Types Of Cationic Lipids Research Articles

Effect of cationic and neutral lipids in cationic liposomes on antibody production induced by systemic administration of mRNA lipoplexes into mice

We have previously developed a modified ethanol (EtOH) injection (MEI) method for mRNA transfection wherein cationic liposome/mRNA complexes (mRNA lipoplexes) were prepared by mixing a small volume of a lipid-EtOH solution with phosphate-buffered saline (PBS) containing mRNA. In the present study, five types of cationic lipids, namely N,N,N-trimethyl-2,3-bis(oleoyloxy)propan-1-aminium methyl sulfate salt (DOTAP), dimethyldioctadecylammonium bromide (DDAB), N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), 2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-6-14), and 11-[(1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino]-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12); two types of neutral lipid, cholesterol (Chol) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and polyethylene glycol-cholesteryl ether (PEG-Chol) as a PEG-lipid were selected. Ten types of mRNA lipoplexes composed of each cationic and neutral lipid and PEG-Chol were prepared using the MEI method. mRNA lipoplexes containing DC-1-16 or DDAB with DOPE and PEG-Chol (LP-DC116/DOPE and LP-DDAB/DOPE, respectively) were injectable and their systemic injection with ovalbumin (OVA) mRNA induced high anti-OVA IgG1 levels. Furthermore, systemic injection of the lipoplexes with luciferase (Luc) mRNA resulted in high Luc expression in the lungs and spleen of mice. These findings suggest that LP-DC116/DOPE and LP-DDAB/DOPE lipoplexes effectively induce antibody production via increased protein expression in the spleen.

Optimization of transfection into cultured cells with siRNA lipoplexes prepared using a modified ethanol injection method

To simplify the preparation of cationic liposome/small interfering RNA (siRNA) complexes (siRNA lipoplexes), we previously developed a modified ethanol injection (MEI) method, in which siRNA-containing phosphate-buffered saline was rapidly poured into a small volume of a lipid-ethanol (EtOH) solution. In this study, to optimize the lipid composition and solvents for dissolving lipids and siRNA in the transfection of siRNA lipoplexes prepared using the MEI method, we selected five types of cationic lipids, three types of neutral lipids, and five types of polyethylene glycol (PEG)-lipids and prepared siRNA lipoplexes via the MEI method using a lipid-EtOH solution of cationic lipids, neutral lipids, and PEG-lipids. Regardless of the cationic lipid type, siRNA lipoplexes composed of cationic lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and poly(ethylene glycol) cholesteryl ether showed high gene knockdown efficiency in human breast tumor MCF-7 cells. Regardless of the PEG-lipid type, the size of the siRNA lipoplexes decreased with increasing amounts of PEG-lipid in the lipid-EtOH solution. However, the gene knockdown activity of the siRNA lipoplexes was not abolished via PEGylation when siRNA lipoplexes were PEGylated with PEG-lipids having short lipid anchors. Furthermore, the preparation of siRNA lipoplexes by mixing a lipid-dimethyl sulfoxide solution with a phosphate-buffered saline solution of siRNA increased the size of the siRNA lipoplexes compared with that using a lipid-EtOH solution. However, these lipoplexes showed high gene knockdown efficiency in cells. When prepared by mixing a lipid-EtOH solution with an isotonic glucose or sucrose solution of siRNA, the siRNA lipoplexes tended to reduce gene knockdown efficiency in the cells. These results indicate that the lipid composition and solvents used for dissolving the lipids and siRNA affect the transfection efficiency and size of the siRNA lipoplexes. This study provides information on the optimal preparation of siRNA lipoplexes using the MEI method.

Effect of the combination of cationic lipid and phospholipid on gene-knockdown using siRNA lipoplexes in breast tumor cells and mouse lungs.

Previously, using three types of cationic lipids, the effect of phospholipids in liposomal formulations on gene-knockdown efficacy was determined after invitro and invivo transfection with small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) containing various cationic lipids and phospholipids. In the present study, six other types of cationic lipids, namely N,N-dimethyl-N-tetradecyltetradecan-1-aminium bromide, N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), 2-[bis{2-(tetradecanoyloxy)ethyl}amino]-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-6-14), 1,2-di-O-octadecenyl-3-trimethylammonium propane chloride (DOTMA), 1,2-distearoyl-3-trimethylammonium-propane chloride (DSTAP) and 1,2-dioleoyl-3-dimethylammonium-propane were selected, and the effect of phospholipids in liposomal formulations containing each cationic lipid on gene-knockdown was evaluated. A total of 30 types of cationic liposomes composed of each cationic lipid with phosphatidylethanolamine containing unsaturated or saturated diacyl chains (C14, C16 or C18) were prepared. Regardless of the type of cationic lipid, the inclusion of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in the liposomal formulations resulted in injectable size of siRNA lipoplexes after mixing of siRNA and cationic liposomes. Transfection of their lipoplexes with luciferase (Luc) siRNA into human breast cancer MCF-7-Luc cells stably expressing Luc led to a strong knockdown of Luc. Furthermore, the systemic injection of siRNA lipoplexes composed of DC-1-16, DC-6-14, DOTMA or DSTAP with DOPE resulted in siRNA accumulation in the lungs. Significant gene-knockdown was observed in the lungs of mice following the systemic injection of siRNA lipoplexes containing DC-1-16 and DOPE. Cationic liposomes composed of DC-1-16 and DOPE serve as potential carriers for invitro and invivo siRNA transfection.

In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic Liposome-DNA Complexes.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems.

Cuboplex-Mediated Nonviral Delivery of Functional siRNA to Chinese Hamster Ovary (CHO) Cells.

Lipid nanoparticles of internal cubic symmetry, termed cuboplexes, are potential nonviral delivery vehicles for gene therapy due to their "topologically active" nature, which may enhance endosomal escape and improve delivery outcomes. In this study, we have used cationic cuboplexes, based on monoolein (MO) doped with a cationic lipid, for the encapsulation and delivery of antisense green fluorescent protein (GFP)-small interfering RNA (siRNA) into Chinese Hamster Ovary (CHO)-GFP cells. Agarose gel electrophoresis has confirmed the successful encapsulation of siRNA within cationic cubosomes, while synchrotron small-angle X-ray scattering (SAXS) demonstrated that the underlying cubic nanostructure of the particles was retained following encapsulation. The cationic cubosomes were shown to be reasonably nontoxic against the CHO-GFP cell line. Fluorescence-activated cell sorting (FACS) provided evidence of the successful transfection to CHO-GFP cells. Knockdown efficiency was strongly linked to the type of cationic lipid used, although all cubosomes had essentially the same internal nanostructure. The gene knockdown efficiency for some cationic cubosomes was shown to be higher than lipofectamine, which is a commercially available liposome-based formulation, while the controlled release of the siRNA from the cubosomes over a 72 h period was observed using confocal microscopy. This combination exemplifies the potential of cationic cuboplexes as a novel, nonviral, controlled-release delivery vector for siRNA.

Effects of PEG anchors in PEGylated siRNA lipoplexes on invitro gene‑silencing effects and siRNA biodistribution in mice.

Polyethylene glycol (PEG)-modifications (PEGylations) of cationic liposome/small interfering RNA complexes (siRNA lipoplexes) can enhance their systemic stability. The present study determined the effects of PEG anchors in PEGylated siRNA lipoplexes on in vitro gene-silencing effects and siRNA biodistribution after intravenous injection. Three types of dialkyl or trialkyl cationic lipids were used in the current study for the preparation of cationic liposomes. Additionally, various PEGylated siRNA lipoplexes that contained PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), PEG-1,2-distearoyl-rac-glycero-3-methylpolyoxyethylene (DSG), PEG-cholesterol (PEG-Chol) and PEG-chondroitin sulfate conjugate (PEG-CS) were prepared. The results revealed that PEGylation of siRNA lipoplexes with PEG-DSPE strongly decreased gene-silencing effects in cells. In contrast, those with PEG-DSG, PEG-Chol and PEG-CS did not largely decrease gene-silencing effects. However, regardless of the PEG-derivative type, PEGylation of siRNA lipoplexes decreased their agglutination with erythrocytes. Furthermore, intravenous injection of PEGylated siRNA lipoplexes markedly decreased the accumulation of siRNA in the lungs, regardless of the type of PEG-derivative. However, non-PEGylated siRNA lipoplexes accumulated mainly in the lungs regardless of the siRNA lipoplex cationic lipid type. The results indicated that PEGylation of siRNA lipoplexes with PEG-DSG, PEG-Chol and PEG-CS may improve systemic stability without losing transfection activity by PEGylation.

Synthesis of Cationic and Ionizable Amphiphiles Based on Heminal Aminodiol as Potential siRNA Delivery Systems

Research methods based on the use of RNA interference mechanisms are now included among the basic methods of molecular biology. Drugs based on siRNA are being developed for the treatment of cancer, infectious diseases and other pathologies that are associated with impaired Junctions of specific genes. One of the main problems of siRNA-based drug development is their efficient and safe delivery to target cells. Modern delivery strategies are based on the use of chemical compounds or biological carriers, such as viruses. Lipid nanoparticles (liposomal agents) are the most advanced platform among non-viral vectors for the delivery of gene materials into cells. In this paper, a scheme has been proposed and the synthesis of new cationic amphiphiles has been carried out as the basis for the means of delivering siRNA to target cells. Synthesized amphiphiles belong to two types of cationic lipids: with a permanently charged amino group in the form of a salt and with an ionizable polar block. Ionized amphiphiles are a new generation of cationic lipids that exhibit reduced toxicity and immunogenicity. They undergo ionization only in the acidic environment of endosomes during intracellular transport in the cytosol, which leads to the release of the encapsulated cargo. The structure of the target compounds is based on diethanolamine derivatives, which is a spacer between the hydrophobic block and the polar fragment. The hydrophobic block contains palmitoyl residues, and the polar one - ethylenediamine and 3-diethylaminopropylamine derivatives. The developed synthetic scheme is distinguished by the simplicity and versatility of the proposed approach, which allows it to be used in the preparation of a series of samples in preparative quantities necessary for the subsequent physicochemical and biochemical studies.

Effects of cationic lipids in cationic liposomes and disaccharides in the freeze-drying of siRNA lipoplexes on gene silencing in cells by reverse transfection

RNA interference is a promising technology to inhibit the production of target proteins, and screening with synthetic small interfering RNA (siRNA) libraries has become a crucial research tool used to study gene function in cells. Reverse (Rev) transfection with freeze-dried siRNA/cationic liposome complexes (siRNA lipoplexes) can simplify and speed up siRNA transfection without the preparation of siRNA lipoplexes just before transfection. In this study, we examined the effects of cationic lipids in cationic liposomes and disaccharides in freeze-drying of siRNA lipoplexes on gene silencing in cells by Rev-transfection. We used three types of cationic cholesterol derivatives and three types of dialkyl or trialkyl cationic lipids for the preparation of cationic liposomes, and we prepared six types of freeze-dried siRNA lipoplexes in the presence of trehalose or sucrose solution in multi-well plates. Increasing concentrations of trehalose or sucrose included during freeze-drying of siRNA lipoplexes resulted in increased gene silencing activity upon Rev-transfection. Strong gene silencing activity was observed regardless of the type of cationic lipid in cationic liposomes when siRNA lipoplexes were freeze-dried with the disaccharides at concentrations of more than 25 mM or 100 mM. In addition, siRNA lipoplexes freeze-dried with 100 mM trehalose or sucrose showed long-term (1 month) stability without apparent loss of gene silencing activity. These findings suggested that Rev-transfection with freeze-dried siRNA lipoplexes may have potential applications in the screening of gene function using siRNA libraries.

Antigen Priming with Enantiospecific Cationic Lipid Nanoparticles Induces Potent Antitumor CTL Responses through Novel Induction of a Type I IFN Response.

Certain types of cationic lipids have shown promise in cancer immunotherapy, but their mechanism of action is poorly understood. In this study, we describe the properties of an immunotherapeutic consisting of the pure cationic lipid enantiomer R-1,2-dioleoyl-3-trimethyl-ammonium-propane (R-DOTAP) formulated with modified viral or self-peptide Ags. R-DOTAP formulations with peptide Ags stimulate strong cross-presentation and potent CD8 T cell responses associated with a high frequency of polyfunctional CD8 T cells. In a human papillomavirus tumor model system, a single s.c. injection of tumor-bearing mice with R-DOTAP plus human papillomavirus Ags induces complete regression of large tumors associated with an influx of Ag-specific CD8 T cells and a reduction of the ratio of regulatory/Ag-specific CD8 T cells. R-DOTAP also synergizes with an anti-PD1 checkpoint inhibitor, resulting in a significant inhibition of B16 melanoma tumor growth. We found that R-DOTAP stimulates type I IFN production by dendritic cells in vivo and in vitro. s.c. injection of R-DOTAP results in an IFN-dependent increase in draining lymph node size and a concomitant increase in CD69 expression. Using knockout mice, we show that type I IFN is required for the induction of CD8 T cell activity following administration of R-DOTAP plus Ag. This response requires Myd88 but not TRIF or STING. We also show that R-DOTAP stimulates both TLR7 and 9. Collectively, these studies reveal that R-DOTAP stimulates endosomal TLRs, resulting in a Myd88-dependent production of type I IFN. When administered with Ag, this results in potent Ag-specific CD8 T cell responses and antitumor activity.

Optimized combination of cationic lipids and neutral helper lipids in cationic liposomes for siRNA delivery into the lung by intravenous injection of siRNA lipoplexes

In this study, we examined the effect of neutral helper lipids in cationic liposomes on gene silencing efficacy in the lungs after intravenous injection of cationic liposomes/siRNA complexes (siRNA lipoplexes) into mice. We used 6 kinds of dialkyl or trialkyl cationic lipids as cationic lipids and prepared cationic liposomes composed of each cationic lipid with 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), DOPE/cholesterol (Chol), or Chol. Regardless of the type of cationic lipid in the cationic liposomes, the increase in Chol content in the formulation decreased the in vitro gene silencing efficacy, and it increased siRNA accumulation in the lungs after intravenous injection of siRNA lipoplexes. However, gene silencing efficacies in the lungs were not greatly affected by the type of neutral helper lipid in cationic liposomes composed of cationic lipids with saturated dialkyl chains (C16–C18), although they were increased by the inclusion of Chol into formulations of cationic liposomes composed of cationic lipids with unsaturated dialkyl chains (C18). These findings indicated that siRNA biodistribution and in vivo knockdown efficiency after the intravenous injection of siRNA lipoplexes were strongly affected by combination of a cationic lipid and neutral helper lipid in cationic liposomes.

Effect of Cationic Lipid Type in Folate-PEG-Modified Cationic Liposomes on Folate Receptor-Mediated siRNA Transfection in Tumor Cells.

In this study, we examined the effect of cationic lipid type in folate (FA)-polyethylene glycol (PEG)-modified cationic liposomes on gene-silencing effects in tumor cells using cationic liposomes/siRNA complexes (siRNA lipoplexes). We used three types of cationic cholesterol derivatives, cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol), N-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide (OH-Chol), and cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol), and we prepared three types of FA-PEG-modified siRNA lipoplexes. The modification of cationic liposomes with 1–2 mol % PEG-lipid abolished the gene-silencing effect in human nasopharyngeal tumor KB cells, which overexpress the FA receptor (FR). In contrast, FA-PEG-modification of cationic liposomes restored gene-silencing activity regardless of the cationic lipid type in cationic liposomes. However, the optimal amount of PEG-lipid and FA-PEG-lipid in cationic liposomes for selective gene silencing and cellular uptake were different among the three types of cationic liposomes. Furthermore, in vitro transfection of polo-like kinase 1 (PLK1) siRNA by FA-PEG-modified liposomes exhibited strong cytotoxicity in KB cells, compared with PEG-modified liposomes; however, in in vivo therapy, intratumoral injection of PEG-modified PLK1 siRNA lipoplexes inhibited tumor growth of KB xenografts, as well as that of FA-PEG-modified PLK1 siRNA lipoplexes. From these results, the optimal formulation of PEG- and FA-PEG-modified liposomes for FR-selective gene silencing might be different between in vitro and in vivo transfection.

Effect of cationic lipid type in PEGylated liposomes on siRNA delivery following the intravenous injection of siRNA lipoplexes

Effect of cationic lipid type in PEGylated liposomes on siRNA delivery following the intravenous injection of siRNA lipoplexes

Effect of cationic lipid type in cationic liposomes for siRNA delivery into the liver by sequential injection of chondroitin sulfate and cationic lipoplex

Previously, we reported that intravenous injection of chondroitin sulfate (CS), followed by intravenous injection of cationic liposome/siRNA complex (cationic lipoplex) could deliver siRNA into the liver. In this study, we examined the effects of the type of cationic lipid in the liposome on siRNA delivery into the liver. We used 6 types of cationic cholesterol derivatives and 11 types of dialkyl or trialkyl cationic lipids, and prepared 17 types of cationic liposomes to evaluate the gene-silencing effect of siRNA in mouse liver following sequential injection of CS plus cationic lipoplex. In 14 types of cationic liposomes, siRNA was accumulated in the liver when cationic lipoplexes were injected immediately after CS. However, suppression of apolipoprotein B (ApoB) mRNA expression in the liver and reduction of low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) cholesterol level in the serum were observed only after sequential injection of CS plus ApoB siRNA lipoplex with cationic liposomes composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide (DC-1-18)/DOPE. Based on these findings, the type of cationic lipid strongly affected the gene-silencing effect of siRNA in the liver by sequential injection of CS plus cationic lipoplex.

Effect of cationic lipid in cationic liposomes on siRNA delivery into the lung by intravenous injection of cationic lipoplex

Cationic liposomes composed of dialkyl cationic lipid such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) can efficiently deliver siRNA to the lungs following the intravenous injection of cationic liposome/siRNA complexes (lipoplexes). In this study, we examined the effect of cationic lipid of cationic liposomes on siRNA delivery to the lungs after intravenous injection. We used six kinds of cationic cholesterol derivatives and 11 kinds of dialkyl or trialkyl cationic lipids as cationic lipids, and prepared 17 kinds of cationic liposomes composed of a cationic lipid and 1,2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) for evaluation of siRNA biodistribution and in vivo gene silencing effects. Among cationic liposomes, those composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide (DC-1-18), 2-((1,5-bis(octadecyloxy)-1,5-dioxopentan-2-yl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-3-18D), 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12), or cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol) with DOPE exhibited high accumulation of siRNA in the lung and significant suppression of Tie2 mRNA expression after the intravenous injection of cationic lipoplexes with Tie2 siRNA. Furthermore, DC-1-16/DOPE and DC-1-18/DOPE lipoplexes with protein kinase N3 (PKN3) siRNA could suppress the tumour growth when intravenously injected into mice with lung LLC metastasis. These findings indicate that the siRNA biodistribution and in vivo knockdown efficiency after the intravenous injection of cationic lipoplexes were strongly affected by the type of cationic lipid of cationic liposomes.

Hyaluronate Targeted Solid Lipid Nanoparticles of Etoposide: Optimization and In Vitro Characterization

The aim of the present study was preparation of hyaluronan (HA) targeted solid lipid nanoparticles (SLNs) of etoposide. SLNs were prepared by an emulsification‐solvent evaporation method and physically coated with HA. Four variables, including the ratio of cetyl alcohol to cationic lipid, cationic lipid type (stearylamine (SA) or dodecylamine (DDA)), lipid to HA ratio, and organic to aqueous phase ratio, were studied in an irregular fraction factorial design. Four responses, including particle size, zeta potential, drug loading, and 24‐hour release efficiency percent, were measured for each formulation and then the optimization was carried out. The percent of HA coated on the SLNs was calculated by CHN elemental analysis which was shown to be about 55.89%. The cationic lipid type and the ratio of cetyl alcohol to cationic lipid had the highest influence on particle size and zeta potential, respectively. The highest effects of the ratio of lipid to HA and the organic to aqueous phase ratio were on the drug loading efficiency of SLNs. The optimized formulation of SLNs was obtained by SA, the equal proportion of cetyl alcohol and cationic lipid, the ratio of 1.5 for lipid to HA, and 10% of organic phase to aqueous phase.

PDNA-loaded Bubble liposomes as potential ultrasound imaging and gene delivery agents

We have developed polyethyleneglycol (PEG)-modified liposomes (Bubble liposomes; BLs) that entrap ultrasound (US) contrast gas, and we have reported that the combination of BLs and US exposure was an effective tool for delivering pDNA and siRNA in vitro and in vivo. In this study, we prepared pDNA-loaded BLs using three types of cationic lipids to enhance the US imaging effect and the transfection efficiency via systemic injection. We investigated the US imaging abilities of these BLs, their protective effects on pDNA from serum component, and their transfection effects in vitro and in vivo. As a result, we demonstrated that the US imaging ability and transfection effect varied with lipid component and that p-BLs containing DSDAP could be the most stable and effective tool the among three types of p-BLs. Indeed, in ischemic muscle, p-BLs containing DSDAP could be detected using diagnostic US and could deliver bFGF-expressing pDNA using therapeutic US, leading to the induction of angiogenic factors and the improvement of blood flow. These results suggest that combining p-BLs with US exposure may be useful for stable US imaging and efficient gene delivery and may lead to the establishment of a theranostic approach, which is a combination of disease diagnosis and therapy.

Trimethylarsonium-Based Cationic Phospholipids for Gene Delivery

Lipophosphoramide-based cationic lipids are a class of synthetic vectors used for gene delivery that can be produced in multigram scale. The use of trimethylarsonium moiety as a cationic polar head was beneficial to produce efficient gene delivery vectors for in vivo applications. Moreover, this type of cationic lipid can also exhibit some bactericidal effects.

Influence of Biological Media on the Structure and Behavior of Ferrocene-Containing Cationic Lipid/DNA Complexes Used for DNA Delivery

Biological media affect the physicochemical properties of cationic lipid-DNA complexes (lipoplexes) and can influence their ability to transfect cells. To develop new lipids for efficient DNA delivery, the influence of serum-containing media on the structures and properties of the resulting lipoplexes must be understood. To date, however, a clear and general picture of how serum-containing media influences the structures of lipoplexes has not been established. Some studies suggest that serum can disintegrate lipoplexes formed using certain types of cationic lipids, resulting in the inhibition of transfection. Other studies have demonstrated that lipoplexes formulated from other lipids are stable in the presence of serum and are able to transfect cells efficiently. In this article, we describe the influence of serum-containing media on lipoplexes formed using the redox-active cationic lipid bis(n-ferrocenylundecyl)dimethylammonium bromide (BFDMA). This lipoplex system promotes markedly decreased levels of transgene expression in COS-7 cells as serum concentrations are increased from 0 to 2, 5, 10, and 50% (v/v). To understand the cause of this decrease in transfection efficiency, we used cryogenic transmission electron microscopy (cryo-TEM) and measurements of zeta potential to characterize lipoplexes in cell culture media supplemented with 0, 2, 5, 10, and 50% serum. Cryo-TEM revealed that in serum-free media BFDMA lipoplexes form onionlike, multilamellar nanostructures. However, the presence of serum in the media caused disassociation of the intact multilamellar lipoplexes. At low serum concentrations (2 and 5%), DNA threads appeared to separate from the complex, leaving the nanostructure of the lipoplexes disrupted. At higher serum concentration (10%), disassociation increased and bundles of multilamellae were discharged from the main multilamellar complex. In contrast, lipoplexes characterized in serum-free aqueous salt (Li(2)SO(4)) medium and in OptiMEM cell culture medium (no serum) did not exhibit significant structural changes. The zeta potentials of lipoplexes in serum-free media (salt medium and cell culture medium) were similar (e.g., approximately -35 mV). Interestingly, the presence of serum caused the zeta potentials to become less negative (about -20 mV in OptiMEM and -10 mV in Li(2)SO(4)), even though serum contains negatively charged entities that have been demonstrated to lead to more negative zeta potentials in other lipoplex systems. The combined measurements of zeta potential and cryo-TEM are consistent with the proposition that DNA threads separate from the lipoplex in the presence of serum, resulting in a decrease in the net negative charge of the surface of the lipoplex.

Synthesis of AZT-based cationic lipids and in vitro evaluation of siRNA delivery

New types of cationic lipids based on AZT were evaluated for their biological effects in terms of their cytotoxicity and their cellular delivery of siRNA.

Synthesis of a polyamidoamine dendron-bearing lipid having sugar moieties and its use for preparation of nonviral gene vectors

We have so far developed a new type of cationic lipid that consists of an amine-terminated poly(amidoamine) dendron and two long alkyl chains. In this study, we designed a dendron-bearing lipid (DL) modified with sugar moieties as the ligand at the terminal groups of the dendron to improve the transfection activities. We combined lactobionic acid at the end of every dendritic branch of the DL to produce galactose moieties-attached DL (Gal-DL). The lipoplexes containing Gal-DL exhibited enhancement of transfection of HepG2 cells. Moreover, Gal-DL significantly promoted the transfection activity of the lipoplexes including poly(ethylene glycol)-attached dendron-bearing lipid (PEG-DL).