1.Enhancing transfection efficiency of polyethylenimine by a hydrophobic peptide from bee venom.
Ying-Li WANG ; Yang-Pei ZHANG ; Shou-Ping JI
Journal of Experimental Hematology 2007;15(6):1266-1269
The study was aimed to investigate the possibility of enhancing transfection efficiency of branched polyethylenimine (BPEI) in HeLa cells by hydrophobic tail of bee venom peptide (melittin). Hydrophobic tail of melittin was synthesized and its membrane permeable activity was evaluated by hemolysis test. The peptide was mixed with BPEI and the transfection efficiency was determined in HeLa cells by using green fluorescent protein gene (GFP) as a reporter gene. The cytotoxicity of the mixture was analyzed by MTT assay at 24 hours after transfection. The results indicated that the synthesized peptide had permeable activity leading to hemolysis in both neutral and acidic solution. At optimal condition, the peptide could significantly improve the transfection efficiency of BPEI and the cytotoxicity of the mixture was lower than BPEI itself. It is concluded that hydrophobic tail of melittin may be a potential enhancer to improve transfection efficiency mediated by cationic polymers in difficult to transfect cells.
HeLa Cells
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Humans
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Hydrophobic and Hydrophilic Interactions
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Melitten
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chemistry
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genetics
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Peptides
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chemistry
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Polyethyleneimine
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pharmacology
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Transfection
2.Peptide MC10 mediated PEI-beta-CyD as a gene delivery vector targeting to Her-2 receptor.
Jun LIU ; Yi-Ping HU ; Qi-Ying JIANG ; Dan CHEN ; Hai YU ; Qing-Qing WANG ; Gu-Ping TANG
Journal of Zhejiang University. Medical sciences 2009;38(1):7-14
OBJECTIVETo develop a novel non-viral gene delivery vector based on polyethylenimine and beta-cyclodextrin targeting to Her-2 receptor (MC10-PEI-beta-CyD).
METHODSThe PEI-beta-CyD was synthesized by low molecular weight polyethylenimine (PEI, Mw 600) cross-linked beta-cyclodextrin (beta-CyD) via N, N-carbonyldiimidazole (CDI). The chemical linker[N-succinimidy-3-(2-pyridyldithio) propionate, SPDP] was used to bind peptide MC10 (MARAKEGGGC) to PEI-beta-CyD to form the vector MC10-PEI-beta-CyD. The (1)H-NMR was used to confirm the structure of vector. The DNA condensing ability,and the particle size of MC10-PEI-beta-CyD/DNA complexes were demonstrated by gel retardation assay and electron microscope observation (TEM). Cell viability was tested by MTT assay. The transfection efficiency was determined on cultured SKOV-3, A549 and MCF-7 cells.
RESULTMC10 was linked onto PEI-beta-CyD successfully. The vector was able to condense DNA at N/P ratio of 5 and particle size was about (170 +/-35)nm. The vector showed low cytotoxicity and high transfection efficiency in cultured SKOV-3, A549 and MCF-7 cells.
CONCLUSIONA novel non-viral vector MC10-PEI-beta-CyD with low cytotoxicity and high transfection efficiency has been successfully synthesized.
Cell Line ; Gene Targeting ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Peptides ; chemistry ; Polyethyleneimine ; chemistry ; pharmacology ; Receptor, ErbB-2 ; genetics ; beta-Cyclodextrins ; chemistry
3.Angiogenic activity of alginate-graft-PEI/pVEGF complexes in vivo.
Zhonghui HUANG ; Wei TENG ; Ying CHEN ; Qinmei WANG
Chinese Journal of Biotechnology 2013;29(12):1817-1827
To study the angiogenic activity of amphoteric brush-type copolymer complex of alginate-graft-PEI/pVEGF (Alg-g-PEI/pVEGF) in vivo, we evaluated the toxicity of Alg-g-PEI/pVEGF complexes to rMSCs and zebra fish first. Then, we used gel retardation assay to investigate the protection of complex to pDNA against DNase I, serum and heparin. For in vivo study, we evaluated the angiogenic activity of Alg-g-PEI/pVEGF complexes by using CAM and zebra fish as animal models, PEI 25K/pVEGF and saline as positive and negative controls. Our results show that Alg-g-PEI protected pVEGF from enzymolysis and displacement of heparin in some degree, and its complexes with pVEGF were less toxic to rMSCs and zebra fish. Alg-g-PEI/pVEGF complexes induced significant angiogenesis, which was dosage-dependent. In CAM, when the dosage of pVEGF was 2.4 microg/CAM, Alg-g-PEI group achieved the maximum of angiogenesis, and the area ratio of vessel to the total surface was 44.04%, which is higher than PEI 25K group (35.90%) and saline group (24.03%) (**P < 0.01). In zebra fish, the angiogenesis increased with the increase of N/P ratios of Alg-g-PEI/pVEGF complexes in our studied range; when N/P ratio was 110, the optimal angiogenesis was obtained with vessel length of 1.11 mm and area of 1.70 x 10(3) pixels, which is higher than saline group (0.69 mm and 0.94 x 10(3) pixels) (**P < 0.01) and PEI 25k group (0.82 mm and 1.11 x 10(3) pixels) (**P < 0.01). Our results demonstratethat Alg-g-PEI/pVEGF significantly induces angiogenesis in CAM and zebra fish, and has a great potential in therapeutic angiogenesis.
Alginates
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chemistry
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Angiogenesis Inducing Agents
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pharmacology
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Animals
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Chick Embryo
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Drug Carriers
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chemistry
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Genetic Vectors
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genetics
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Glucuronic Acid
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chemistry
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Hexuronic Acids
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chemistry
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Mesenchymal Stromal Cells
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cytology
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drug effects
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Polyethyleneimine
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chemistry
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Polymers
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pharmacology
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toxicity
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Vascular Endothelial Growth Factor A
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chemistry
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Zebrafish
4.Intratumoral Injection of (188)Re labeled Cationic Polyethylenimine Conjugates: A Preliminary Report.
Eun Mi KIM ; Hwan Jeong JEONG ; Young Jun HEO ; Hyung Bae MOON ; Hee Seung BOM ; Chang Guhn KIM
Journal of Korean Medical Science 2004;19(5):647-651
(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.
Animals
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Burkitt Lymphoma/pathology/*radiotherapy
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Cations
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Female
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Injections, Intralesional
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Mice
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Mice, Inbred BALB C
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Pilot Projects
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Polyethyleneimine/chemistry/*pharmacology
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Radioisotopes/chemistry/*pharmacology
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Research Support, Non-U.S. Gov't
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Rhenium/chemistry/*pharmacology
5.Transfection with a novel cationic gene carrier: PEI-PBLG.
Ji-Zheng WEI ; Lei LIN ; Wei XIONG ; Qing-Yu ZHU
Chinese Journal of Biotechnology 2007;23(2):229-234
This study mainly deals with cell transfection and cytotoxicity for PEI(10kD)-PBLG, a novel cationic copolymer, to observe its potential as a gene carrier. Size measurement and SEM were used to show the modality of the PEI-PBLG/pDNA complexes. Cytotoxicity of PEI (10kD)-PBLG was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and compared with PEI(25kD)-PBLG, PEI(10kD), and PEI(25kD). Furthermore, pEGFP that can express the enhanced green fluorescent protein was chosen as a reporter to observe the transfection efficiency directly. Then, PEI (10kD)-PBLG/pEGFP complexes were transfected into several cell lines, such as Hela, COS-7, Vero-E6, and ECV-304, and effects of the transfection conditions were evaluated. The efficiencies were measured by FACS. Size measurement of complex particles indicated that PEI-PBLG/pDNA tended to form smaller nanoparticles compared with PEI/pDNA. The representative size of the PEI(10kD)-PBLG/pDNA complex was approximately 100 - 200 nm. SEM images showed that the particles were condense and compact. This can be suitable for their entry into cells. Cytotoxicity studies suggested that PEI (10kD)-PBLG had considerably lower toxicity than the other three materials. In the transfection tests, PEI (10kD)-PBLG/pDNA complexes could be transfected into all the cell lines that were tested. These provided the highest level of EGFP expression (45.02%) in Hela cells, which was considerably higher than that of PEI(10kD)/pEGFP (29.16%). Being less affected by the serum during transfection, PEI-PBLG/pDNA complexes offered greater biocompatibility than PEI. PEI-PBLG copolymer reduces the cytotoxicity of PEI, improves the transfection efficiency, and offers greater biocompatibility than PEI. It shows considerable potential as an efficient nonviral carrier for gene delivery.
Animals
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COS Cells
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Cell Line
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Cell Survival
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drug effects
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Cercopithecus aethiops
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DNA
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chemistry
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genetics
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ultrastructure
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Flow Cytometry
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Green Fluorescent Proteins
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genetics
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metabolism
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HeLa Cells
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Humans
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Microscopy, Electron, Scanning
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Microscopy, Fluorescence
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Molecular Weight
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Plasmids
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chemistry
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genetics
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Polyethyleneimine
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analogs & derivatives
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chemistry
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pharmacology
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Polyglutamic Acid
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analogs & derivatives
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chemistry
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pharmacology
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Transfection
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methods
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Vero Cells
6.Receptor-mediated gene delivery using polyethylenimine (PEI) coupled with polypeptides targeting FGF receptors on cells surface.
Da LI ; Qing-qing WANG ; Gu-ping TANG ; Hong-liang HUANG ; Fen-ping SHEN ; Jing-zhong LI ; Hai YU
Journal of Zhejiang University. Science. B 2006;7(11):906-911
OBJECTIVETo construct a novel kind of nonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability.
METHODSThe synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo.
RESULTSThe polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides.
CONCLUSIONThe synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.
Animals ; Binding Sites ; Carcinoma ; therapy ; Cell Line, Tumor ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Female ; Fibroblast Growth Factors ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; chemical synthesis ; chemistry ; pharmacology ; Humans ; In Vitro Techniques ; Ligands ; Liver Neoplasms ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Particle Size ; Peptides ; chemistry ; metabolism ; pharmacology ; Polyethyleneimine ; chemistry ; metabolism ; pharmacology ; Prostatic Neoplasms ; therapy ; Receptors, Fibroblast Growth Factor ; drug effects ; genetics ; metabolism ; Structure-Activity Relationship ; Surface Properties ; Transfection ; Transplantation, Heterologous ; Xenograft Model Antitumor Assays
7.Effect of nanosize delivery system for ASODN against hTERT on the expression of telomerase in the esophageal cancer EC9706 cells.
Jin WANG ; Zhen-zhong ZHANG ; Tian-yang ZHOU ; Yu-qiong LIU ; Hui-xiang LI
Chinese Journal of Oncology 2008;30(8):566-572
OBJECTIVETo investigate the inhibitory effect of nanoparticle-mediated antisense oligodeoxynucleotide (ASODN) of human telomerase reverse transcriptase (hTERT) on telomerase in the esophageal cancer EC9706 cells.
METHODSLine-polyethylenimine (L-PEI) was used to condense ASODN into nanoparticle and to couple NGR peptides into targeting nanoparticle, and the prepared L-PEI/ASODN complexes were transfected into the EC9706 cells. Cellular uptake of L-PEI/ASODN complexes was detected by laser confocal scanning microscopy. MTT assay was used to detect the inhibitory rate of EC9706 cell growth. The level of hTERT mRNA and its protein expression were measured by RT-PCR and immunohistochemistry, respectively. Annexin V FITC/PI double labeling was used to detect cell apoptosis. The distribution of drug in nude mice was observed by laser confocal scanning microscopy, and the growth and morphology of the tumor was examined.
RESULTSThe L-PEI-mediated ASODN uptake was enhanced. After transfection, the inhibitory rate of EC9706 cells was time-dependant and there was a significant difference between control cell group and L-PEI/ASODN group (P < 0.05). At 48 h after transfection, the level of hTERT mRNA was decreased significantly compared with that of control cell group (P < 0.05), and the expression of hTERT protein was negative. There was apparent apoptosis in EC9706 cells after transfection with L-PEI/ASODN complexes. For the two NGR/L-PEI/ASODN groups, fluorescence was observed in the liver, kidney, lung and tumor tissues of nude mice, and their uptake intensity was time-dependent. The mean volume of tumors in the two NGR/L-PEI/ASODN groups was significantly smaller than those in blank control group and SODN group (P < 0.05). Apoptotic bodies were detected in the tumors of L-PEI/ASODN group.
CONCLUSIONThe NGR/L-PEI/ASODN nanoparticles can effectively reach into the human esophageal cancer xenograft and inhibit the tumor growth in nude mice, and this may provide a theoretical and experimental basis for gene therapy for human esophageal squamous cell carcinoma.
Animals ; Apoptosis ; Cell Line, Tumor ; Esophageal Neoplasms ; metabolism ; pathology ; Female ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nanoparticles ; Neoplasm Transplantation ; Oligodeoxyribonucleotides, Antisense ; genetics ; pharmacology ; Oligopeptides ; chemistry ; pharmacokinetics ; Polyethyleneimine ; chemistry ; pharmacokinetics ; RNA, Messenger ; metabolism ; Telomerase ; genetics ; metabolism ; Tissue Distribution ; Transfection ; Tumor Burden