OBJECTIVETo evaluate the feasibility of using iron oxide nanoparticles as gene vector and the effect of magnetic field on efficiency of transfection.

METHODSIron oxide nanoparticles were prepared by alkaline precipitation of divalent and trivalent iron chloride. The surface of iron oxide nanoparticles was modified by self-assembled poly-L-lysine to form particle complexes (IONP-PLL). Transfection was determined by delivering reporter gene, PGL2-control encoding luciferase, to different cell lines using IONP-PLL as vector. The effect of magnetic field on efficiency of transfection was determined using Nd-Fe-B permanent magnet.

RESULTSForeign gene could be delivered to various cell lines by IONP-PLL and expressed with high efficiency, but the transfection efficiency and time course varied in the different cell lines studied. Magnetic field could enhance the efficiency of transfection by 5 - 10 fold.

CONCLUSIONIONP-PLL can be used as a novel non-viral gene vector in vitro, which offers a basis for gene delivery in vivo.

Animals ; COS Cells ; Ferric Compounds ; administration & dosage ; Genetic Vectors ; Magnetics ; Polylysine ; administration & dosage ; Transfection ; methods

Alginate-(Poly-L-Lysine)-Alginate(APA) microcapsules were prepared by Electrostatic Droplet Generator(EDG) technique and the thickness of microcapsule membrane, which was composed by polyelectrolyte complex, were studied in this paper. The theoretical formula was given for the measurement of membrane thickness of APA microcapsules by element analysis of membrane and calculation. The membrane thickness was 7-10 microns by theoretical calculation. On the other hand, the thickness of membrane was measured by SEM and optical microscopy and the results were 7 microns and 12 microns, respectively. The results showed that theoretical calculation is in good accordance with experimental determoination of mermbrane thickness and the membrane thickness of APA microcapsule is about 7-10 microns. The optical microscopy is an easy way to measure membrane thickness.
Alginates ; Capsules ; Mathematical Computing ; Membranes, Artificial ; Polylysine ; analogs & derivatives ; Spectrum Analysis

During the production of ε-poly-L-lysine (ε-PL) in fed-batch fermentation, the decline of ε-PL synthesis often occurs at middle or late phase of the fermentation. To solve the problem, we adopted two strategies, namely pH shift and feeding yeast extract, to improve the productivity of ε-PL. ε-PL productivity in fermentation by pH shift and feeding yeast extract achieved 4.62 g/(L x d) and 5.16 g/(L x d), which were increased by 27.3% and 42.2% compared with the control ε-PL fed-batch fermentation, respectively. Meanwhile, ε-PL production enhanced 36.95 g/L and 41.32 g/L in 192 h with these two strategies, increased by 27.4% and 42.48% compared to the control, respectively. ε-PL production could be improved at middle or late phase of fed-batch fermentation by pH shift or feeding yeast extract.
Batch Cell Culture Techniques ; Fermentation ; Industrial Microbiology ; Nitrogen ; chemistry ; Polylysine ; biosynthesis

To enhance the production of ε-poly-L-lysine (ε-PL) by improving dissolved oxygen level of the fermentation system, different oxygen-vectors were added to broth and n-dodecane was screened as the best oxygen-vector. The best amount of n-dodecane was 0.5% (V/V) and the best time was at start of the fermentation. In a fed-batch fermentation in a 5 L bioreactor, ε-PL concentration reached a maximum of (30.8 ± 0.46) g/L and the dry cell weight obtained was (33.8 ± 0.29) g/L, increasing by 31.6% and 20.7% compared with the control group, respectively. This improvement can be related to 0.5% n-dodecane could maintain dissolved oxygen concentration > 32% of air concentration compared with 23.8% in ε-PL production phase, and the production of a main by-product, poly-L-diaminopropionic acid, fell by 31%. These results indicated that the dissolved oxygen level in the broth was improved by adding n-dodecane, which can inhibit the by-product production and improve the biosynthesis of ε-PL.
Alkanes ; chemistry ; Batch Cell Culture Techniques ; Bioreactors ; Fermentation ; Oxygen ; chemistry ; Polylysine ; biosynthesis

Using lactoferrin as the specific ligand, we developed a simplified method for preparation of molecular conjugate for gene delivery. Replacement of column chromatography and dialysis by one step centrifugal filtration (Centricon, cut off size : 30,000), resulted in the rapid purification of bovine lactoferrin/polylysine (bLf/pL) and human lactoferrin/polylysine (hLf/pL) conjugates and easy separation of unconjugated polylysine. The Lf/pL conjugates prepared by this method efficiently transferred the reporter genes, CAT and LacZ gene, to HeLa and hepatic cells. The bLf/pL and hLf/pL conjugates could transfer the reporter genes to various hepatocytes including primary mouse hepatocyte, Hepa 1-6, SK-Hep1 and Chang liver, but not to NIH 3T3 mouse fibroblast cells, indicating that the Lf/pL conjugates conferred hepatocyte-specific gene transfer. The bLf/pL and hLf/pL conjugates prepared in the present study exhibited higher transfection efficiencies for mouse and human hepatocytes than the commercially available transferrin/polylysine (Tf/pL) conjugate.
Animals ; Cats ; Chromatography ; Dialysis ; Fibroblasts ; Filtration ; Genes, Reporter ; Genes, vif* ; Hepatocytes* ; Humans ; Lac Operon ; Lactoferrin* ; Liver ; Mice ; Polylysine ; Transfection

PURPOSE: To assess the signal enhancement by gadolinium-DTPA-polylysine (Gd-polylysine) as compared to gadopentetate (Gd-DTPA) in MR imaging of heart that have undergone ischemia-reperfusion, and to estimate the extent of myocardial damage covered bythe MR signal enhancement. MATERIALS AND METHODS: A series of contrast enhanced cardiac MR images were obtained from 17 cats subjected to a 90 minutes of occlusion of the left anterior descending coronary artery (LAD) followed by a 90 minutes of raperfusion. Time courses of changes in the signal intensity (Sl) of the ischemic area were measu red in Gd-polylysine group (8 cats) and Gd- DTPA group (9 cats). The size of U R signal enhanced area was then compared to the sizes of infarction and the area at risk revealed byTTC histochemical staining. RESULTS: Maximum Sis were obtained at 60 minutes and 30 minutes after injection of the contrast material, respectively for Gd-polylysine group and Gd-DTPA group. Signal enhancement was stronger and persistent for a longer period in Gd-polylysine group than in GD-DTPA group. Sizes of the enhanced area, the infarction, and the area at risk were about 30%, 15%, and 50% of the total left ventricle (LV) area; the difference between the groups was statistically insignificant. CONCLUSION: Gd-polylysine can be used better for a blood pool marker than Gd-DTPA in MR imaging of myocardial ischemia, due to its strong and persistent signal enhancement. The MR signal enhanced area includes both the infarcted area and a portion of the area at risk.
Animals ; Cats* ; Coronary Vessels ; Gadolinium DTPA ; Gadolinium* ; Heart ; Heart Ventricles ; Infarction ; Magnetic Resonance Imaging* ; Myocardial Ischemia ; Pentetic Acid ; Polylysine*

OBJECTIVETo establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for therapeutic usage in hepatocyte transplantation and bioartificial liver support systems for the treatment of acute and chronic liver diseases,and for experimental usage as an in vitro model of the liver.

METHODSAdult hepatocytes from 20 human donors undergoing partial hepatectomy were isolated using a two-step extracoporeal collagenase perfusion technique.Seven preincubation time points (2h,6h,12h,24h,36h,48h and 72h) were selected for optimization.After pre-incubation at 4 degrees C for 12-24h in HepatoZYME-SFM (the optimal condition),hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules,transferred to a complete medium containing 10% dimethyl sulphoxide and immediately placed into an isopropanol progressive freezing container for overnight freezing at -80 degrees C followed by immersion in liquid nitrogen the next day.During the post-thawing culture period,the cells were tested for albumin secretion,urea synthesis,cell cycling,transcription and protein synthesis (measuring mRNA and protein levels),and the morphological structure and pathology,for comparison with the features from before microencapsulated cryopreservation (PMC).

RESULTSThe viability and plating efficiency of the hepatocytes isolated using the two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0%,respectively.The pre-incubation times of 12h and 24h (viability:61.4+/-4.8% and 62.0+/-5.6%; plating efficiency:3.2+/-5.8% and 62.6+/-3.6%,respectively) showed significantly higher albumin secretion than all other time points tested (F =40.3,all P less than 0.05).Compared with the immediate cryopreservation (immediately frozen control) hepatocytes,the PMC hepatocytes showed significantly better transcription and protein synthesis and higher albumin secretion and urea levels.The PMC group did not show a significantly different level of albumin production from the directly cultured hepatocytes (culture day 2:ll9.2ng/ml vs.131.36ng/ml,P =0.051; day 3:110ng/ml vs.120.4ng/ml,P=0.063; day 4:98.2ng/ml vs.109.8ng/ml,P more than 0.05).However,over culturing days 2,3 and 4,comparison of the PMC hepatocytes to the immediate cryopreservation hepatoeytes showed the former to have significantly higher secretion of albumin (119.2ng/ml vs.101.2ng/ml,110.0ng/ml vs.87.6ng/ml and 98.2ng/ml vs.73.8ng/ml; all P less than 0.05) and urea level (7.83 mug/ml vs.6.79 mug/ml,6.83 mug/ml vs.5.89 mug/ml and 5.85 mug/ml vs.4.83 mug/ml; all P less than 0.05).The post-thawed PMC hepatoeytes showed preservation of the morphological structure,while the immediate cryopreservation hepatocytes did not.

CONCLUSIONThe two-step extracorporeal collagenase perfusion technique after partial hepatectomy is a novel,simple,and reliable method for hepatocyte isolation.Pre-incubation at 4 degrees C for 12-24h before the microencapsulation cryopreservation allows for efficient recovery of functional and morphological integrity after thawing and provides viable hepatoeytes that may be useful for clinical applications in pharmacotoxicology,bioartificial liver therapy and cell therapy in humans.

Albumins ; Alginates ; Capsules ; Cell Cycle ; Cell Survival ; Cryopreservation ; Dimethyl Sulfoxide ; Hepatectomy ; Hepatocytes ; cytology ; Humans ; Perfusion ; Polylysine ; analogs & derivatives

We have constructed the immune microsphere against tumor necrosis factor-alpha (TNF-alpha) prospectively, hoping to establish the experiment groundwork in more researches which could be used in specific elimination of the TNF-alpha by blood purification method for the future. The recombinant human tumor necrosis factor-alpha monoclonal antibody (rHTNF-alpha McAb) was wrapped on the polystyrene microsphere (PSM) carrier connecting poly-L-lysine (PLL) beforehand. They were earmarked by the fluorescein isothiocyanate (FITC) respectively. The packing conditions were examined using the inversted and fluorescence microscopes and the spectrophotometer. The results showed that the best conditions for wrapping were 20 degrees C, pH9.5 and 60 minutes. The PLL content was not changed in the washing fluid after coating, which indicated the wrapping was quite firm. At the same temperature and same coating time, the rHTNF-alpha McAb coated on the PLL was obviously substantial when the concentration of glutaraldehyde solution was 0.2%. The findings demonstrated that the built immune microsphers can be used as a novel adsorption material. This method is simple and economic, and it offers a new approach to the related studies.
Antibodies, Monoclonal ; immunology ; Fluorescein ; chemistry ; Humans ; Microspheres ; Polylysine ; chemistry ; Polystyrenes ; chemistry ; Recombinant Proteins ; immunology ; Tumor Necrosis Factor-alpha ; immunology

GH3 cells are derived from rat pituitary tumor cells that secrete prolactin and growth hormone, and important in studying prolactin-secreting pitutary tumors. This study was performed to examine the effects of polylysine on growth and differentiation of GH3 cells by means of (a) cell attachment assay (b) cell count and bromodeoxyuridine labeling and (c) immunohistochemistry for prolactin in the absence or presence of epidermal growth factor (EGF). Cell shape, attachment to the culture surface and growth of GH3 cells were not affected by polylysine coating. The percentages of prolactin-immunoreactive cells were higher in the cells cultured on the polylysine-coated surface compared to those on the plastic surface. Cell number and BrdU incorporation were lower in the EGF-treated cells on both culture surfaces. The results provided basic information on the effects of polylysine coating on GH3 cells in culture and suggested that polylysine coating was useful for the study on GH3 cells because it enhanced cell differentiation as well as it provided stronger attachment than plastic surfaces.
Animals ; Bromodeoxyuridine ; Cell Count ; Cell Differentiation ; Cell Shape ; Epidermal Growth Factor ; Growth Hormone ; Immunohistochemistry ; Pituitary Neoplasms* ; Plastics ; Polylysine* ; Prolactin ; Rats*

Several polymers were used to delivery genes to diabetic animals. Polyaminobutyl glycolic acid was utilized to deliver IL-10 plasmid DNA to prevent autoimmune insulitis of non-obese diabetic (NOD) mouse. Polyethylene glycol grafted polylysine was combined with antisense glutamic acid decarboxylase (GAD) MRNA to represent GAD autoantigene expression. GLP1 and TSTA (SP-EX4) were delivered by bioreducible polymer to stop diabetic progression. Fas siRNA delivery was carried out to treat diabetic NOD mice animal.
Animals ; Antigens, Neoplasm ; DNA ; Glutamate Decarboxylase ; Glycolates ; Histocompatibility Antigens ; Interleukin-10 ; Mice ; Mice, Inbred NOD ; Plasmids ; Polyethylene Glycols ; Polylysine ; Polymers ; RNA, Messenger ; RNA, Small Interfering ; Transplants