polymer with proper molecular weight to enhance the gene expression efficiency of Adenovirus (Ad).Methods Ad was resepcitively complexed with PEI-600,PEI-1 800 and PEI 25 000 by electrostatic adsorption.Afterwards,MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was used to investigate the cytotoxicity of the complexes with a serial concentration,followed by cell transduction assay to select the best amount of PEI with fixed dose of Ad.In the further,the physicochemical characteristics of the optimized complexes,Zeta potential,and size distribution were studied.Results MTT showed the higher the PEI moderate molecular weight was,the greater the cytotoxity was.PEI-25 000 showed a greater cytotoxicity than that of PEI-600 and PEI-1 800.As compared with PEI-600 and PEI-25 000,PEI-1 800 significanlty promoted expression of Ad gene in A549 cells.PEI turned the Zeta potential of Ad from negative to positive charge.Conclusion In the three kinds of polymer/adenovirus compounds,the cytotoxicity of the compound is positively correlated with the polymer molecular weight and concentration.The greater molecular weight the polymer as well as the higher the concentration are,the higher the cytotoxicity is.
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Objective:To transfect the non-viral vector polyethylenimine (PEI)mediated miR-2861 mimic into the MC3T3-E1 cell line,and to explore the transfection efficiency of PEI/miR-2861 complex and its effects on the proliferation and osteogenesis differentiation in pre-osteoblasts. Methods:The proper amount of PEI was blended with miR-2861 mimic and negative control (NC)separately in a ratio of N∶P=10∶1,and they were divided into experiment group and NC group. The NC/PEI complex acted as the NC group was used to eliminate the interference of osteogenesis from the addition of double-stranded RNA mimic.MTT assay was used to determine the optimal concentration of PEI/miR-2861 mimic complex.The fluorescence imaging technique and bulge-loop RT-PCR were used to detect the transfection efficiency and mRNA expression of miRNA-2861 in the cells with different concentrations (10,30, 50,and 100 nmol · L-1 ), separately.The osteogenesis ability of MC3T3 cells was identified with RT-PCR and Alizarin red staining with the selected concentration of PEI/miR-2861 by transient transfection.Results:Compared with blank control group,the proliferation rates of MC3T3 cells in 100 nmol·L-1 PEI/miR-2861 group was decreased significantly at 72 h (P < 0. 05 ). With the increasing of transfected concentration the transfection efficiency of miRNA/PEI complex was increased gradually.The results of Alizarin red staining and quantitative analysis showed that calcium deposits were more and bigger in experiment group after induced for 21 d,while both in blank control group and NC group they were less.Conclusion:The miRNA-2861 mimic can be effectively transfected into the MC3T3-E1 cell line and expresses with a high level,which is mediated by PEI as the gene vector.miR-2861 mimic has a certain ability of promoting osteogenesis differentiation of MC3T3-E1 cells.
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ABSTRACT Lentiviral vector-mediated gene transfer offers several advantages over other gene delivery vectors when considering gene and cell therapy applications. However, using these therapies in clinical applications involves large-scale vector production in an efficient and cost-effective manner. Here we describe a high yield production of a lentivirus encoding recombinant factor VIII in a scalable and GMP-compliant culture system, based on serum free suspension cultures and transient transfection with an inexpensive reagent, polyethylenimine (PEI), reaching a total viral yield of 2.48x108 particles.
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Objective To compare the transfection efficiency of cationic polyethylenimine(PEI)with Lipofectamine 2000TM by using the plasmid DNA encoding vascular endothelial cell growth factor (VEGF165 )gene in human embryonic kidney cell line 293T.Methods PEI of different N/P ratio and Lipofectamine 2000TM were used to deliver the vector containing VEGF165 to 293T cells,respectively.Green fluorescent protein(GFP)gene was inserted into the vector as a report gene.Evaluation of cytoactive was performed by CCK-8 assay 24 h after transfection.The cells were observed by fluorescent microscope and the presence of VEGF165 in cell supernatant was detected by ELISA 48 h after transfection.The transfection efficiency was calculated and com-pared.Results Similar cytoactive and best transfection efficiency could be obtained when N/P ratio was 9,the transfection efficien-cy was around 70%.Furthermore,the presence of VEGF165 increased significantly after transfection(P <0.05),but there was no significant difference between the two groups in which different transfection methods were adopted.Conclusion PEI as a novel oli-gofectamine reagent could mediate more efficient transfection compared with lipofectamine.It also has low cell-toxicity and low price and could be an ideal vector in gene delivery technology.
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Objective To synthesize miRNA carrier PEG-b-PLL and to testify the stability , encapsulation efficiency and cyto-toxicity of its complexes .Methods H1 NMR was used to determine the degree of polymerization of PLL , 4%agarose gel electrophore-sis was used to determine entrapment of the polyer to the miRNA;then dynamic light scattering ( DLS) was used to measure the hydro-dynamic parameter such as size , polydispersity index ( PDI) and zeta potential of the polyplexes .The entrapment efficiency was deter-mined by ultraviolet-visible spectrophotometer , and finally , the cyto-toxicity of PEG-b-PLL was evaluated by CKK-8 kit with K562 cell lines.Results The characteristics indicated polyplexes prepared by PEG-b-PLL and miRNA fulfill the demand of being the gene carri-er of miRNA because of low cyto-toxicity , high encapsulation efficiency and stability .Conclusion The miRNA carrier PEG-b-PLL had good character and low cyto-toxicity.It showed considerable potential as an efficient miRNA carrier .
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Objective: To observe polyethylenimine-mediated BMP-7 gene transfection in promoting fracture healing in elderly rats. Methods: Male SD rats (20-month-old) were randomly divided into 3 groups: group A received BMP-7 gene therapy mediated by polyehtylenimine, group B was treated with normal saline and group C was treated with BMP-7 plasmid without polyethylenimine. Right femoral shaft fracture model was established in all rats. Animals in group A received transdermal injection of pcDNA3. 1-BMP-7/PET; those in group B and C received the same volume of normal saline and pcDNA3. 1-BMP-7, respectively. X-ray photography, histological observation (H-E staining), immunohistochemical staining of collagen type I, biomechanical and bone mineral density test were employed to assess the healing of fracture 2, 4, and 8 weeks after treatment. Results: The results of X ray and H-E staining showed no fracture healing in the 3 groups during the 8th week after fracture; however, the growth of coloboma in group A was better than that of the other 2 groups, with partial bone union between the fracture ends. Staining of collagen type I showed deeper, wider staining in group A compared with the other 2 group. Anti-bending intensity and bone mineral density tests showed that the parameters in group A were better than those of the other 2 groups (P<0.05 or 0.01). Conclusion: Polyethylenimine-mediated hBMP-7 gene transfer can effectively promote fracture healing in elderly rats.
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In our previous study,we identified a novel testis-specific expressed gene 2(TSEG-2)from mouse testis.To further investigate its functions,35 male Balb/c mice(8 weeks old)were divided into cryptorchidism group(n=20),sham group(n=10),and control group(n=5).In cryptorchidism group,the right testes were anchored to the inner lateral abdominal wall.In situ hybridization(ISH)was applied to measure the localization of TSEG-2 in mouse testis.Real-time quantitative PCR was performed to detect the expression of TSEG-2 gene.Meanwhile,under the mediation of polyethylenimine(PEI),the recombinant vector pEGFP-TSEG-2(n=5)or empty vector(mock,n=5)was transfected into the testis of male mice.The transfection efficiencies were measured under a fluorescence microscope.The apoptosis of spermatogenic cells was detected by terminal deoxynuleotidyl-mediated nick end labeling(TUNEL).The results showed that TSEG-2 was expressed in convoluted seminiferous tubules,more precisely,in spermatogonia and spermatocytes.As compared with sham and control groups,the TSEG-2 transcription was significantly enhanced(P<0.05)and was correlated with apoptosis of spermatogenic cells in cryptorchid testes(P<0.05).PEI was efficient in mediating transfection of TSEG-2 into seminiferous tubules of testis.One week post-transfection,intratesticular injection of TSEG-2 resulted in increased apoptosis of spermatogenic cells in vivo (P<0.05).These results indicate that TSEG-2 may participate in the apoptosis of spermatogenic cells and the pathogenesis of cryptorchidism.
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Using a micro-column packed with polyethylenimine aminated glycidyl methacrylatE-grafted-polytetrafluoroethylene fiber made by ourselves as the adsorption material, the adsorption behaviors of Pb and Cd under different conditions were studied by inductively coupled plasma optical emission spectrometry. The conditions for the preconcentration of Pb and Cd such as pH, flow-rate, time and eluent acidity were optimized. The results show that the PTFE-g-GMA-PEI Fiber has excellent adsorption property and high adsorption capacity for Pb2+ and Cd2+. The Enrichment factors for Pb2+ and Cd2+ were 30 and 80, respectively and the detection limits were 3.5 μg/L and 0.15 μg/L, respectively, the relative standard derivations were 1.5% and 0.6%(n=9)respectively. When the single ion concentration was 50 μg/L, the sampling frequency was 9 and 18 times/h, respectively. The method was applied to the simultaneous determination of trace lead and cadmium of several Chinese traditional medicine samples, the standard recovery of samples was 90%-108%.
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Summary: The aim of present study was to evaluate the feasibility and efficiency of enhanced green fluorescent protein (EGFP) gene delivery to myocardium in vivo by ultrasound targeted microbubble destruction (UTMD) and polyethylenimine (PEI). SonoVue/DNA and PEI/DNA/SonoVue complexes were prepared. Gel electrophoresis analysis was performed to determine the structural integrity of plasmid DNA or PEI/DNA after UTMD. Solutions of plasmid DNA, SonoVue/DNA, PEI/DNA complexes or PEI/DNA/SonoVue complexes were respectively transduced into BALB/c mice hearts by means of transthoracic ultrasound irradiation. Mice undergoing PBS injection, plasmid injection or PEI/DNA complexes injection without ultrasound irradiation served as controls. Gene expression in myocardium was detected 4 days after treatment. Cryosections and histological examinations were conducted. Electrophoresis gel assay showed no damage to DNA or PEI/DNA complexes after UTMD. When the heart was not exposed to ultrasound, the expression of EGFP was observed in the subendocardial myocardium obviously. The strongest expression was detected in the anterior wall of the left ventricle when the heart was exposed to ultrasound alone. Injection of PEI/DNA complexes and UTMD resulted in the highest transfection efficiency and the distributional difference of EGFP was not obvious. No tissue damage was seen histologically. In conclusion, a combination of UTMD and PEI was highly effective in transfecting mice hearts without causing any apparently adverse effect. It provides an alternative to current clinical gene therapy and opens a new concept of non-viral gene delivery for the treatment of cardiac disease.
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Aim To avoid the limitation of the use of cationic polyethlenimine (PEI)-complexed plasmid DNA use for in vitro or in vivo gene delivery due to its cytotoxicity and lower efficiency in the presence of serum. Methods A polyplex with decreased positive charge on the complex surface was designed. The PEI/DNA (PD) complexes coated with an anionic biodegradable polymer, alginate were prepared and their gene delivery behavior with PD was compared. Results The alginate-coated PD polyplex, where alginate: PEI: DNA [alginate: DNA, 0. 15 (w/w); PEI: DNA, N: P = 10] showed about 10 -30 fold-increased transfection efficiency compared to corresponding non-coated complexes to C3 cells in the presence of 50% serum. The surface charge of the alginate-coated complex was approximately half of that of the alginate-lacking complex. The size of alginate-coated complex was slightly smaller than that of the corresponding complex without alginate. The former complex also showed a reduced erythrocyte aggregation activity and decreased cytotoxicities to C3 cells in comparison with PD complex. Conclusion The alginate-coated PD polyplexes as a new gene delivery system can improve transfection efficiency in high serum concentration with low cytotoxicity to C3 cells.
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(188)Re(Rhenium) is easily obtained from an in-house (188)W/(188)Re generator that is similar to the current (99)Mo/(99m)Tc generator, making it very convenient for clinical use. This characteristic makes this radionuclide a promising candidate as a therapeutic agent. Polyethylenimine (PEI) is a cationic polymer and has been used as a gene delivery vector. Positively charged materials interact with cellular blood components, vascular endothelium, and plasma proteins. In this study, the authors investigated whether intratumoral injection of (188)Re labeled transferrin (Tf)-PEI conjugates exert the effect of radionuclide therapy against the tumor cells. When the diameters of the Ramos lymphoma (human Burkitt's lymphoma) xenografted tumors reached approximately 1 cm, 3 kinds of (188)Re bound compounds (HYNIC-PEI-Tf, HYNIC-PEI, (188)Re perrhenate) were injected directly into the tumors. There were increases in the retention of (188)Re inside the tumor when PEI was incorporated with (188)Re compared to the use of free 188Re. The (188)Re HYNIC-Tf-PEI showed the most retention inside the tumor (retention rate=approximately 97%). H&E stain of isolated tumor tissues showed that (188)Re labeled HYNIC-PEI-Tf caused extensive tumor necrosis. These results support (188)Re HYNIC-PEI-Tf as being a useful radiopharmaceutical agent to treat tumors when delievered by intratumoral injection.
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Animals , Female , Mice , Burkitt Lymphoma/pathology , Cations , Injections, Intralesional , Mice, Inbred BALB C , Pilot Projects , Polyethyleneimine/chemistry , Radioisotopes/chemistry , Rhenium/chemistryABSTRACT
0.01). The tumor volume and the tumor weight were also significantly decreased in the treated group (P
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Objective To establish an efficient, simple, low cytotoxicity and cheap transfaction system. Methods We have used the cationic polymer polyethylenimine(PEI) to study transient transfection in MCF-7 cells by testing different conditions, including cell concentrations, DNA concentrations, the ratio of PEI nitrogen to DNA phosphate(PEI-N∶DNA-P) and the time of cells grown in serum-free culture together with PEI-DNA complex. Results The optimized cell concentrations were 2?10 5 cells seeded per well in 24-well dishes 18-24?h before transfection. The DNA concentrations and ratio of PEI-N∶DNA-P are very important for optimal transfection and they affect each other. For 1??g DNA per well, the optimal PEI-N∶DNA-P is about 33∶1, however, as for 4??g DNA, it is 9∶1. The best time of cells grown in serum-free culture together with PEI-DNA complex is about 5-7?h.Conclusion With optimized conditions, we can establish an efficient, simple, low cytotoxicity and cheap transfection system by using PEI.