Transfection efficiency boost by designer multicomponent lipoplexes

Cationic liposome-DNA complexes (lipoplexes) have emerged as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. We investigated the correlation between structural evolution of multicomponent lipoplexes when interacting with cellular lipids, the extent of DNA release and the efficiency in transfecting mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines. We show, for the first time, that the transfection pattern increases monotonically with the number of lipid components and further demonstrate by means of synchrotron small angle X- ray scattering (SAXS) that structural changes of lipoplexes induced by cellular lipids correlate with the transfection efficiency. Specifically, inefficient lipoplexes either fused too rapidly upon interaction with anionic lipids or, alternatively, are found to be extremely resistant to solubilization. The most efficient lipoplex formulations exhibited an intermediate behaviour. The extent of DNA unbinding (measured by electrophoresis on agarose gel) correlates with structural evolution of the lipoplexes but DNA-release does not scale with the extent of transfection. The general meaning of our results is of broad interest in the field of non-viral gene delivery: rational adjusting of lipoplex composition to generate the proper interaction between lipoplexes and cellular lipids may be the most appropriate strategy in optimizing synthetic lipid transfection agents.     

Transfection efficiency boost by designer multicomponent lipoplexes

Cationic liposome-DNA complexes (lipoplexes) have emerged as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. We investigated the correlation between structural evolution of multicomponent lipoplexes when interacting with cellular lipids, the extent of DNA release and the efficiency in transfecting mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines. We show, for the first time, that the transfection pattern increases monotonically with the number of lipid components and further demonstrate by means of synchrotron small angle X- ray scattering (SAXS) that structural changes of lipoplexes induced by cellular lipids correlate with the transfection efficiency. Specifically, inefficient lipoplexes either fused too rapidly upon interaction with anionic lipids or, alternatively, are found to be extremely resistant to solubilization. The most efficient lipoplex formulations exhibited an intermediate behaviour. The extent of DNA unbinding (measured by electrophoresis on agarose gel) correlates with structural evolution of the lipoplexes but DNA-release does not scale with the extent of transfection. The general meaning of our results is of broad interest in the field of non-viral gene delivery: rational adjusting of lipoplex composition to generate the proper interaction between lipoplexes and cellular lipids may be the most appropriate strategy in optimizing synthetic lipid transfection agents.     

Transfection efficiency of lipoplexes for site-directed delivery

Targeted gene delivery is a promising strategy to cure disease on its basic level at the site of interest. The ultrastructure, internalization, and transfection efficiency of lipoplexes was investigated. We found that at a charge ratio (ρ) of 4.0 lipoplexes had optimum characteristics for gene delivery in vitro. To decrease the size of lipoplexes, we used a method of continuous-flow microfluidics. PEGylation of lipoplexes did not hinder internalization, but was found to hamper transfection. To discriminate between uptake and transfection efficiency of lipoplexes, we used fluorescence-based approaches: microscopy and FACS. To this end, GFP plasmid was labeled with Alexa 594, and, in parallel experiments, GFP plasmid was combined with rhodamine-labeled lipid. Our studies confirm that cellular uptake does not imply transfection efficiency, and that hurdles in cellular processing have to be taken before targeted gene delivery becomes an established therapeutic option.     

Transfection with fluorinated lipoplexes based on fluorinated analogues of DOTMA, DMRIE and DPPES

Fluorinated double-chain (poly)cationic lipids (one or both of these chains being ended by a highly fluorinated tail) which are close analogues of DOTMA, DMRIE or DPPES were designed as synthetic vectors for gene delivery. For N/P ratios (N=number of amine functions of the lipid; P=number of DNA phosphates) from 0.8 to 5, these fluorinated cationic lipids condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. No specific cell toxicity was evidenced for these new fluorinated lipoplexes. The efficiency of some of the fluorinated lipoplexes to transfect lung epithelial A549 cells was comparable to that of the first generation of fluorinated lipoplexes made from fluorinated analogues of DOGS (Transfectam) [Bioconjug. Chem. 12 (2001) 114]. These results, combined with the higher in vivo transfection potential found for fluorinated lipoplexes than for conventional lipoplexes or PEI polyplexes [J. Gene Med. 3 (2001) 109], confirm that fluorinated lipoplexes are very promising gene transfer systems.     

Transfection with fluorinated lipoplexes based on fluorinated analogues of DOTMA,DMRIE and DPPES

Fluorinated double-chain (poly)cationic lipids (one or both of these chains being ended by a highly fluorinated tail) which are close analogues of DOTMA, DMRIE or DPPES were designed as synthetic vectors for gene delivery. For N/P ratios (N=number of amine functions of the lipid; P=number of DNA phosphates) from 0.8 to 5, these fluorinated cationic lipids condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. No specific cell toxicity was evidenced for these new fluorinated lipoplexes. The efficiency of some of the fluorinated lipoplexes to transfect lung epithelial A549 cells was comparable to that of the first generation of fluorinated lipoplexes made from fluorinated analogues of DOGS (Transfectam) [Bioconjug. Chem. 12 (2001) 114]. These results, combined with the higher in vivo transfection potential found for fluorinated lipoplexes than for conventional lipoplexes or PEI polyplexes [J. Gene Med. 3 (2001) 109], confirm that fluorinated lipoplexes are very promising gene transfer systems.     

Translocation boost protein-folding efficiency of double-barreled chaperonins

Incorrect folding of proteins in living cells may lead to malfunctioning of the cell machinery. To prevent such cellular disasters from happening, all cells contain molecular chaperones that assist nonnative proteins in folding into the correct native structure. One of the most studied chaperone complexes is the GroEL-GroES complex. The GroEL part has a "double-barrel" structure, which consists of two cylindrical chambers joined at the bottom in a symmetrical fashion. The hydrophobic rim of one of the GroEL chambers captures nonnative proteins. The GroES part acts as a lid that temporarily closes the filled chamber during the folding process. Several capture-folding-release cycles are required before the nonnative protein reaches its native state. Here we report molecular simulations that suggest that translocation of the nonnative protein through the equatorial plane of the complex boosts the efficiency of the chaperonin action. If the target protein is correctly folded after translocation, it is released. However, if it is still nonnative, it is likely to remain trapped in the second chamber, which then closes to start a reverse translocation process. This shuttling back and forth continues until the protein is correctly folded. Our model provides a natural explanation for the prevalence of double-barreled chaperonins. Moreover, we argue that internal folding is both more efficient and safer than a scenario where partially refolded proteins escape from the complex before being recaptured.     

Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency.

Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was 相似文献   

Transfection with fluorinated lipoplexes based on new fluorinated cationic lipids and in the presence of a bile salt surfactant

The synthesis of two fluorinated cationic lipids, which are analogues of frequently used synthetic gene carrier agents (including the cationic 2,3-dioleoyloxy-N-[2-(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium (DOSPA) component of the commercially available liposomal Lipofectamine), and the disintegration and DNA accessibility (evaluated by the ethidium bromide (BET) intercalation assay) as well as the in vitro transfection efficacy of cationic lipoplexes formulated with these new lipids in conjunction with conventional or fluorinated helper lipids, in the absence or presence of sodium taurocholate (STC), a powerful anionic bile salt detergent, is reported. A higher stability, with respect to the STC lytic activity and DNA accessibility, of the fluorinated cationic lipoplexes as compared with their respective lipofectamine-based ones was demonstrated. Indeed, while the Lipofectamine lipoplexes were fully disintegrated at a [STC]/[lipid] molar ratio of 2000, only 40-60% of the DNA intercalation sites of the lipoplexes based on the fluorinated analogue of DOSPA were accessible to ethidium bromide. A higher transfection potential in the presence of STC was further found for the lipoplexes formulated with the fluorinated analogue of DOSPA as compared with the Lipofectamine preparation. For a STC concentration of 7.5 mM, lipofection mediated with these fluorinated lipoplexes was significantly higher (nearly 30- to 50-fold, p < 0.05) than with the Lipofectamine ones. These results confirm the remarkable transfection potential of fluorinated lipoplexes.     

Small-molecule compounds boost genome-editing efficiency of cytosine base editor

Cytosine base editor (CBE) enables targeted C-to-T conversions at single base-pair resolution and thus has potential therapeutic applications in humans. However, the low efficiency of the system limits practical use of this approach. We reported a high-throughput human cells-based reporter system that can be harnessed for quickly measuring editing activity of CBE. Screening of 1813 small-molecule compounds resulted in the identification of Ricolinostat (an HDAC6 inhibitor) that can enhance the efficiency of BE3 in human cells (2.45- to 9.21-fold improvement). Nexturastat A, another HDAC6 inhibitor, could also increase BE3-mediated gene editing by 2.18- to 9.95-fold. Ricolinostat and Nexturastat A also boost base editing activity of the other CBE variants (BE4max, YE1-BE4max, evoAPOBEC1-BE4max and SpRY-CBE4max, up to 8.32-fold). Meanwhile, combined application of BE3 and Ricolinostat led to >3-fold higher efficiency of correcting a pathogenic mutation in ABCA4 gene related to Stargardt disease in human cells. Moreover, we demonstrated that our strategy could be applied for efficient generation of mouse models through direct zygote injection and base editing in primary human T cells. Our study provides a new strategy to improve the activity and specificity of CBE in human cells. Ricolinostat and Nexturastat A augment the effectiveness and applicability of CBE.     

Transfection of embryonal carcinoma cells at high efficiency using liposome-mediated transfection

Embryonal carcinoma (EC) cells are recognized as an excellent model system for studying the early stages of mammalian development. Many studies performed with EC cells involve transient transfection with promoter/reporter gene constructs and/or mammalian expression vectors. One of the limitations of working with EC cells is their inability to be transfected at high efficiency. In most cases, EC cells are transfected using the calcium phosphate method. The objective of this study was to identify protocols and culture conditions that significantly increase the transfection efficiency of EC cells. F9 EC cells were used for this purpose, because they are the EC cell line studied most commonly. We show that the transfection efficiency of F9 EC cells using the calcium phosphate method is less than 5%; whereas, their transfection efficiency can be improved approximately 15-fold using optimized culture conditions and liposome-based transfection reagents. Specifically, we demonstrate that more than 50% of F9 EC cells can be transfected using LipofectAMINE 2000. In addition to higher levels of transfection, there is much less plate-to-plate variation with liposome-based reagents as compared to transfection with calcium phosphate. Interestingly, transfection efficiency using these reagents was found to be inversely related to cell density. This contrasts sharply with the recommendation that transfection with LipofectAMINE 2000 or LipofectAMINE in conjunction with the PLUS reagent be performed at high cell densities. Given the improvements in transfection efficiency reported here, it will now be possible to perform studies with F9 EC cells that require transfection at significantly higher levels than that achieved using the calcium phosphate method. Overall, the highest transfection efficiencies were consistently obtained using LipofectAMINE 2000.     

Biophysical characterization of anionic lipoplexes

Transfection efficiency of liposomal gene delivery vectors depends on an optimal balance in the electro-chemical and structural properties of the transfection-capable complexes. We have recently reported a novel anionic lipoplex DNA delivery system composed of a ternary complex of endogenous occurring non-toxic anionic lipids, physiological Ca²⁺ cations, and plasmid DNA encoding a gene of interest with high transfection efficiency and low toxicity. In this work, we investigate the electro-chemical and structural properties anionic lipoplexes and compare them with those of Ca²⁺-DNA complexes. Biophysical characterization is used to explain the transfection efficiency of anionic lipoplexes in mammalian CHO-K1 cells. Circular dichroism and fluorescence spectroscopy showed that the plasmid DNA underwent conformational transition from native B-DNA to Z-DNA due to compaction and condensation upon Ca²⁺-mediated complexation with anionic liposomes. Zeta potential measurements and gel electrophoresis studies demonstrated that Ca²⁺ interaction with plasmid DNA during the formation of lipoplexes also led to increased association of supercoiled plasmid DNA with the lipoplexes, leading to charge neutralization which is expected to facilitate transfection. However, even 10-fold higher concentrations of Ca²⁺ alone (in the absence of the anionic liposomes) were unable to induce these changes in plasmid DNA molecules. A model explaining the possible mechanism of anionic lipoplex formation and the correlation of high transfection efficiency to biophysical properties was proposed. These studies confirm the utility of biophysical studies to identify optimal formulation conditions to design efficient liposomal gene delivery vectors.     

Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency.

Clinical applications of gene therapy mainly depend on the development of efficient gene transfer vectors. Large DNA molecules can only be transfected into cells by using synthetic vectors such as cationic lipids and polymers. The present investigation was therefore designed to explore the physicochemical properties of cationic lipid-DNA particles, with plasmids ranging from 900 to 52 500 bp. The colloidal stability of the lipoplexes formed by complexing lipopolyamine micelles with plasmid DNA of various lengths, depending on the charge ratio, resulted in the formation of three domains, respectively corresponding to negatively, neutrally and positively charged lipoplexes. Lipoplex morphology and structure were determined by the physicochemical characteristics of the DNA and of the cationic lipid. Thus, the lamellar spacing of the structure was determined by the cationic lipid and its spherical morphology by the DNA. The main result of this study was that the morphological and structural features of the lipopolyamine-DNA complexes did not depend on plasmid DNA length. On the other hand, their gene transfer capacity was affected by the size of plasmid DNA molecules which were sandwiched between the lipid bilayers. The most effective lipopolyamine-DNA complexes for gene transfer were those containing the shortest plasmid DNA.     

Transfection of oral cancer cells mediated by transferrin-associated lipoplexes: mechanisms of cell death induced by herpes simplex virus thymidine kinase/ganciclovir therapy

The Herpes Simplex Virus thymidine kinase (HSV-tk) suicide gene/ganciclovir (GCV) approach has been used for the treatment of a variety of cancers. The purpose of the present study was to evaluate the cytotoxic effect of ganciclovir in oral squamous cancer cells, previously transfected with HSV-tk gene delivered by transferrin-associated complexes (Tf-lipoplexes), as well as to investigate the mechanisms involved in the bystander effect and in the process of cell death. The delivery of HSV-tk gene to the oral cancer cells, HSC-3 and SCC-7, mediated by Tf-lipoplexes followed by ganciclovir treatment resulted in essentially 100% cytotoxicity, the observed toxic effect being dependent both on GCV dose and incubation time. Cell death was shown to occur mainly by an apoptotic process. Different experimental approaches demonstrated that the observed cytotoxicity was mainly due to diffusion of the toxic agent into neighbouring, non-transfected cells, via gap junctions. Preliminary in vivo studies in a murine model for oral squamous cell carcinoma have shown a significant inhibition of tumor growth upon injection of Tf-lipoplexes carrying HSV-tk followed by intraperitonal injection of GCV, as compared to controls.     

Transfection of oral cancer cells mediated by transferrin-associated lipoplexes: Mechanisms of cell death induced by herpes simplex virus thymidine kinase/ganciclovir therapy

The Herpes Simplex Virus thymidine kinase (HSV-tk) suicide gene/ganciclovir (GCV) approach has been used for the treatment of a variety of cancers. The purpose of the present study was to evaluate the cytotoxic effect of ganciclovir in oral squamous cancer cells, previously transfected with HSV-tk gene delivered by transferrin-associated complexes (Tf-lipoplexes), as well as to investigate the mechanisms involved in the bystander effect and in the process of cell death. The delivery of HSV-tk gene to the oral cancer cells, HSC-3 and SCC-7, mediated by Tf-lipoplexes followed by ganciclovir treatment resulted in essentially 100% cytotoxicity, the observed toxic effect being dependent both on GCV dose and incubation time. Cell death was shown to occur mainly by an apoptotic process. Different experimental approaches demonstrated that the observed cytotoxicity was mainly due to diffusion of the toxic agent into neighbouring, non-transfected cells, via gap junctions. Preliminary in vivo studies in a murine model for oral squamous cell carcinoma have shown a significant inhibition of tumor growth upon injection of Tf-lipoplexes carrying HSV-tk followed by intraperitonal injection of GCV, as compared to controls.     

Transfection efficiency and cytotoxicity of polyethyleneimine-coated magnetic iron oxide nanoparticles in rooster sperm cells

Since the morphology of the rooster spermatozoa is different to other animal spermatozoa, the aim of the current study was to investigate the transfection efficiency and cytotoxicity of polyethyleneimine (PEI) coated magnetic iron oxide nanoparticles (MION) on these cells. Liposome/nucleic acid (NA) complexes and PEI-coated MION linked to the labeled oligonucleotides were used. Viability and percentage of exogenous nucleic acid uptake of spermatozoa were measured by flow cytometry analyses. The results showed a significant increase in exogenous nucleic acid uptake by rooster spermatozoa (P < 0.001) when treated with MION-NA complexes in comparison to liposome/NA. There were no significant differences between efficiency of lipid-based transfection agent and MION (P > 0.05) during short incubation period. MION with or without magnetic field, did not show significant cytotoxicity while the lipid-based transfection agent significantly decreased (P < 0.05) the viability of rooster spermatozoa. Results of this study showed that magnetofection and lipofection were both effective methods which increased exogenous nucleic acid uptake by rooster spermatozoa. However, the magnetofection method was more successful in maintaining the cell's survival than lipofection method.     

Complex formation of cholesterol-containing polymers in water solutions

The dynamic light scattering method was used to study the process of complex formation of cholesterol-containing polymers of vinylsaccharide 2-deoxy-2-methacrylamido-D-glucose (double and ternary copolymers) in water solutions in the different polymer mole ratio. Compared to the initial ternary cholesterol-containing copolymer the conditions of compaction of the complex formed are found.     

Lateral diffusion of saturated phosphatidylcholines in cholesterol-containing bilayers

A pulsed field gradient NMR was used to study lateral diffusion in the cholesterol-containing oriented bilayers of saturated (dipalmitoyl- and dimyristoyl-) phosphatidylcholines, upon their limiting hydration. Similar dependences of lateral diffusion coefficients on temperature and cholesterol concentration were observed, which agree with phase diagram showing the presence of the regions of disordered and ordered liquid-crystalline phases and a two-phase region. Under the same conditions, the lateral diffusion coefficient of dipalmitoylphosphatidylcholine is lower, which agrees qualitatively with its larger molecular weight. The comparison of data for dipalmitoylphosphatidylcholine with previous results for dipalmitoylsphingomyelin-cholesterol bilayers under the same conditions, in spite of similarity of phase diagrams, shows large (two–three times) differences in the lateral diffusion coefficient and a different profile of its dependence on cholesterol concentration. The comparison of data for dimyristoylphosphatidylcholine with previous results shows that the values of lateral diffusion coefficient and the shape of its dependence on cholesterol concentration coincide at high concentrations (>15 mol%) but differ at lower concentrations The revealed disagreement may be caused by the fact that the measurements were carried out at different water content in the system. At limiting hydration (more than 35% of water), the lateral diffusion coefficient for lipids decreases when cholesterol concentration rises, while at water content about 25% (as a result of equilibrium hydration from vapors) the lateral diffusion coefficient of phosphatidylcholine may be independent of cholesterol concentration. This is the consequence of the denser packing of molecules in the bilayer at reduced water content, an effect that competes with the ordering effect of cholesterol.     

Synthesis of cholesterol-containing cationic amphiphiles with heterocyclic bases]

A number of cationic derivatives of cholesterol containing polar residues of N-methylimidazole, pyridine, N-methylmorpholine, and 4-N,N-dimethyaminopyridine were synthesized by the interaction of the corresponding heterocyclic bases with cholesterol 5-bromopentanoate followed by the treatment with methyl iodide in the case of tertiary amines. In addition, N-(4-cholesteryloxycarbonylbutyl)piperazine was obtained for the preparation of pH-sensitive liposomes.     

Lateral diffusion of saturated phosphatidylcholines in cholesterol-containing bilayers

Lateral diffusion in oriented bilayers of saturated cholesterol-containing phosphatidylcholines, dipalmitoylphosphatidylcholine and dimyrilstoylphosphatidylcholine upon their limiting hydration has been studied by NMR with impulse gradient of magnetic field. For both systems, similar dependences of the coefficient of lateral diffusion on temperature and cholesterol concentration were observed, which agree with the phase diagram showing the presence of regions of ordered and unordered liquid-crystalline phases and a two-phase region. Under similar conditions, the coefficient of lateral diffusion for dipalmytoylphosphatidylcholine has lower values, which is in qualitative agreement with its greater molecular mass. A comparison of data for dipalmytoylphosphatidylcholine with the results obtained earlier for dipalmytoylsphyngomyelin/cholesterol under the same conditions shows, despite a similarity in phase diagrams, greater (two- to threefold) differences in the values of the coefficient of lateral diffusion and a different mode of dependence of the coefficient on cholesterol concentration. A comparison of data for dimyrilstoylphosphatidylcholine with the results obtained previously shows that the values of the coefficient of lateral diffusion and the mode of its dependence on cholesterol concentration coincide in the region of higher concentrations (more than 15 mole %) and differ in the region of lower concentrations (below 15 mole %). The discrepancies may be explained by different contents of water in the systems during the measurements. At a limiting hydration (more than 35%) of water, the coefficient of lateral diffusion decreases with increasing cholesterol concentration. If the content of water is about 25% (as a result of equilibrium hydration from vapors), the coefficient of lateral diffusion of phosphatidylcholine is probably independent of cholesterol concentration. This results from a denser packing of molecules in the bilayer at a lower water concentration, an effect that competes with the ordering effect of cholesterol.