Healing process in scarring inevitably produces a considerable amount of non-organized dense collagen-rich matrix called scar thus impairing the native structure of skin. Connective tissue growth factor (CTGF) overexpression within healing tissues is known to play an imperative role in collagen production stimulated by transforming growth factor-beta in cutaneous wound healing. Undoubtedly, the knockdown of CTGF expression through siRNA-mediated gene silencing could simply impede the scarring process. However, the less stability and low transfection of siRNAs themselves urge a safe carrier to protect and transfect them into cells at a high rate avoiding toxicities. Here, we developed a degradable poly(sorbitol-co-PEI) (PSPEI), prepared by polymerization of sorbitol diacrylate with low molecular weight polyethylenimine, which has high transfection efficiency but low cytotoxicity, and utilized it in siCTGF delivery to silence the expression of CTGF in an animal model of cutaneous wound healing. Unlike contracted scar in normal healing, there was no or less contraction in the healed skin of mice treated with siCTGF using PSPEI. Histologically, the healed tissues also had distinct papillary structures and dense irregular connective tissues that were lacking in the control scar tissues. This study exemplifies a successful treatment of cutaneous wound healing using a polymer system coupled with RNA interference. Hence, the approach holds a great promise for developing new treatments with novel targets in regenerative medicines.
Animals ; Cicatrix* ; Collagen ; Connective Tissue ; Connective Tissue Growth Factor ; Gene Silencing ; Mice ; Models, Animal ; Molecular Weight ; Polyethyleneimine ; Polymerization ; Polymers ; Regenerative Medicine ; RNA Interference ; RNA, Small Interfering* ; Skin ; Sorbitol ; Transfection ; Wound Healing* ; Wounds and Injuries*

OBJECTIVEThis study aims to synthesize MTO1 (a kind of oligodeoxynucleotides) and N-isopropylacrylamide-modified polyethylenimines (PEN) complexes (MT01/PEN) as well as to investigate the effect of the complexes on the expression of osteoprotegerin (OPG) and the receptor activator of nuclear factor κB ligand (RANKL) in the human osteoblast-like cell line MG63.

METHODSMG63 cells were transfected by MT01/PEN complexes formed with three different mass ratios (1:2, 1:4, 1:6) of MT01 to PEN. MT01 and MT01-s were used as positive control. Enzyme-linked immunosorbent assay and real-time polymerase chain reaction were performed to estimate the amount of OPG and RANKL released into the culture media and in MG63 at 24, 48, 72 h.

RESULTSMG63 responded to the MT01/PEN complexes by significantly upregulating the OPG on the protein and mRNA levels (P < 0.05). The protein and mRNA levels of RANKL were lower in most of the groups with complexes, and the OPG/RANKL ratio were higher (P < 0.05). MG63 were affected by the MT01/PEN complexes with different mass ratios, particularly when the ratio was 1:6.

CONCLUSIONMT01 can enhance the promotion of ossification by establishing the delivery system with PEN.

Acrylamides ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Humans ; Oligodeoxyribonucleotides ; Osteoblasts ; Osteoprotegerin ; Polyethyleneimine ; RANK Ligand ; RNA, Messenger ; Real-Time Polymerase Chain Reaction

BACKGROUND AND OBJECTIVES: MicroRNA 145 is known to be responsible for cellular proliferation, and its enhanced expression reportedly inhibits the retardation of vascular smooth muscle cell growth specifically. In this study, we developed a microRNA 145 nanoparticle immobilized, hyaluronic acid (HA)-coated stent. MATERIALS AND METHODS: For the gene therapy, we used disulfide cross-linked low molecular polyethylenimine as the carrier. The microRNA 145 was labeled with YOYO-1 and the fluorescent microscopy images were obtained. The release of microRNA 145 from the stent was measured with an ultra violet spectrophotometer. The downstream targeting of the c-Myc protein and green fluorescent protein was determined by Western blotting. Finally, we deployed microRNA 145/ssPEI nanoparticles immobilized on HA-coated stents in the balloon-injured external iliac artery in a rabbit restenosis model. RESULTS: Cellular viability of the nanoparticle-immobilized surface tested using A10 vascular smooth muscle cells showed that MSN exhibited negligible cytotoxicity. In addition, microRNA 145 and downstream signaling proteins were identified by western blots with smooth muscle cell (SMC) lysates from the transfected A10 cell, as the molecular mechanism for decreased SMC proliferation that results in the inhibition of in-stent restenosis. MicroRNA 145 released from the stent suppressed the growth of the smooth muscle at the peri-stent implantation area, resulting in the prevention of restenosis at the post-implantation. We investigated the qualitative analyses of in-stent restenosis in the rabbit model using micro-computed tomography imaging and histological staining. CONCLUSION: MicroRNA 145-eluting stent mitigated in-stent restenosis efficiently with no side effects and can be considered a successful substitute to the current drug-eluting stent.
Blotting, Western ; Cell Proliferation ; Drug-Eluting Stents ; Genetic Therapy ; Hyaluronic Acid ; Iliac Artery ; MicroRNAs* ; Microscopy ; Muscle, Smooth ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle ; Nanoparticles ; Polyethyleneimine ; Stents* ; Viola

In the present study, packaging system composed of pAAV-CMV-GFP, pAAV-RC and pHelper were transfected into human embryonic kidney 293 cells (HEK293 cells) mediated by polyethyleneimine (PEI) to explore an optimal transfection condition. Different total plasmid DNA dosages (1, 2, 3, 4, 5, 6 μg) and different PEI/Plasmid ratios (1:1, 3:1, 5:1, 7:1) were tested with detection of green fluorescence protein (GFP) with ImagePro Plus6. 0 Software. Then transfection efficiency of the optimized transfection system was further observed for different time periods(12, 24, 36, 48, 60, 72 h). The results showed that total plasmid dosage of 4 μg/well with PEI/plasmid ratio of 3 : 1-5 : 1 was an efficient transfection condition. Transfection efficiency-time curve was an S-shaped curve. Transfection efficiency reached a plateau at 60 h after transfection. The optimized conditions for PEI-mediated transfection at the optimal time result in enhanced transfection efficiency of triple plasmid into HEK293 cells.
Green Fluorescent Proteins ; HEK293 Cells ; Humans ; Plasmids ; Polyethyleneimine ; Transfection ; methods

We prepared silver nanoparticles/polyethyleneimine-reduction graphene oxide (AgNP/rGO-PEI) composite materials, and evaluated their quality performance in our center. Firstly, we prepared AgNP/rGO-PEI, and then analysed its stability, antibacterial activity, and cellular toxicity by comparing the AgNP/rGO-PEI with the silver nanoparticles (PVP/AgNP) modified by polyvinylpyrrolidone. We found in the study that silver nanoparticles (AgNP) distributed relatively uniformly in AgNP/rGO-PEI surface, silver nanoparticles mass fraction was 4.5%, and particle size was 6-13 nm. In dark or in low illumination light intensity of 3 000 lx meter environment (lux) for 10 days, PVP/AgNP aggregation was more obvious, but the AgNP/rGO-PEI had good dispersibility and its aggregation was not obvious; AgNP/rGO-PEI had a more excellent antibacterial activity, biological compatibility and relatively low biological toxicity. It was concluded that AgNP/rGO-PEI composite materials had reliable quality and good performance, and would have broad application prospects in the future.
Anti-Bacterial Agents ; chemistry ; Graphite ; chemistry ; Light ; Nanoparticles ; chemistry ; Oxides ; chemistry ; Particle Size ; Polyethyleneimine ; chemistry ; Silver Compounds ; chemistry

Cell Adhesion ; Cell Proliferation ; Cells, Cultured ; Chitosan ; chemistry ; Coated Materials, Biocompatible ; Collagen ; chemistry ; Humans ; Materials Testing ; Polyethyleneimine ; chemistry ; Static Electricity ; Surface Properties ; Titanium ; chemistry

OBJECTIVETo explore the feasibility of magnetic resonance cell imaging technology by using polyethylene imine (PEI)-coated magnetic nanoparticles of Fe₄O₄ (PEI-Fe₄O₄-MNPs) to track cell biology behavior.

METHODSEndocytic PEI-Fe₄O₄-MNPs in SHI-1 cells were observed by transmission electron microscopy (TEM) . Iron contents of nano-labeled cells were analyzed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and Prussian blue staining. The proliferation ability of labeled cells was detected by cell counting kit-8 (CCK-8) assay; the differentiation and colony-forming abilities were also observed. SHI-1 cells without endocytosing PEI-Fe₄O₄-MNPs were used as control.

RESULTSOur data showed that PEI-Fe₄O₄-MNPs could label SHI-1 cells. The labeling efficiency depended on the nanoparticles' concentration and the duration of cells treating. Inhibition rates of SHI-1cells labeled by 60-100 μg Fe/ml PEI-Fe₄O₄-MNPs were much higher than of 5-50 μg Fe/ml ones following treating by 5-100 μg Fe/ml PEI-Fe₄O₄-MNPs for 48 hrs. The expressions of CD11b and CD14 were (78.4±18.5)% and (18.7±2.9)% in control vs (83.3±14.2)% and (20.4±2.1)% in cells fractions treated by 30 μg Fe/ml PEI-Fe₄O₄-MNPs. Clony-forming rates of SHI-1 cells labeled by 0, 20 , 50 μg Fe/ml PEI-Fe₄O₄-MNPs were (25.20±7.22)%, (25.93±13.15)%, (23.37±9.33)%, respectively. Differentiation and colony-forming potentials of labeled cells were similar with control in the certain range of PEI-Fe₄O₄-MNPs concentration.

CONCLUSIONSHI-1 cells were efficiently labeled by PEI-Fe₄O₄-MNPs with well biocompatibilities in proper range of concentration, the latter could be coupled with magnetic resonance imaging (MRI) to track cells in vivo.

Cell Line, Tumor ; Coated Materials, Biocompatible ; chemistry ; Ferric Compounds ; chemistry ; Humans ; Magnetic Resonance Imaging ; Magnetics ; Microscopy, Electron, Transmission ; Nanoparticles ; chemistry ; Polyethyleneimine ; chemistry

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 ; chemistry ; Angiogenesis Inducing Agents ; pharmacology ; Animals ; Chick Embryo ; Drug Carriers ; chemistry ; Genetic Vectors ; genetics ; Glucuronic Acid ; chemistry ; Hexuronic Acids ; chemistry ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Polyethyleneimine ; chemistry ; Polymers ; pharmacology ; toxicity ; Vascular Endothelial Growth Factor A ; chemistry ; Zebrafish

OBJECTIVETo synthesize a biodegradable non-viral gene carrier with a high transfection efficiency and a low cytotoxicity.

METHODSPoly(ethylene glycol)-block-(poly(L-glutamic acid)-graft-polyethylenimine) was prepared via ammonolysis of poly(ethylene glycol)-block-poly (γ-benzyl L-glutamate) with the low-molecular-mass polyethylenimine (600 Da). The synthesized copolymer was characterized by 1H nuclear magnetic resonance spectroscopy and gel permeation chromatography. The polyplex micelle from PEG-b-(PG-g-PEI) and plasmid DNA (pDNA) was studied using dynamic light scattering, zeta-potential measurements, and gel retardation assay. The in vitro cytotoxicity and transfection efficiency of PEG-b-(PG-g-PEI) were tested by MTT assay and luciferase assay in HEK 293T cells using PEI (25 kDa) as the control.

RESULTSPEG-b-(PG-g-PEI) could efficiently condense DNA into nanosized particles with positive surface charges when the N/P ratio of polymer and DNA was above 5:1. The zeta potential of the polyplexes was about 25 mV, and the particle size was 120 nm at a N/P ratio of 10. The cell toxicity and gene transfection evaluations showed a lower cytotoxicity and a higher gene transfection efficiency of the copolymer than PEI 25000 in HEK 293T cells.

CONCLUSIONSThe polymer can be used as a potential non-viral gene carrier for gene therapy.

Cell Survival ; Gene Transfer Techniques ; Genetic Vectors ; Glutamic Acid ; chemistry ; HEK293 Cells ; Humans ; Particle Size ; Plasmids ; Polyethylene Glycols ; chemical synthesis ; chemistry ; Polyethyleneimine ; analogs & derivatives ; chemical synthesis ; chemistry ; Polyglutamic Acid ; analogs & derivatives ; chemical synthesis ; chemistry ; Polymers ; Transfection

OBJECTIVETo explore the transgenic efficiency of non-viral vector Tf-PEG-PEI and the cell specific silencing effect of plasmid pPSMAe/p-shNS-ploy(A) on prostate cancer cells.

METHODSPolyethyleneimine (PEI) was modified by using polyethylene glycol and transferrin to synthesize the non-viral vector Tf-PEG-PEI. NS-specific plasmids pPSMAe/p-shNS-ploy(A) and Tf-PEG-PEI were used to transfect prostate cancer LNCap and PC-3 cells. The changes of cell morphology, proliferation ability and cell cycle were studied after down-regulating the NS gene level.

RESULTSTf-PEG-PEI was successfully modified. After transfection, the PSMA-expressing LNCaP cells became larger and showed more pseudopodia, having a tendency to differentiate. Their cell proliferation ability was reduced, and the detection of cell cycle showed a decrease of S phase and an increase of G(1) phase after knocking down NS gene. These targets were not changed in non-PSMA-expresing PC-3 cells.

CONCLUSIONSThe non-viral vector Tf-PEG-PEI has a high ability to transfer targeted gene into target cells. The cellular specificity of short-hairpin RNA transcription driven by PSMAe/p is confirmed by silencing NS gene. The use of cell specific promoter may be an effective strategy of gene therapy for prostate cancer.

Antigens, Surface ; genetics ; metabolism ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; GTP-Binding Proteins ; genetics ; metabolism ; Genetic Vectors ; Glutamate Carboxypeptidase II ; genetics ; metabolism ; Humans ; Male ; Nuclear Proteins ; genetics ; metabolism ; Plasmids ; Polyethylene Glycols ; Polyethyleneimine ; analogs & derivatives ; Promoter Regions, Genetic ; Prostatic Neoplasms ; pathology ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; Transferrin ; genetics