Cationic Lipoplexes Research Articles

PLAUR Confers Resistance to Gefitinib Through EGFR/P-AKT/Survivin Signaling Pathway

Background/Aims: Tyrosine kinase inhibitor gefitinib significantly improves the survival of patients with non-small-cell lung cancer (NSCLC) by inhibiting epidermal growth factor receptor (EGFR) tyrosine kinase. However, patients eventually develop resistance to gefitinib through uncharacterized mechanisms. It is known that plasminogen activator urokinase receptor (PLAUR) plays an important role in cell proliferation, migration and apoptosis. However, the role of PLAUR, particularly exosomal PLAUR in gefitinib resistance in NSCLC has not been reported. The aim of this study is to determine the relationship between PLAUR and gefitinib resistance. Methods: In this study, a tethered cationic lipoplex nanoparticle (TCLN) biochip containing molecular beacons was used as probes to detect PLAUR mRNA in plasma exosomes from patients with gefitinib-sensitive and -resistant NSCLC. In vitro, Real-time PCR was used to examine the expression of PLAUR mRNA and Western blot was applied to examine the expression of related proteins. The gene knockdown was achieved by Lentivirus based RNA silence technique. The cell counting kit-8 assay and EdU incorporation were used to examine cell proliferation. The flow cytometry was applied to determine cell apoptosis and cell cycle, while the mitochondrial membrane potential was measured by JC-1 dye assay. Signaling pathway affected by PLAUR knockdown was identified by cDNA Microarray. The effect of PLAUR knockdown on tumorigenesis was analyzed in vivo. Results: We found that the exosomal PLAUR mRNA in the plasma of gefitinib-resistant NSCLC patients was significantly increased compared to that of gefitinib-sensitive NSCLC patients. The PLAUR mRNA and soluble PLAUR protein were also significantly increased in gefitinib-resistant human lung adenocarcinoma PC9R cells compared to gefitinib-sensitive PC9 cells. Silencing PLAUR in PC9R cells impaired mitochondrial membrane potential and increased cell apoptosis via EGFR/p-AKT/survivin signaling pathway. Furthermore, EGFR was upregulated in the geftinib-resistant PC9R cells, and knockdown of EGFR significantly increased cell apoptosis. Conclusions: Taken together, our results demonstrated that PLAUR induces geftinib-resistance through EGFR/p-AKT/survivin signaling pathway in gefitinib-resistant human lung adenocarcinoma cells. PLAUR could be a novel therapeutic target for gefitinib-resistant NSCLC patients.

Quantitative contributions of processes by which polyanion drugs reduce intracellular bioavailability and transfection efficiency of cationic siRNA lipoplex

RNA Interference (RNAi) is a potentially useful tool to correct the detrimental effects of faulty genes; several RNAi are undergoing clinical evaluation in various diseases. The present study identified the relative contributions of three mechanisms by which polyanion drugs reduced the gene silencing activity of Lipoplex, a complex of small interfering RNA (siRNA) and cationic liposomes. The study used a siRNA against the chemoresistance gene survivin and two model polyanion drugs (suramin, heparin). Products of Lipoplex destabilization were separated, identified, and/or quantified using ultrafiltration, gel electrophoresis, and RT-qPCR (quantitative reverse transcription polymerase chain reaction). Cell binding and endocytosis of fluorescence-labeled Lipoplex and the amount of siRNA at its site of action RISC (RNA-induced silencing complex) were evaluated using endocytosis markers, confocal microscopy, quantitative image analysis, immunoprecipitation, and RT-qPCR. The results show suramin and heparin exerted multiple concentration-dependent effects. First, these agents altered several Lipoplex properties (i.e., reduced particle size, changed surface charge, modified composition of protein biocorona). Second, both caused Lipoplex destabilization to release double- and single-strand siRNA and/or smaller siRNA-lipid complexes with reduced siRNA cargo. Third, both prevented the cell surface binding and internalization of Lipoplex, diminished the siRNA concentration in RISC, and retarded the mRNA knockdown. Suramin and heparin yielded qualitatively and quantitatively different results. Analysis of the experimental results of suramin using quantitative pharmacology (QP) modeling indicated the major cause of gene silencing activity loss depended on drug concentration, changing from inhibition of endocytosis at lower concentration (accounting for 60% loss at ~9μM) to inhibition of cell surface binding and loss of siRNA cargo at higher concentrations (accounting for 64% and 27%, respectively, at 70μM). In summary, the present study demonstrates the complex and dynamic interactions between polyanions and Lipoplex, and the use of QP modeling to delineate the contributions of three mechanisms to the eventual loss of gene silencing activity.

Transfection of 3′,3′′-bis-peptide-siRNA conjugate by cationic lipoplexes mixed with a neutral cytosin-1-yl-lipid

Transfection of 3′,3′′-bis-peptide-siRNA conjugate by cationic lipoplexes mixed with a neutral cytosin-1-yl-lipid

Impact of the Charge Ratio on the In Vivo Immunogenicity of Lipoplexes.

The present study investigated the immunogenic potential of different cationic liposome formulations with a DNA plasmid encoding Pfs25, a malaria transmission-blocking vaccine candidate. Pfs25 plasmid DNA was complexed with cationic liposomes to produce lipoplexes at different charge ratios of the cationic lipid head group to the nucleotide phosphate (N:P). The formation of lipoplexes was visualized by Cryogenic-TEM. Confocal microscopy of lipoplexes formed with GFP encoding plasmid DNA, and flow cytometry was used to determine their in vitro transfection capability. Two different lipoplex formulations using plasmid DNA encoding Pfs25 were evaluated for in vivo immunogenicity after intramuscular administration in Balb/c mice. Immune sera were analyzed by ELISA. The results demonstrated that the cationic liposome-mediated DNA immunization with an N:P charge ratio of 1:3 (anionic lipoplexes) is more effective than the use of naked plasmid DNA alone. No antibody response was observed when lipoplexes with a higher N:P charge ratio of 10:3 (cationic lipoplexes) were used. Trehalose was added to some lipoplex formulations as a cryoprotectant and adjuvant, but it did not yield any further improvement of immunogenicity in vivo. The results suggest that Pfs25 plasmid DNA delivered as lipoplexes at a charge ratio of 1:3 elicited strong immunogenicity in mice and may be improved further to match the immune responses of DNA vaccines administered by in vivo electroporation.

Polyanionic Drugs Reduce Intracellular Bioavailability and Transfection Efficiency of Cationic siRNA Lipoplex: Quantitative Pharmacology Studies

RNA Interference (RNAi) is a potentially useful tool to correct the detrimental effects of faulty genes, and several such agents are undergoing clinical evaluation. The present study evaluated the effects of polyanionic drugs (suramin, heparin) on the in vitro stability, cellular processing, and biological activity of Lipoplex, a complex between small interfering RNA (siRNA) and cationic liposome (50% DOTAP, 30% Cholesterol, 19% DOPE, 1% DSPE‐PEG). The resulting Lipoplex had a diameter of 195 nm and surface charge of +33.9 mV. Two siRNA, against survivin and metadherin, were used. The destabilization products of Lipoplex were separated, identified, and quantified using ultrafiltration, gel electrophoresis, and RT‐qPCR (quantitative reverse transcription polymerase chain reaction). Binding and endocytosis of Lipoplex in cells, and the amount of siRNA located in its target site RISC (RNA‐induced silencing complex) were evaluated using endocytosis markers, confocal microscopy, quantitative image analysis, immunoprecipitation, and RT‐qPCR. The results show suramin and heparin exerted several effects that were dependent on their concentrations as well as Lipoplex concentration. These agents altered the Lipoplex physical properties (i.e., reduced particle size, changed surface charge from positive to negative, removed protein‐biocorona or modified its composition). These changes are associated with Lipoplex destabilization and release of lipids and siRNA (ds/ss siRNA) and formation smaller Lipoplex with reduced siRNA content. The Lipoplex destabilization products due to suramin and heparin were different from each other and from those caused by serum proteins, both qualitatively and quantitatively. In addition, suramin and heparin prevented the cell surface binding and internalization of Lipoplex, diminished the intracellular bioavailability of siRNA in RISC, and retarded the mRNA knockdown by Lipoplex. Quantitative pharmacology analysis using experimental results and computational simulations indicated that polyanion:siRNA molar concentration ratio (Ratio) determined the relative contribution of two mechanisms to the reduction of Lipoplex‐conferred gene silencing; the Lipoplex destabilization and loss of siRNA cargo had a greater contribution compared to the inhibition of Lipoplex cellular binding and endocytosis at lower Ratio (e.g., 17:1), whereas the latter had a greater contribution at higher Ratio (e.g., 128:1). In summary, interactions with polyanionic drugs destabilize Lipoplex result in different products for different drugs, and the three‐way interactions between polyanionic drugs, Lipoplex and cell membrane binding sites diminish the Lipoplex biological activity.Support or Funding InformationSupported in part by research grants, R01CA158300 (GW, JA) and R01CA163015 (GW, JA) from National Cancer Institute and R01EB015253 (JA, GW) from National Institute of Biomedical Imaging and Bioengineering, NIH, DHHS.

Gene transfection efficiency into dendritic cells is influenced by the size of cationic liposomes/DNA complexes

Cationic liposomes have attracted recent attention as DNA vaccine carriers that can target dendritic cells (DCs). In general, cationic liposome/DNA complexes (lipoplexes) are taken up by various cells via clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, or phagocytosis, with the mode of endocytosis determining further intracellular trafficking pathways. Moreover, the physicochemical properties of cationic lipoplexes, including lipid composition, shape, size, and charge, influence transfection efficiency, affecting uptake and subsequent intracellular pathways. To develop cationic liposomes as potential DNA vaccine carriers, the objective of this study was to study the effect of lipoplex size on DNA transfection efficiency in DCs. We explored the size-dependent endocytosis pathway and the intracellular trafficking of cationic lipoplexes using bone marrow derived dendritic cells (BMDCs). Our results indicated that small-sized lipoplexes (approximately 270nm diameter) were taken up by BMDCs via caveolae-mediated endocytosis, which led to a non-degradative pathway, whereas larger-sized lipoplexes (approximately 500nm diameter) were taken up by BMDCs via clathrin-mediated endocytosis and micropinocytosis, which led to a lysosomal degradation pathway. These findings suggest that, by regulating the size of lipoplexes, it may be possible to develop cationic liposomes as DNA vaccine therapies for targeting DCs.

Anionic pH-Sensitive Lipoplexes.

To provide long circulating nanoparticles able to carry a gene to tumors, we have designed anionic pegylated lipoplexes which are pH sensitive. Anionic pegylated lipoplexes have been prepared from the combined formulation of cationic lipoplexes and pegylated anionic liposomes. The neutralization of the particle surface charge as a function of the pH was monitored by light scattering in order to determine the ratio between anionic and cationic lipids that would give pH sensitive complexes. This ratio has been optimized to form particles sensitive to pH change in the range 5.5-6.5. Compaction of DNA into these newly formed anionic complexes is checked by DNA accessibility to picogreen. The transfection efficiency and pH sensitive property of these formulations has been shown in vitro using bafilomycin, a vacuolar H+-ATPase inhibitor.

Analyzing the miRNA content of extracellular vesicles by fluorescence nanoparticle tracking

We present a method that takes advantage of the fluorophore loading dependence of fluorescence nanoparticle tracking (fNTA) to determine the content of specific miRNA targets in extracellular vesicles (EVs) and their stoichiometry across the entire EV population. The method is based on an assay for detecting EV miRNA by hybridization to fluorescently labeled, miRNA-specific molecular beacons encapsulated in cationic lipoplex nanoparticles that fuse non-specifically with negatively charged EVs. To demonstrate the method, we carry out a stoichiometric analysis of miR-21 in EVs released from A549 lung cancer cells. We find approximately 2.3% of the A549 EVs have an average copy number of ~44 miR-21/A549 EV and contain at least a threshold number of 33 miR-21 copies/A549 EV required for fluorescence tracking. Potential applications of sizing, enumerating, and phenotyping EVs using this method include specifying dosages for therapeutic applications and identifying specific EV subpopulations in patient samples for diagnostic applications.

Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy

Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

Evaluation of the injection route of an anionic polymer for small interfering RNA delivery into the liver by sequential injection of anionic polymer and cationic lipoplex of small interfering RNA

Previously, we developed a novel small interfering RNA (siRNA) transfer method for the liver by sequential intravenous injection of an anionic polymer and cationic liposome/siRNA complex (cationic lipoplex). In this study, we examined the effects of the type of anionic polymer and injection route of the anionic polymer on the biodistribution of siRNA after sequential injection of anionic polymer and cationic lipoplexes. When cationic lipoplexes were injected intravenously into mice, siRNA largely accumulated in the lungs. In contrast, sequential injection of cationic lipoplex after intravenous injection of 1 mg chondroitin sulfate C and A (CS-C and CS-A) or polyaspartic acid decreased the accumulation of siRNA in the lungs and increased it in the liver, compared with injection of cationic lipoplex. Regarding the injection route of the anionic polymer, intramuscular, intraperitoneal, or subcutaneous injection of 10 mg CS-C before intravenous injection of cationic lipoplex resulted in siRNA accumulation mainly in the liver. Furthermore, the injection of cationic lipoplex of apolipoprotein B (ApoB) siRNA after intravenous or intramuscular injection of CS-C could suppress ApoB mRNA levels in the liver. Sequential injection of CS-C plus cationic lipoplex could deliver siRNA efficiently into the liver regardless of the injection route of CS-C.

What the cell surface does not see: The gene vector under the protein corona

The fate of lipid-based nanovectors, used in genetic targeting inside cells, depends on their behavior in biological media. In fact, during both in vitro and in vivo transfection, nanovectors come in contact with proteins that compete for their surface and build the protein corona, their true biological identity while engaging the cell membrane. Nonetheless, after cell internalization, the efficacy of transfection may depend also on structural modifications that occurred under the protein cover, following interaction with biological fluids. Here, based on previous in vivo experiments, two widely used lipid mixtures, namely DOTAP/DOPC and DC-Chol/DOPE, were identified as paradigms to investigate the impact of the inner structure of nanovectors on the transfection efficiency, all being proficiently internalized. The evolution of the inner structure of cationic lipoplexes and nanoparticles based on such lipid mixtures, following interaction with human plasma, could be unraveled. Particles were investigated in high dilution, approaching the biosimilar conditions. Data have demonstrated that the modulation of their inner structure depends on their lipid composition and the plasma concentration, still preserving the genetic payload. Interestingly, protein contact induces a variety of inner structures with different perviousness, including reshaping into cubic phases of different porosity, sometimes observed upon interaction between carrier-lipids and cell-lipids. Cubic reshaping is of biological relevance, as lipid cubic phases have been recently associated to both fusogenicity and to the readiness in releasing the payload to the final target via endosomal escape.

Delivery of Plasmid DNA into Tumors by Intravenous Injection of PEGylated Cationic Lipoplexes into Tumor-Bearing Mice

For systemic injection of cationic liposome/plasmid DNA (pDNA) complexes (cationic lipoplexes), polyethylene glycol (PEG)-modification (PEGylation) of lipoplexes can enhance their systemic stability. In this study, we examined whether intravenous injection of PEGylated cationic lipoplexes into tumor-bearing mice could deliver pDNA into tumor tissues and induce transgene expression. PEGylation of cationic liposomes could prevent their agglutination with erythrocytes. However, when PEGylated cationic lipoplexes were injected intravenously into tumor-bearing mice, they accumulated in tumor vascular vessels and did not exhibit transgene expression in tumors with both poor and well-developed vascularization. Furthermore, PEGylated cationic lipoplexes of CpG- free pDNA could not increase transgene expression in tumors after intravenous injection. These results suggested that PEGylation could not extravasate cationic lipoplexes from vascular vessels in tumors and abolished transgene expression although it enhanced the systemic stability of cationic lipoplexes by avoiding interactions with blood components such as erythrocytes. Successful delivery of pDNA to tumors by PEGylated cationic liposomes will require a rational strategy and the design of liposomal delivery systems to overcome the issue associated with the use of PEG.

Physical Chemical and Biomolecular Methods for the Optimization of Cationic Lipid-Based Lipoplexes In Vitro for the Gene Therapy Applications.

Preparation and application protocols play a very important role while optimizing the cationic lipid-based lipoplexes in vitro. These protocols serve as the basis for the betterment of the lipoplexes with regard to their successful application in animals and eventually human subjects. Starting from the chemical structures of used cationic lipids (CLs), optimization of the additive inclusions, methods of nanoparticle (lipoplex) formation, presence of blood serum, time intervals of lipoplex incubation, and type of efficiency read-outs in various conditions play important roles in reaching insightful conclusions. Such steps of summarizing protocols and requirements of the pertinent events focus on getting improved lipoplexes for achieving optimal effects in terms of post transfection gene and protein expression. The progression of optimization and efficiency evaluation lead to predictable structure-method-activity relationship with involvement of various feedback principles including physical chemical and biomolecular evaluations before and after the use of lipoplexes in biological systems. This chapter discusses some of the focused strategies for the establishment of lipoplexes for a better post transfection activity with reduced risk of failure.

SiRNA delivery to lung-metastasized tumor by systemic injection with cationic liposomes

Context: Cationic liposomes can efficiently deliver siRNA to the lung by intravenous injection of cationic liposome/siRNA complexes (lipoplexes).Objective: The aim of this study was to examine a formulation of cationic liposomes for siRNA delivery to lung metastasis of breast tumor.Materials and methods: For the preparation of cationic liposomes, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or dimethyldioctadecylammonium bromide (DDAB) as a cationic lipid and cholesterol (Chol) or 1,2-dioleoyl-l-α-glycero-3-phosphatidylethanolamine (DOPE) as a neutral lipid were used. In vitro and in vivo gene silencing effects by cationic lipoplexes were evaluated after transfection into stably luciferase-expressing human breast tumor MCF-7-Luc cells and after intravenous injection into mice with lung MCF-7-Luc metastasis, respectively. Intracellular localization of siRNA after transfection into MCF-7 cells by cationic lipoplexes and biodistribution of siRNA after intravenous injection of cationic lipoplexes into the mice with lung metastasis were examined by confocal and fluorescent microscopy analyses, respectively.Results: In in vitro transfection, DOTAP/DOPE and DDAB/DOPE lipoplexes of luciferase siRNA strongly suppressed luciferase activity in MCF-7-Luc cells, but DOTAP/Chol and DDAB/Chol lipoplexes did not, although DOTAP/Chol and DDAB/Chol lipoplexes exhibited higher cellular uptake than DOTAP/DOPE and DDAB/DOPE lipoplexes. When their cationic lipoplexes were intravenously injected into mice with lung MCF-7-Luc metastasis, siRNAs were mainly accumulated in the lungs; however, the reduced luciferase activities in the lung-metastasized tumors were observed only by injections of DOTAP/Chol and DOTAP/DOPE lipoplexes, but not by DDAB/Chol and DDAB/DOPE lipoplexes.Conclusions: DOTAP-based liposomes might be useful as an in vivo siRNA delivery carrier that can induce gene silencing in lung-metastasized tumors.

Therapeutic effect for liver-metastasized tumor by sequential intravenous injection of anionic polymer and cationic lipoplex of siRNA

Previously, we developed a novel siRNA transfer method to the liver by sequential intravenous injection of anionic polymer and cationic liposome/siRNA complex (cationic lipoplex). In this study, we investigated whether siRNA delivered by this sequential injection could significantly suppress mRNA expression of the targeted gene in liver metastasis and inhibit tumor growth. When cationic lipoplex was intravenously injected into mice bearing liver metastasis of human breast tumor MCF-7 at 1 min after intravenous injection of chondroitin sulfate C (CS) or poly-l-glutamic acid (PGA), siRNA was accumulated in tumor-metastasized liver. In terms of a gene silencing effect, sequential injections of CS or PGA plus cationic lipoplex of luciferase siRNA could reduce luciferase activity in liver MCF-7-Luc metastasis. Regarding the side effects, sequential injections of CS plus cationic lipoplex did not exhibit hepatic damage or induction of inflammatory cytokines in serum after repeated injections, but sequential injections of PGA plus cationic lipoplex did. Finally, sequential injections of CS plus cationic lipoplex of protein kinase N3 siRNA could suppress tumor growth in the mice bearing liver metastasis. From these findings, sequential injection of CS and cationic lipoplex of siRNA might be a novel systemic method of delivering siRNA to liver metastasis.

590. Tumor-Targeted Delivery of Therapeutic siRNA by EGFR Aptamer-Conjugated Liposomes

Important considerations for effective cancer gene therapy are the targeted delivery of therapeutic genes and efficient endosomal escape of the genes in the cytoplasm. To address the first issues, target-specific aptamers and cationic lipids have been adopted for formulation of tumor-specific lipidic nanoparticles containing therapeutic genes. First of all we have developed EGF receptor (EGFR) aptamer-conjugated liposomes containing siRNA. The liposomal vehicles were optimized in terms of lipid constituents, ratio of lipid/siRNA, and aptamer conjugation process, and their biophysical characteristics were then determined by dynamic light scattering. Compared to the nonspecific cationic lipoplexes of siRNA, EGFR aptamer-conjugated liposomes encapsulating siRNA showed a relatively lower binding to EGFR-expressing cancer cells, resulting in a lower siRNA transfection. However, the EGFR aptamer-conjugated liposomes exhibited efficient transfection only to EGFR-expressing cancer cells, but not to EGFR-negative cancer cells. This research suggests that aptamer-conjugated lipidic nanoparticles can be utilized for target-specific delivery of therapeutic siRNAs.

Optimization of siRNA Delivery Method into the Liver by Sequential Injection of Polyglutamic Acid and Cationic Lipoplex

Previously, we developed a novel siRNA transfer method to the liver by sequential intravenous injection of poly-L-glutamic acid (PGA) and cationic liposome/siRNA complex (cationic lipoplex). In this study, we examined the effects of the charge ratio (+/-) of cationic liposome/siRNA, molecular weight of PGA and cationic lipid of cationic liposome on the biodistribution of siRNA after sequential injection of PGA plus cationic lipoplex. When 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (Chol) lipoplex was intravenously injected into mice, the accumulation of siRNA was mainly observed in the lungs. In contrast, when DOTAP/Chol lipoplex was intravenously injected at 1 min after intravenous injection of PGA, siRNA was largely accumulated in the liver. The charge ratio (+/-) of DOTAP/Chol liposome/siRNA did not affect the biodistribution of siRNA after sequential injection. As regards the molecular weight of PGA, the accumulation of siRNA was observed mainly in the liver after the sequential injection of PGA of 20.5, 38, 64 or 200 kDa plus DOTAP/Chol lipoplex. Furthermore, to examine the effect of cationic lipid of cationic liposome on the biodistribution of siRNA, we prepared other cationic liposomes composed of 1,2-di-O-octadecenyl-3-trimethylammonium propane chloride (DOTMA)/Chol, dimethyldioctade-cylammonium bromide (DDAB)/Chol and O,O’-ditetradecanoyl-N-(α-trimethylammonioacetyl)di-ethanolamine chloride (DC-6-14)/Chol. For the cationic liposomes, the accumulation of siRNA was observed mainly in the liver when their cationic lipoplexes were sequentially injected after injection of PGA into mice. From these findings, sequential injection of PGA plus cationic lipoplex could deliver siRNA efficiently into the liver regardless of the charge ratio (+/-) of lipoplex, lengths of PGA and cationic lipid of liposome.

Relating toxicity to transfection: using sphingosine to maintain prolonged expression in vitro.

Cationic reagents are commonly used to facilitate DNA delivery, and transfection experiments are typically initiated in cell culture where the optimal charge ratio is determined. While transfection rates are often enhanced at higher +/– charge ratios, the cellular toxicity associated with the greater amounts of cationic components at elevated charge ratios is often not considered. In addition, the prolonged effects of cationic lipid uptake on cell viability are not evident in a typical 24–48 h transfection experiment. In this study, we compare the transfection efficiency of cationic lipoplexes to effects on viability of cultured cells in both the short and long term (7 days). Our results indicate that, while minimal toxicity is evident 24 h after exposure to DOTAP-based lipoplexes, cell viability continues to decline and ultimately compromises reporter gene expression at longer times. Substitution of a naturally occurring cationic amphiphile, sphingosine, for DOTAP greatly reduces toxicity and allows high expression to be maintained over prolonged periods.

Fine tuning of mixed ionic and hydrogen bond interactions for plasmid delivery using lipoplexes

Non viral gene transfection has been mostly reached via cationic polymer and lipid, required for DNA complexation and cell internalisation. However, cationic charges often induce cytotoxicity and limit the efficacy of the lipoplexes in vivo due to their fast elimination from the blood stream. Few years ago, we had developed noncationic lipid interacting with DNA via hydrogen bond interactions. To take advantage of both the internalisation efficacy of cationic complexes and the higher DNA release efficacy of non cationic lipids, we chose to mix both ionic and hydrogen bond interactions within one lipoplex. The idea behind this strategy would be to reduce the overall charge while maintaining a high level of transfection. Four mixed formulations of cationic lipid and thiourea lipid were prepared. We found that decreasing ionic interactions and increasing hydrogen bond interactions improved cationic lipoplexes properties. Indeed, we showed that replacement of net positive charges by hydrogen bond interactions with DNA phosphates led to efficient lipoplexes for in vitro DNA transfection at lower cationic charge content, which consequently reduced lipoplex cytotoxicity.

Sequential intravenous injection of anionic polymer and cationic lipoplex of siRNA could effectively deliver siRNA to the liver

In this study, we developed novel siRNA transfer method to the liver by sequential intravenous injection of anionic polymer and cationic liposome/cholesterol-modified siRNA complex (cationic lipoplex). When cationic lipoplex was intravenously injected into mice, the accumulation of siRNA was mainly observed in the lungs. In contrast, when cationic lipoplex was intravenously injected at 1min after intravenous injection of poly-l-glutamic acid (PGA) or chondroitin sulfate C (CS), siRNA was accumulated in the liver. In terms of suppression of gene expression in vivo, apolipoprotein B (ApoB) mRNA in the liver and low-density-lipoprotein (LDL) and very low-density-lipoprotein (VLDL) cholesterol level in serum were reduced at 48h after single sequential injection of PGA or CS plus cationic lipoplex of cholesterol-modified ApoB siRNA. Furthermore, sequential injections of PGA plus cationic lipoplex of cholesterol-modified luciferase siRNA could reduce luciferase activity in tumor xenografts bearing liver metastasis of human breast tumor MCF-7-Luc. From these findings, sequential injection of anionic polymer and cationic lipoplex of siRNA might produce a systemic vector of siRNA to the liver.