Role of liposome and peptide in the synergistic enhancement of transfection with a lipopolyplex vector.
ABSTRACT: Lipopolyplexes are of widespread interest for gene therapy due to their multifunctionality and high transfection efficiencies. Here we compared the biological and biophysical properties of a lipopolyplex formulation with its lipoplex and polyplex equivalents to assess the role of the lipid and peptide components in the formation and function of the lipopolyplex formulation. We show that peptide efficiently packaged plasmid DNA forming spherical, highly cationic nanocomplexes that are taken up efficiently by cells. However, transgene expression was poor, most likely due to endosomal degradation since the polyplex lacks membrane trafficking properties. In addition the strong peptide-DNA interaction may prevent plasmid release from the complex and so limit plasmid DNA availability. Lipid/DNA lipoplexes, on the other hand, produced aggregated masses that showed poorer cellular uptake than the polyplex but contrastingly greater levels of transgene expression. This may be due to the greater ability of lipoplexes relative to polyplexes to promote endosomal escape. Lipopolyplex formulations formed spherical, cationic nanocomplexes with efficient cellular uptake and significantly enhanced transfection efficiency. The lipopolyplexes combined the optimal features of lipoplexes and polyplexes showing optimal cell uptake, endosomal escape and availability of plasmid for transcription, thus explaining the synergistic increase in transfection efficiency.
Project description:Multifunctional, lipopolyplex formulations comprising a mixture of cationic liposomes and cationic, receptor-targeting peptides have potential use in gene therapy applications. Lipopolyplex formulations described here are typically far more efficient transfection agents than binary lipoplex or polyplex formulations. It has been shown previously that the peptide component mediates both DNA packaging and targeting of the nanoparticle while in this report we investigate the contribution of the lipid component. We hypothesised that the lipid components synergise with the peptides in the transfection process by promoting endosomal escape after lipid bilayer fusion. Lipopolyplexes were prepared with cationic liposomes comprising DOTAP with either neutral lipid DOPE or DOPC. DOPE promotes fusogenic, inverted hexagonal lipid structures while DOPC promotes more stable laminar structures. Lipopolyplexes containing DOPE showed substantially higher transfection efficiency than those formulated with DOPC, both in vitro and in vivo. DOPE-containing lipopolyplexes showed rapid endosomal trafficking and nuclear accumulation of DNA while DOPC-containing formulations remained within the late endo-lysosomal compartments. These findings are consistent with previous finding for the role of DOPE in lipoplexes and support the hypothesis regarding the function of the lipid components in lipopolyplexes. These findings will help to inform future lipopolyplex design, strategies and clinical development processes.
Project description:BACKGROUND: Effective gene transfection without serum deprivation is a prerequisite for successful stem cell-based gene therapy. Polyethylenimine (PEI) is an efficient nonviral gene vector, but its application has been hindered by serum sensitivity and severe cytotoxicity. METHODS: To solve this problem, a new family of lipopolyplexes was developed by coating PEI/DNA polyplexes with three serum-resistant cationic lipids, namely, lysinylated, histidylated, and arginylated cholesterol. The physical properties, transfection efficiency, cellular uptake, subcellular distribution, and cytotoxicity of the lipopolyplexes was investigated. RESULTS: The outer coat composed of lysinylated or histidylated cholesterol remarkably improved the transfection efficiency of the polyplex with a low PEI/DNA ratio of 2 in the presence of serum. The resulting lysinylated and histidylated cholesterol lipopolyplexes were even more efficient than the best performing polyplex with a high PEI/DNA ratio of 10. Results from cellular uptake and subcellular distribution studies suggest that their higher transfection efficiency may result from accelerated DNA nuclear localization. The superiority of the lipopolyplexes over the best performing polyplex was also confirmed by delivering the therapeutic gene, hVEGF(165). Equally importantly, the lipid coating removed the necessity of introducing excess free PEI chains into the transfection solution for higher efficiency, generating lipopolyplexes with no signs of cytotoxicity. CONCLUSION: Noncovalent modification of polyplexes with lysinylated and histidylated cholesterol lipids can simultaneously improve efficiency and reduce the toxicity of gene delivery under serum conditions, showing great promise for genetic modification of bone marrow stem cells.
Project description:Complexes combining nucleic acids with lipids and polymers (lipopolyplexes) show great promise for gene therapy since they enable compositional, physical and functional versatility to be optimized for therapeutic efficiency. When developing lipopolyplexes for gene delivery, one of the first evaluations performed is an in vitro transfection efficiency experiment. Many different in vitro models can be used, and the effect of the model on the experiment outcome has not been thoroughly studied. The objective of this work was to compare the insights obtained from three different in vitro models, as well as the potential limitations associated with each of them. We have prepared a series of lipopolyplex formulations with three different cationic polymers (poly-l-lysine, bioreducible poly-l-lysine and polyethyleneimine), and assessed their in vitro biological performance in 2D monolayer cell culture, 3D spheroid culture and microdroplet-based single-cell culture. Lipopolyplexes from different polymers presented varying degrees of transfection efficiency in all models. The best-performing formulation in 2D culture was the polyethyleneimine lipopolyplex, while lipoplexes prepared with bioreducible poly-l-lysine were the only ones achieving any transfection in microdroplet-enabled cell culture. None of the prepared formulations achieved significant gene transfection in 3D culture. All of the prepared formulations were well tolerated by cells in 2D culture, while at least one formulation (poly-l-lysine polyplex) delayed 3D spheroid growth. These results highlight the need for selecting the appropriate in vitro model depending on the intended application.
Project description:The design, synthesis and formulation of non-viral gene delivery vectors is an area of renewed research interest. Amongst the most efficient non-viral gene delivery systems are lipopolyplexes, in which cationic peptides are co-formulated with plasmid DNA and lipids. One advantage of lipopolyplex vectors is that they have the potential to be targeted to specific cell types by attaching peptide targeting ligands on the surface, thus increasing both the transfection efficiency and selectivity for disease targets such as cancer cells. In this paper, we have investigated two different modes of displaying cell-specific peptide targeting ligands at the surface of lipopolyplexes. Lipopolyplexes formulated with bimodal peptides, with both receptor binding and DNA condensing sequences, were compared with lipopolyplexes with the peptide targeting ligand directly conjugated to one of the lipids. Three EGFR targeting peptide sequences were studied, together with a range of lipid formulations and maleimide lipid structures. The biophysical properties of the lipopolyplexes and their transfection efficiencies in a basal-like breast cancer cell line were investigated using plasmid DNA bearing genes for the expression of firefly luciferase and green fluorescent protein. Fluorescence quenching experiments were also used to probe the macromolecular organisation of the peptide and pDNA components of the lipopolyplexes. We demonstrated that both approaches to lipopolyplex targeting give reasonable transfection efficiencies, and the transfection efficiency of each lipopolyplex formulation is highly dependent on the sequence of the targeting peptide. To achieve maximum therapeutic efficiency, different peptide targeting sequences and lipopolyplex architectures should be investigated for each target cell type.
Project description:Brain capillary endothelial cells of the blood-brain barrier (BBB) are difficult targets for nonviral transfection even for the most potent transfection agents. Efficient protection and nuclear delivery of plasmid DNA are the key requirements for enhancing the transfection. We designed novel DNA intercalating conjugates of PEG-tris(acridine) with a short nuclear localization signal (NLS) peptide and investigated the effect of their complexes with luciferase-encoded plasmid DNA on lipoplex- and polyplex-mediated transfection of murine brain capillary endothelial bEnd.3 cells. These intercalation complexes protected DNA from nucleolytic degradation forming a protective PEG layer around plasmid DNA and could be efficiently condensed by Lipofectamine2000 or Exgen500 into nanosized particles. Complexation of plasmid DNA with a PEG-acridine/NLS-PEG-acridine mixture (9:1 w/w), taken in an amount equal to 5-6 NLS peptides per DNA molecule, significantly enhanced both lipo- and polyplex transfection efficacies and increased the number of transfected bEnd.3 endothelial cells in the presence of serum. Comparative transgene expression efficiency was significantly higher at longer PEG linker and optimal conjugate-to-DNA weight ratio, especially, at lower N/P ratio for both transfection agents, reaching 15-16-fold for lipoplexes and 10-11-fold for polyplexes. In addition, the NLS-PEG-acridine conjugates did not increase cytotoxicity of lipoplexes and polyplexes to bEnd.3 cells. These conjugates can serve as promising components for development of systemic nonviral transfecting approach to the transfection of the BBB and temporary modulation of its drug permeability.
Project description:Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of complex factors such as pH-induced changes in polymer characteristics (e.g., proton buffering capacity and ionization), polyplex characteristics (e.g., size, surface charge, and decomplexation), and cellular characteristics (e.g., cellular uptake, cell cycle phases, and intracellular pH environment). Notably, acidic medium delayed endocytosis, endosomal acidification, cytosolic release, and decomplexation of polyplexes, thereby negatively affecting gene expression. However, acidic medium inhibited mitosis and reduced dilution of gene expression, resulting in increased transfection efficiency. Compared to pH 7.4 medium, acidic transfection medium reduced gene expression 1.6-7.7-fold whereas acidic culture medium enhanced transfection efficiency 2.1-2.6-fold. Polymeric transfection was affected more by the culture medium than by the transfection medium. Understanding the effects of extracellular pH during polymeric transfection may stimulate new strategies for determining effective and safe polymeric gene carriers.
Project description:In regard to gene vectors for cancer gene therapy, their percolation into the tumor tissue should be essential for successful outcome. Here, we studied the tumor penetrability of nonviral vectors (polyplexes) after incubation with the multicellular tumor spheroid (MCTS) models and intratumoral (i.t.) injection into subcutaneous tumors. As a result, polyethylene glycolated (PEGylated), core-shell type polyplexes (polyplex micelles) showed facilitated percolation and improved transfection inside the tumor tissue, whereas conventional polyplexes from cationic polymers exhibited limited percolation and localized transfection. Furthermore, the transfection of hypoxia-responsive plasmid demonstrated that polyplex micelles allowed the transfection to the hypoxic region of the tumor tissue in both in vitro and in vivo experiments. To the best of our knowledge, our results demonstrated for the first time that polyplex micelles might show improved tumor penetrability over cationic polyplexes, thereby achieving transfection into the inside of the tumor tissue.
Project description:Nonviral gene therapy has high potential for safely promoting tissue restoration and for treating various genetic diseases. One current limitation is that conventional transfection reagents such as polyethylenimine (PEI) form electrostatically stabilized plasmid DNA (pDNA) polyplexes with poor colloidal stability. In this study, a library of poly(ethylene glycol-b-(dimethylaminoethyl methacrylate-co-butyl methacrylate)) [poly(EG-b-(DMAEMA-co-BMA))] polymers were synthesized and screened for improved colloidal stability and nucleic acid transfection following lyophilization. When added to pDNA in the appropriate pH buffer, the DMAEMA moieties initiate formation of electrostatic polyplexes that are internally stabilized by hydrophobic interactions of the core BMA blocks and sterically stabilized against aggregation by a PEG corona. The BMA content was varied from 0% to 60% in the second polymer block in order to optimally tune the balance of electrostatic and hydrophobic interactions in the polyplex core, and polymers with 40 and 50 mol% BMA achieved the highest transfection efficiency. Diblock copolymers were more stable than PEI in physiologic buffers. Consequently, diblock copolymer polyplexes aggregated more slowly and followed a reaction-limited colloidal aggregation model, while fast aggregation of PEI polyplexes was governed by a diffusion-limited model. Polymers with 40% BMA did not aggregate significantly after lyophilization and produced up to 20-fold higher transfection efficiency than PEI polyplexes both before and after lyophilization. Furthermore, poly(EG-b-(DMAEMA-co-BMA)) polyplexes exhibited pH-dependent membrane disruption in a red blood cell hemolysis assay and endosomal escape as observed by confocal microscopy.Lyophilized polyplexes made with the lead candidate diblock copolymer (40% BMA) also successfully transfected cells in vitro following incorporation into gas-foamed polymeric scaffolds. In summary, the enhanced colloidal stability, endosomal escape, and resultant high transfection efficiency of poly(EG-b-(DMAEMA-co-BMA))-pDNA polyplexes underscores their potential utility both for local delivery from scaffolds as well as systemic, intravenous delivery.
Project description:A major challenge for non-viral gene delivery is gaining a mechanistic understanding of the rate-limiting steps. A critical barrier in polyplex-mediated gene delivery is the timely unpacking of polyplexes within the target cell to liberate DNA for efficient gene transfer. In this study, the component plasmid DNA and polymeric gene carrier were individually labeled with quantum dots (QDs) and Cy5 dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled sensitive detection of discrete changes in polyplex stability. The intracellular uptake and dissociation of polyplexes through QD-FRET was captured over time by confocal microscopy. From quantitative image-based analysis, distributions of released plasmid within the endo/lysosomal, cytosolic, and nuclear compartments formed the basis for constructing a three-compartment first-order kinetics model. Polyplex unpacking kinetics for chitosan, polyethylenimine, and polyphosphoramidate were compared and found to correlate well with transfection efficiencies. Thus, QD-FRET-enabled detection of polyplex stability combined with image-based quantification is a valuable method for studying mechanisms involved in polyplex unpacking and trafficking within live cells. We anticipate that this method will also aid the design of more efficient gene carriers.
Project description:Polyplex formation (complexation) and gene release from the polyplexes (decomplexation) are major events in polymeric gene delivery; however, the effect of the decomplexation rate on transfection has been rarely investigated. This study employed mixed polymers of poly((L)-lysine) (PLL: MW ~7.4 kDa) and reducible PLL (RPLL) (MW ~6.7 kDa) to design decomplexation rate-controllable PLL(100-x)RPLL(x)/pDNA complexes (PRL(x) polyplexes). The transfection efficiency of a model gene (luciferase) in MCF7 and HEK293 cell lines increased with increasing x (RPLL content) in the PRL(x) polyplexes until peaking at x = 2.5 and 10, respectively, after which point transfection efficiency declined rapidly. In MCF7 cells, PRL(2.5) polyplex produced 3 or 223 times higher gene expression than PLL or RPLL polyplexes, respectively. Similarly, the transfection efficiency of PRL(10) polyplex-transfected HEK293 cells was 3.8 or 67 times higher than that of PLL or RPLL polyplexes, respectively. The transfection results were not apparently related to the particle size, surface charge, complexation/compactness, cellular uptake, or cytotoxicity of the tested polyplexes. However, the decomplexation rate varied by RPLL content in the polyplexes, which in turn influenced the gene transfection. The nuclear localization of pDNA delivered by PRL(x) polyplexes showed a similar trend to their transfection efficiencies. This study suggests that an optimum decomplexation rate may result in high nuclear localization of pDNA and transfection. Understanding in decomplexation and intracellular localization of pDNA may help develop more effective polyplexes.