Marika Ruponen

Interactions of polymeric and liposomal gene delivery systems with extracellular glycosaminoglycans: physicochemical and transfection studies.

Complexes of DNA with cationic lipids and cationic polymers are frequently used for gene transfer. Extracellular interactions of the complexes with anionic glycosaminoglycans (GAGs) may interfere with gene transfer. Interactions of GAGs with the carrier-DNA complexes were studied using tests for DNA relaxation (ethidium bromide intercalation), DNA release (electrophoresis), and transfection (pCMVbetaGal transfer into RAA smooth muscle cells). Several cationic lipid formulations (DOTAP, DOTAP/Chol, DOTAP/DOPE, DOTMA/DOPE, DOGS) and cationic polymers (fractured dendrimer, polyethylene imines 25 kDa and 800 kDa, polylysines 20 kDa and 200 kDa) were tested. Polycations condensed DNA more effectively than the monovalent lipids. Hyaluronic acid did not release or relax DNA in any complex, but it inhibited the transfection by some polyvalent systems (PEI, dendrimers, DOGS). Gene transfer by the other carriers was not affected by hyaluronic acid. Sulfated GAGs (heparan sulfate, chondroitin sulfates B and C) completely blocked transfection, except in the case of the liposomes with DOPE. Sulfated GAGs relaxed and released DNA from some complexes, but these events were not prerequisites for the inhibition of transfection. In conclusion, polyvalent delivery systems with endosomal buffering capacity (DOGS, PEI, dendrimer) were most sensitive to the inhibitory effects of GAGs on gene transfer, while fusogenic liposomes (with DOPE) were the most resistant systems.

Structure-activity relationships of poly(L-lysines): effects of pegylation and molecular shape on physicochemical and biological properties in gene delivery.

The influence of shape, molecular weight and pegylation of linear, grafted, dendritic and branched poly-L-lysines on their DNA delivery properties were investigated. DNA binding, condensation, complex size and morphology, cell uptake and transfection efficiency were determined. Most polylysines condense DNA, linear polymers being more efficient than most dendritic ones. At low molecular weights of PLL DNA binding and condensation were less efficient, particularly with dendrimers. Pegylation did not decrease DNA condensation of PLLs at less than 60% (fraction of M(w)) of PEG. Pegylation stabilized the complexes sterically, but did not protect them from interaction with polyanionic chondroitin sulfate. Cell uptake of polylysine/DNA complexes was high and pegylation increased the transfection efficacy. However, overall transfection level of polylysines is low possibly due to inadequate escape of the complexes from endosomes or poor release of DNA from the complexes. Physicochemical and biological structure-property relationships of poly-L-lysines were demonstrated, but no clear correlations between the tested physicochemical determinants (size of complexes, zeta-potentials, condensation of DNA and the shape of complexes) and biological activities were seen. Transfection activity may be ultimately determined by intracellular factors and/or still unknown features of DNA complexation with the carriers.

Cell-surface glycosaminoglycans inhibit cation-mediated gene transfer

Cationic polymers and liposomes are used to wrap DNA into complexes that promote its cellular uptake. The mechanisms of the uptake and the intracellular fate of these complexes are obscure, as are reasons for an unpredictable and sometimes poor efficiency of the transgene expression. Polyanionic glycosaminoglycans (GAGs) on the cell surface interact with the cationic DNA complexes and influence transfection.

Vitreous is a barrier in nonviral gene transfer by cationic lipids and polymers.

AbstractPurpose. To investigate the role of vitreous in nonviral gene delivery into retinal pigment epithelial (RPE) cells. Methods. Human RPE cell line D407 was cultured in six-well plates. Bovine vitreous, hyaluronan, or DMEM was added on the cells. Complexes of DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted onto the vitreous, hyaluronan, or DMEM. Cellular uptake of DNA was studied with ethidium monoazide DNA and gene expression with GFP-plasmid complexes. FITC-dextrans and FITC-polylysines were used to probe the effects of the size and cationic charge on permeation in the vitreous in a similar experimental setup. Fluorescent cells were analyzed by flow cytometry. Results. Vitreous decreased the cellular uptake of DNA complexes 2-30 times, and GFP expression was also impaired. In hyaluronan solutions the cellular uptake of the complexes was also decreased significantly in most cases. In vitreous, cellular uptake of all FITC-dextrans decreased slightly, and uptake of poly-L-lysines was decreased substantially, whereas in hyaluronan solutions the effects were mild or nonexistent. Conclusions. Polymeric and liposomal gene delivery is substantially limited by the vitreous. This is probably because of the size and charge of the retinal gene delivery after intravitreal injections.

Polyplex‐mediated gene transfer and cell cycle: effect of carrier on cellular uptake and intracellular kinetics, and significance of glycosaminoglycans

Here we report on studies that probe whether the intracellular kinetics of plasmid DNA (pDNA) and cell surface glycosaminoglycans (GAGs) are modified during the cell cycle in a way that can be correlated with changes in gene transfer efficiency with poly(ethyleneimine) (PEI) and poly‐L‐lysine (PLL) polyplexes.

DIFFERENT SYNERGISTIC ROLES OF SMALL POLYETHYLENIMINE AND DOSPER IN GENE DELIVERY

Low-molecular-weight PEIs and cationic liposomes can be combined resulting in a synergistic increase in transfection efficiency as we have reported earlier. Here, we have further investigated the potential mechanisms of this synergy. Complex morphology, complex sizes and DNA condensation were studied using transmission electron microscopy, light scattering methods and ethidium bromide exclusion, respectively. Cellular uptake, transfection efficiency, and effect of proton pump inhibitor bafilomycin A1 were examined in cell cultures. The cellular uptake of DNA was negligible with PEI2K-DNA complexes, whereas the uptake of the PEI2K-DNA-Dosper or the Dosper-DNA complexes was maximally about 40%. The number of transfected cells was two times higher with PEI2K-DNA-Dosper complexes than with Dosper-DNA complexes. The PEI2K-DNA-Dosper combination was slightly less sensitive to bafilomycin A1 than the PEI25K-DNA or Dosper-DNA complexes. There were no differences between PEI2K and PEI25K in DNA condensation. Dosper condensed DNA slightly more in PEI2K complexes. The PEI25K-DNA complexes were much smaller (<250 nm) than the PEI2K-DNA complexes (0.5-12 micro m) which were also rather polydisperse. It is suggested that two independent mechanisms would lead to synergistic transfection efficiency: (1) Dosper improves the cellular uptake of PEI2K-DNA complexes, and (2) PEI2K improves a transfer of the complexes from lysosomes to nucleus.

Neural retina limits the nonviral gene transfer to retinal pigment epithelium in an in vitro bovine eye model

We investigated the permeation of liposomal and polymeric gene delivery systems through neural retina into retinal pigment epithelium (RPE) and determined the roles of various factors in permeation and subsequent uptake of the delivery systems by RPE. Anterior parts and vitreous of fresh bovine eyes were removed. Retina was left intact or peeled away. Complexes of ethidium monoazide (EMA)-labeled plasmid DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted on the retina or RPE. Two hours later the neural retina was removed, if present, and the RPE cells were detached. Contaminants were removed by sucrose centrifugation, and the RPE cells were analyzed for DNA uptake by flow cytometry. Cellular uptake of FITC-dextrans (molecular weight [mw] 20 000, 500 000 and 2 000 000), FITC-poly-L-lysine (mw 20 000), FITC-labeled oligonucleotide (15-mer), and naked EMA-labeled plasmid DNA was determined after pipetting the solutions on the RPE or neural retina. Location of the fluorescent materials in the retina was visualized with fluorescence microscopy. Neural retina decreased the cellular uptake of DNA complexes by an order of magnitude, the uptake of FITC-dextrans slightly, whereas delivery of polycationic FITC-poly-L-lysine to RPE was almost completely inhibibited. Neural retina decreased the cellular uptake of FITC-oligonucleotides, while the uptake of uncomplexed plasmid was always negligible. conclusions from FACS and fluorescence microscopy were similar: delivery of polymeric and liposomal DNA complexes into RPE are limited by the neural retina. This is due to the size and positive charge of the complexes.

Intracellular DNA release and elimination correlate poorly with transgene expression after non-viral transfection

The intracellular limiting steps in non-viral gene delivery are still unclear. The purpose of this study was to quantize intracellular DNA release and elimination kinetics after transfection with various non-viral carriers (calcium phosphate precipitates, branched poly(ethyleneimine), poly-L-lysine, DOTAP, DOTAP/DOPE) and to correlate these factors with transgene expression. Intracellular kinetics of DNA was determined by novel quantitative method based on qRT-PCR and DNase treatment. Intracellular elimination of DNA after calcium phosphate transfection was rapid (half-life of 0.37 h) whereas the amount of DNA in the cells was stable for at least 136 h after poly(ethyleneimine) mediated transfection. Intracellular elimination half-lives for DNA delivered by other carrier systems ranged from 9 to 27 h. Calcium phosphate precipitates are not able to protect DNA, which explains the short elimination half-life. In the case of other carriers DNA is after complex removal mostly carrier bound but after 24 h the major fraction of DNA is in the released or loosened state. Overall, neither total nor released amount of intracellular DNA correlates with the transgene expression.

Long-Lasting Secretion of Transgene Product from Differentiated and Filter-Grown Retinal Pigment Epithelial Cells After Nonviral Gene Transfer

Purpose: The purpose of this study was to investigate the extent, duration, and direction of transgene expression after nonviral gene transfer to differentiated retinal pigment epithelial (RPE) cells. Methods: Polarized human RPE cells (ARPE-19) were transfected with nonviral vectors [DOTAP/DOPE with and without protamine sulfate (PS), DOTAP, PEI (polyethyleneimine), DHP-12] using secreted alkaline phosphatase (SEAP) as a reporter gene. Cellular uptake was studied by flow cytometry. Results: Up to 80-fold differences were observed in the peak reporter gene expression. The highest peak levels and the longest lifetime of SEAP expression (> 69 days) were obtained with DOTAP/DOPE/PS/pDNA complexes. With PEI, higher expression was seen to the apical side than to the basolateral side. Conclusions: In contrast to most differentiated epithelial cells, the differentiated RPE cells can be transfected at high and prolonged levels with selected lipoplexes.

Chondrogenic differentiation of human pluripotent stem cells in chondrocyte co-culture

Chondrogenic differentiation of human embryonic (hESCs) or induced pluripotent stem cells (hiPSCs) has been achieved in embryoid bodies (EBs) by adding selected growth factors to the medium. Also chondrocyte-secreted factors have been considered to promote the chondrogenic differentiation. Hence, we studied whether co-culture with primary chondrocytes can induce hESCs or hiPSCs to differentiate into chondrocyte lineage. Co-culture of hESCs or hiPSCs was established in a transwell insert system in feeder-free culture conditions, while hESCs or hiPSCs grown alone in the wells were used as controls. After 3-week co-culture with weekly replenished chondrocytes, the chondrogenically committed cells (hCCCs) were evaluated by morphology, immunocytochemistry, quantitative real-time RT-PCR, and analysis of chondrogenic, osteogenic and adipogenic differentiation markers. The expressions of chondrocyte- and pluripotency-associated genes were frequently measured during the monolayer expansion of hCCCs from passage 1 to 10. Human CCCs displayed morphology similar to chondrocytes, and expressed chondrocyte-associated genes, which were declined following passaging, similarly to passaged chondrocytes. They also formed a chondrogenic cell pellet, and differentiated into chondrocytic cells, which secreted abundant extracellular matrix. Human CCCs also proliferated rapidly. However, they did not show osteogenic or adipogenic differentiation capacity. Our results show that co-culture of hESCs or hiPSCs with primary chondrocytes could induce specific chondrogenic differentiation.