Synthesis of novel cholesterol-based cationic lipids for gene delivery

The new cholesterol-based cationic lipids B, C, and D with an ether linked spacer were synthesized by using aminopropyl chain extension with acrylonitrile. The cholesterol-based cationic lipid A with carbamoyl linkage were also synthesized in order to compare the difference in transfection efficiency of the two linkage types. To this end, GFP expression of these cationic lipids was confirmed respectively.     

Facile synthesis of carbon-11-labeled cholesterol-based cationic lipids as new potential PET probes for imaging of gene delivery in cancer

Gene therapy based on gene delivery is a promising strategy for the treatment of various human diseases such as cancer. Cationic lipids represent one of the important synthetic gene delivery systems. There is a great interest in imaging of gene therapy using the biomedical imaging technique positron emission tomography (PET). Carbon-11-labeled cholesterol-based cationic lipids were first designed and synthesized as new potential PET probes for imaging of gene delivery in cancer. The [¹¹C-methyl]quaternary amine target tracers, N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]pyrrolidinium iodide ([¹¹C]4a), N-[¹¹C]methyl-N′-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]imidazolium iodide ([¹¹C]4b), N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]piperidinium iodide ([¹¹C]4c), N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]-4-methylpiperidinium iodide ([¹¹C]4d), and N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]morpholinium iodide ([¹¹C]4e), were prepared from their corresponding tertiary amine precursors with [¹¹C]methyl iodide ([¹¹C]CH₃I) through N-[¹¹C]methylation and isolated by a simplified solid-phase extraction (SPE) method using a Silica Sep-Pak cartridge in 50-60% radiochemical yields decay corrected to end-of-bombardment (EOB), based on [¹¹C]CO₂, and 111-185 GBq/μmol specific activity at the end of synthesis (EOS).     

In vitro delivery of curcumin with cholesterol-based cationic liposomes

A new cholesterol-based cationic lipid was synthesized; liposomes prepared on its basis were evaluated as drug delivery vehicles for curcumin. Free and liposome-encapsulated curcumin cytotoxicity against HeLa, A549, HepG2, K562 and 1301 cell lines was assessed. Liposomal curcumin with ED₅₀ values ranging from 2.5–10 μM exhibited 2–8 times higher cytotoxicity than free curcumin. The synthetic cholesterol-based cationic lipid also enhanced cellular uptake of curcumin into tested cells. Cationic liposome alone showed low cytotoxicity at high doses with ED₅₀ values of 90–210 μM.     

Synthesis and optimization of cholesterol-based diquaternary ammonium Gemini Surfactant (Chol-GS) as a new gene delivery vector

Amongst a number of potential nonviral vectors, cationic liposomes have been actively researched, with both gemini surfactants and bola amphiphiles reported as being in possession of good structures in terms of cell viability and in vitro transfection. In this study, a cholesterol-based diquaternary ammonium gemini surfactant (Chol-GS) was synthesized and assessed as a novel nonviral gene vector. Chol-GS was synthesized from cholesterol by way of four reaction steps. The optimal efficiency was found to be at a weight ratio of 1:4 of lipid:DOPE (1,2-dioleoyl-L-alpha- glycero-3-phosphatidylethanolamine), and at a ratio of between 10:1~15:1 of liposome:DNA. The transfection efficiency was compared with commercial liposomes and with Lipofectamine, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE-C), and N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTAP). The results indicate that the efficiency of Chol-GS is greater than that of all the tested commercial liposomes in COS7 and Huh7 cells, and higher than DOTAP and Lipofectamine in A549 cells. Confirmation of these findings was observed through the use of green fluorescent protein expression. Chol-GS exhibited a moderate level of cytotoxicity, at optimum concentrations for efficient transfection, indicating cell viability. Hence, the newly synthesized Chol-GS liposome has the potential of being an excellent nonviral vector for gene delivery.     

Physicochemical characteristics associated with transfection of cationic cholesterol-based gene delivery vectors in the presence of DOPE

The physicochemical properties of a novel series of cholesterol-based cationic lipids in the presence of DOPE were studied by various techniques in an effort to correlate cationic lipid structure with transfection efficacy. It was found that while DOPE improves the β-gal activity of the active AC and MC derivatives, the overall zeta potential of the particles, pDNA complexation and condensation is not improved. This is in stark contrast with the tertiary amine derivative DC whose dispersion properties were improved and its monolayer surface potential is restored at high molecular surface density in the presence of DOPE. Overall the transfection activity mediated by DC and the quaternary ammonium TC derivative was greatly improved in the presence of DOPE and is attributed to decreased cytotoxicity, improved fusogenicity and cellular association.     

Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-L-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.     

Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-l-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.     

Transfection properties of stabilized plasmid-lipid particles containing cationic PEG lipids

Recent work has shown that plasmid DNA can be efficiently encapsulated in well-defined "stabilized plasmid-lipid particles" (SPLP) that have potential as systemic gene therapy vehicles [Gene Ther. 6 (1999) 271]. In this work, we examine the influence of ligands that enhance cellular uptake on the transfection potency of SPLP. The ligand employed is a cationic poly(ethylene glycol) (PEG) lipid (CPL) consisting of a lipid anchor and a PEG(3400) spacer chain with four positive charges at the end of the PEG (CPL(4)). It is shown that up to 4 mol% CPL(4) can be inserted into preformed SPLP, resulting in up to 50-fold enhancements in uptake into baby hamster kidney (BHK) cells. The addition of Ca(2+) to SPLP-CPL(4) (CPL(4)-incorporated SPLP) results in up to 10(6)-fold enhancements in transgene expression, as compared to SPLP in the absence of either CPL(4) or Ca(2+). These transfection levels are comparable to those observed for plasmid DNA-cationic lipid complexes (lipoplexes) but without the cytotoxic effects noted for lipoplex systems. It is concluded that in the presence of Ca(2+) and appropriate ligands to stimulate uptake, SPLP are highly potent transfection agents.     

Molecular shape of the cationic lipid controls the structure of cationic lipid/dioleylphosphatidylethanolamine-DNA complexes and the efficiency of gene delivery

Pyridinium amphiphiles, abbreviated as SAINT, are highly efficient vectors for delivery of DNA into cells. Within a group of structurally related compounds that differ in transfection capacity, we have investigated the role of the shape and structure of the pyridinium molecule on the stability of bilayers formed from a given SAINT and dioleoylphosphatidylethanolamine (DOPE) and on the polymorphism of SAINT/DOPE-DNA complexes. Using electron microscopy and small angle x-ray scattering, a relationship was established between the structure, stability, and morphology of the lipoplexes and their transfection efficiency. The structure with the lowest ratio of the cross-sectional area occupied by polar over hydrophobic domains (SAINT-2) formed the most unstable bilayers when mixed with DOPE and tended to convert into the hexagonal structure. In SAINT-2-containing lipoplexes, a hexagonal topology was apparent, provided that DOPE was present and complex assembly occurred in 150 mm NaCl. If not, a lamellar phase was obtained, as for lipoplexes prepared from geometrically more balanced SAINT structures. The hexagonal topology strongly promotes transfection efficiency, whereas a strongly reduced activity is seen for complexes displaying the lamellar topology. We conclude that in the DOPE-containing complexes the molecular shape and the nonbilayer preferences of the cationic lipid control the topology of the lipoplex and thereby the transfection efficiency.     

Solid phase synthesis of novel asymmetric hydrophilic head cholesterol-based cationic lipids with potential DNA delivery

Twenty-four asymmetric divalent head group cholesterol-based cationic lipids were designed and synthesized by parallel solid phase chemistry. These asymmetric head groups composed of amino functionality together with trimethylamino, di(2-hydroxyethyl)amino or guanidinyl groups. Spacers between cationic heads and linker were both equal and unequal in length. These lipids were subjected to evaluation for DNA binding affinities by gel retardation assay and were screened for their transfection efficiency on HEK293 cells. Cationic lipids with equal chain length exhibited high transfection efficiency when polar part contained asymmetric polar heads. In contrast, lipids with unequal chain length exhibited high transfection efficiency when polar part contained symmetric heads. According to the optimal formulation, seven lipids exhibited higher transfection efficiency than the commercially available transfection agents, Effectene?, DOTAP and DC-Chol, to deliver DNA into PC3 human prostate adenocarcinoma cells. 3β-[N-(N′-Guanidinyl)-2′-aminoethyl)-N-(2-aminoethyl)carbamoyl] cholesterol (5) bearing amino and guanidinyl polar heads exhibited highest transfection efficiency with minimal toxicity. The morphology of active liposomes was observed by transmission electron microscopy (TEM) and size of liposomes were around 200–700 nm.     

Transfer into a mesothelioma cell line of tumor suppressor gene p16 by cholesterol-based cationic lipids

In this work, the tumor suppressor gene p16 was efficiently transferred into FR cells isolated from a patient with malignant mesothelioma using cationic liposomes prepared from trimethyl aminoethane carbamoyl cholesterol (TMAEC-Chol) and triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol). This transfer was performed after preliminary assays were undertaken to find the optimal transfection conditions. Results showed that an efficient transfer of plasmids containing the reporter gene pCMV-beta galactosidase vectorized by TMAEC-Chol/DOPE and TEAPC-Chol/DOPE liposomes into mesothelioma FR cells was obtained as assessed by luminometric measurements of beta-galactosidase activity. Cytotoxicity studied by MTT test showed that at concentrations used for this study, the cationic liposomes have no effect on cell growth. Transfer into mesothelioma FR cells of a plasmid construct containing the tumor suppressor gene p16 was carried out with these liposomes. Western blotting and immunofluorescence showed the presence of p16 in treated cells. An inhibition of cell growth was observed, indicating that efficient tumor suppressor gene transfer can be performed by using cationic liposomes.     

阳离子聚合物在非病毒基因转染中的研究进展

基因治疗为治疗先天性遗传疾病和严重后天获得性疾病提供了一条新途径.目前,基因载体分为两类:病毒载体和非病毒载体.病毒载体转染效率高,但由于某些病毒载体存在免疫原性、致癌性、宿主DNA插入整合等缺点,从而限制了它们的应用.非病毒载体具有价格低、制备简单、安全有效、无免疫原性等优点,成为基因载体研究的热点.阳离子多聚物是非病毒载体的典型代表.文中综述近年来阳离子多聚物作为基因载体的研究现状和进展,重点介绍了阳离子多聚物基因载体的分类和与DNA的相互作用和传递机制.     

阳离子脂质体运载基因的跨膜机制

阳离子脂质体等非病毒载体以其制备简单、低毒性、低免疫原性、可生物降解等优点,成为近年来基因转运中的常用载体。理解阳离子脂质体运载基因的机制对阳离子脂质体的研究具有重要意义。从跨膜机制和信号调控的角度,介绍了脂质体/DNA复合体以特定构象避免细胞外基质中核酸酶的降解,跨越细胞膜进入细胞的过程;阐明了DNA在信号调控的作用下,逃离溶酶体并安全释放的机制;讨论了基因穿过核被膜进入到细胞核的方式,为进一步阐明阳离子脂质体运载基因的分子机制奠定基础。     

Novel ortho ester-based,pH-sensitive cationic lipid for gene delivery in vitro and in vivo

Context: Cationic lipoplexes are less toxic than viral gene vectors and more convenient to prepare but their efficiencies of gene delivery are generally lower.

Objective: To develop ortho ester-based, pH-sensitive lipoplexes for efficient gene delivery both in cultured cells and in vivo.

Materials and methods: A novel cationic and acid-labile lipid (DOC) containing a cationic headgroup and a cholesterol-derived lipid tail joined together by an acid-labile ortho ester linker was designed and synthesized. DOC was formulated into liposomes with the conical helper lipid DOPE, and then into lipoplexes with plasmid DNA encoding a luciferase reporter gene. The physicochemical properties of the lipoplexes (size, surface charge and pH-sensitivity) were characterized. Gene delivery by DOC/DOPE/DNA lipoplexes was also evaluated in CV-1 cells and in CD-1 mice following intratracheal injection. Lipoplexes consisting of the acid-stable cationic lipid DC-Chol were characterized as a control.

Results: DOC formed cationic lipoplexes with DOPE and DNA. After incubation at acidic pH 4.6, DOC/DOPE/DNA lipoplexes lost their positive charges and aggregated with one another as a result of DOC hydrolysis. Both in CV-1 cell culture and in CD-1 mice, DOC/DOPE/DNA lipoplexes increased the luciferase gene expression by 5- to 10-fold compared with the analogous but acid-stable DC-Chol/DOPE/DNA lipoplexes.

Discussion and conclusion: Incorporation of an acid-labile ortho ester linker into a cationic lipid is a viable approach to enhance gene delivery by the corresponding lipoplexes both in cultured cells and in vivo.     

Shell-cross-linked micelles containing cationic polymers synthesized via the RAFT process: toward a more biocompatible gene delivery system

Block copolymers poly(2-(dimethylamino) ethyl methacrylate)-b-poly(polyethylene glycol methacrylate) (PDMAEMA-b-P(PEGMA)) were prepared via reversible addition fragmentation chain transfer polymerization (RAFT). The polymerization was found to proceed with the expected living behavior resulting in block copolymers with varying block sizes of low polydispersity (PDI <1.3). The resulting block copolymer was self-assembled in an aqueous environment, leading to the formation of pH-responsive micelles. Further stabilization of the micellar system was performed in water using ethylene glycol dimethacrylate and the RAFT process to cross-link the shell. The cross-linked micelle was found to have properties significantly different from those of the uncross-linked block copolymer micelle. While a distinct critical micelle concentration (CMC) was observed using block copolymers, the CMC was absent in the cross-linked system. In addition, a better stability against disintegration was observed when altering the ionic strength such as the absence of changes of the hydrodynamic diameter with increasing NaCl concentration. Both cross-linked and uncross-linked micelles displayed good binding ability for genes. However, the cross-linked system exhibited a slightly superior tendency to bind oligonucleotides. Cytotoxicity tests confirmed a significant improvement of the biocompatibility of the synthesized cross-linked micelle compared to that of the highly toxic PDMAEMA. The cross-linked micelles were taken up by cells without causing any signs of cell damage, while the PDMAEMA homopolymer clearly led to cell death.     

Cationic lipid polymerization as a novel approach for constructing new DNA delivery agents

In vivo gene delivery mediated by cationic lipids is often compromised by aggregation due to complexation with proteins in the blood. To improve the stability of cationic lipid-DNA complexes, the present study aimed to develop a novel approach in which a poly(cationic lipid) (PCL) is utilized to form stable cationic polyplexes for gene transfection. Hydrogenation of the acrylamide analogue of betaAE-DMRI, the polymerizable precursor of PCL, provided a monomeric lipid derivative (MHL) which was used for direct comparison of corresponding lipoplex stability, toxicity, and transfection activity. Various formulations of cationic liposomes, such as MHL, MHL-cholesterol (Chol), PCL, PCL-Chol, DOTAP-Chol, and commercially available lipofectamine were generated and examined in this study. The new poly(cationic lipid) did not display any significant toxicity to rat hepatocytes or Hep G2 cells as indicated by an LDH leakage assay. Furthermore, PCL was significantly less toxic than MHL, DOTAP-Chol or lipofectamine. Suspensions of PCL were resistant to aggregation even after 24 h of exposure to solutions containing 50 and 100% fetal bovine serum (FBS). In contrast, suspensions of lipofectamine extensively aggregated after 24 h of exposure to 50% FBS. To examine the influence of lipid polymerization on gene transfer activity, liposome-mediated transfections of a luciferase vector (pGL3) were performed in Hep G2 and Alexander cell lines. The luciferase activity of the PCL formulations in Hep G2 cells were similar to those of the MHL, DOTAP-Chol and lipofectamine formulations, demonstrating that lipid polymerization does not compromise transfection activity. In comparison to the monomeric precursor MHL and to the industry transfection standards DOTAP and lipofectamine, the novel poly(cationic lipid) exhibited the lowest cytotoxicity, was the most resistant to serum-induced aggregation and had comparable transfection activity when coformulated with cholesterol. This novel polymerization approach for the development of stable and active polyplexes may prove a valuable alternative for in vivo gene delivery.     

Development of a nonviral gene delivery vehicle for systemic application

Polycation vehicles used for in vitro gene delivery require alteration for successful application in vivo. Modification of polycations by direct grafting of additional components, e.g., poly(ethylene glycol) (PEG), either before or after DNA complexation, tend to interfere with polymer/DNA binding interactions; this is a particular problem for short polycations such as linear, beta-cyclodextrin-containing polycations (betaCDPs). Here, a new method of betaCDP polyplex (polycation/DNA composite structures) modification is presented that exploits the ability to form inclusion complexes between cyclodextrins and adamantane. Surface-PEGylated betaCDP polyplexes are formed by self-assembly of the polyplexes with adamantane-PEG conjugates. While unmodified polyplexes rapidly aggregate and precipitate in salt solutions, the PEGylated betaCDP polyplexes are stable at conditions of physiological salt concentration. Addition of targeting ligands to the adamantane-PEG conjugates allows for receptor-mediated delivery; galactosylated betaCDP-based particles reveal selective targeting to hepatocytes via the asialoglycoprotein receptor. Galactosylated particles transfect hepatoma cells with 10-fold higher efficiency than glucosylated particles (control), but show no preferential transfection in a cell line lacking the asialoglycoprotein receptor. Thus, surface modification of betaCDP-based polyplexes through the use of cyclodextrin/adamantane host/guest interactions endows the particles with properties appropriate for systemic application.