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.     

Mitochondria transfection by oligonucleotides containing a signal peptide and vectorized by cationic liposomes.

The progress of research in gene therapy allows hope for treatment of mitochondrial genetic disorders provided that efficient methods for gene transfer into mitochondria can be found. In this work, we have used an oligonucleotide coupled covalently to a mitochondria-targeted peptide at one end and a cationic liposome prepared from trimethyl aminoethane carbamoyl cholesterol iodide (TMAEC-Chol) to carry it in living cells. With a fluorescent probe to label the oligonucleotide at the other end and by means of confocal microscopy, we show that such modified oligonucleotides complexed to liposomes enter into the cytoplasm of human fibroblasts in primary culture, and then, after dissociation from the complexes, they penetrate into the mitochondria. The fluorescence was still observed after 8 days, suggesting the continued presence of oligonucleotides. At the concentrations used for this study, the cationic liposomes have practically no effect on cell growth, as revealed by the MTT assay.     

Cellular pathway of plasmids vectorized by cholesterol-based cationic liposomes.

We investigated by transmission electron microscopy the cellular route in tumor MCF7 cells of DNA labeled with digoxigenin, carried by cationic liposomes (Lip+) prepared from TMAEC-Chol [3 beta(N-(N',N',N'-trimethylaminoethane)-carbamoyl)cholesterol iodide] and TEAPC-Chol [3 beta(N-(N',N',N'-triethylaminopropane)-carbamoyl)cholesterol iodide], two cholesterol-based cationic lipids containing a quaternary ammonium. In a previous work we showed the pathway of cationic lipid/plasmid complexes from the beginning of endocytosis until their entry into the perinuclear area. Beyond this limit, unlabeled exogenous plasmids cannot be distinguished with nuclear DNA. This work dealt with the cellular fate of cationic liposome-vectorized plasmids labeled with digoxigenin using an immunogold procedure. Early after the beginning of transfection (30 min, 1 hr, 5 hr), gold particles were observed only in the cytoplasm and in endosome-like vesicles, whereas after 24 hr gold particles were densely present in the nucleus. These results demonstrate the nuclear localization of plasmids vectorized by the cationic liposomes used. The results are discussed in comparison with transfection efficiency measurements.     

Induction of alloreactive cytotoxic T lymphocytes by intra-splenic immunization with allogeneic class I Major Histocompatibility Complex DNA and DC-chol cationic liposomes

Abstract

A simple strategy for designing a cancer immunotherapeutic system involves modification of tumor cells from tumor-bearing animals in vivo in such a way that the host can evoke a specific immune response against them. We have expressed allogeneic class I major histocompatibility complex (MHC) molecules on tumor cells, through ex vivo DNA-mediated gene transfer. These molecules are potent immuno-modulators for the stimulation of strong immune reactions against certain malignancies. In order to achieve efficient gene delivery to tumor cells in vivo we have compared the efficiencies of gene transfer into mammalian tumor cells by the biolistic particle delivery system and cationic liposomes. In this report, we have demonstrated that cationic liposomes prepared by DC-chol and DOPE gives the best efficiency of transfection for tumor cells in vivo. We also showed that a strong anti-H-2K^b allo-reactive cytotoxic T lymphocyte (CTL) response could be generated following in vivo immunization of AKR/J mouse spleens with the H-2K^b gene and DC-chol cationic liposomes. The direct immunization of mouse spleens to induce cell-mediated immunity against exogenous antigens may allow alternative treatment strategies for cancer immunotherapy.     

Example of fatty acid-loaded lipoplex in enhancing in vitro gene transfer efficacies of cationic amphiphile

Herein, we report on the design and synthesis of a novel nontoxic cationic amphiphile N,N-di-n-tetradecyl-N-[2-[N',N'-bis(2-hydroxyethyl)amino]ethyl]-N-(2-hydroxyethyl)ammonium chloride (lipid 1) whose in vitro gene transfer efficacies in CHO, COS-1, MCF-7, and HepG2 cells are remarkably enhanced when used in combination with 30 mole percent added myristic acid. Reporter gene expression assay using p-CMV-SPORT-beta-gal reporter gene revealed poor gene transfer properties of the cationic liposomes of lipid 1 and cholesterol (colipid). However, the in vitro gene delivery efficacies of lipid 1 were found to be remarkably enhanced when the cationic liposomes of lipid 1 and cholesterol were prepared in the presence of 30 mole percent added myristic acid (with respect to lipid 1) as the third liposomal ingredient. The whole cell histochemical X-gal staining of representative CHO cells further confirmed the significantly enhanced gene transfer properties of the fatty acid-loaded cationic liposomes of lipid 1 and cholesterol. Electrophoretic gel patterns in the gel mobility shift assay supports the notion that better DNA release from fatty acid lipoplexes might play a role in their enhanced gene transfer properties. In addition, such myristic acid-loaded lipoplexes of lipid 1 were also found to be serum-compatible up to 30% added serum. Taken together, our present findings demonstrate that the transfection efficacies of fatty acid-loaded lipoplexes are worth evaluating particularly when traditional cationic liposomes prepared with either cholesterol or DOPE colipids fail to transfect cultured cells.     

Efficient gene transfection by bisguanylated diacetylene lipid formulations

We have previously shown that cationic cholesterol derivatives bearing guanidinium groups were efficient vectors for gene transfer. To further evaluate the potentiality of this novel class of cationic lipids, we undertook to study the transfection efficiency of guanidinium-based lipids with other hydrophobic moieties. Specifically, we synthesized a reagent where two guanidinium groups are linked to a diacetylene lipid which may provide the lipoplexes with favorable structural features. We report here that the cationic lipid bisguanidinium-diacetylene (BGDA) is highly efficient for in vitro gene transfection when formulated with dioleoylphosphatidyl ethanolamine (DOPE). We also show that liposomes composed of BGDA, DOPE, and a neutral diacetylene colipid, hydroxyethylenediacetylene (HEDA), are efficient for transfection. Thus, diacetylene-based lipids provide a novel scaffold for gene transfection and will be particularly useful for gaining new insights into the structure-activity relationships of the lipid/DNA complexes as they offer a means to study the effects of polymerizable domains.     

3β [N-(N′, N′-Dimethylaminoethane)-Carbamoyl] Cholesterol (DC-Chol)-Mediated Gene Delivery to Primary Rat Neurons: Characterization and Mechanism

Cationic lipid formulations consisting of 3 [N-(N, N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the helper lipid dioleoylphosphatidylethanolamine (DOPE) (1.5:1 molar ratio) were prepared by solvent evaporation and sized by high pressure extrusion. Liposomes made of 1:1 molar ratio 1,2-dioleoyl-3-trimethyl-ammonium-propane (DOTAP)/DOPE were used as controls in the study. The two formulations were characterized and evaluated for their efficiency in transfecting SKnSH (neuroblastoma) and primary rat neuronal cell lines. DC-Chol/DOPE liposomes were more efficient at transfecting both the SKnSH and the primary rat neuronal cells and also less toxic compared to the DOTAP/DOPE liposomes. The cellular-associated signal of rhodamine-labeled DC-Chol/DOPE liposomes into SKnSH and primary rat neuronal cells was higher than the rhodamine-labeled DOTAP/DOPE liposomes. These results demonstrate that DC-Chol/DOPE cationic liposomes provide an efficient vehicle for the delivery of plasmids into SKnSH and primary neuronal cells compared to DOTAP/DOPE liposomes. DC-Chol/DOPE liposomes may provide a good non-viral candidate for transfecting primary rat neuronal cells.     

Efficient Gene Delivery Using Anionic Liposome-Complexed Polyplexes (LPDII)

Synthetic gene transfer vectors based on polyplexes complexed to anionic liposomes (LPDII vectors) were characterized for their transfection efficiency in cultured mammalian cells. The effects of polycation to DNA ratio, lipid to DNA ratio, choice of polycation and lipid composition were systematically evaluated in human oral carcinoma KB cells, using a luciferase reporter gene. For LPDII formulations containing poly-L-lysine and dioeoylphosphatidylethanolamine/cholesteryl hemisuccinate (DOPE/CHEMS) anionic liposomes, at a constant lipid to DNA ratio, an increase in the polycation/DNA (N/P) ratio resulted in an increase in transfection activity. Meanwhile, the optimal lipid to DNA ratio for efficient gene delivery was influenced by the N/P ratio used, and was increased at higher N/P ratios. For the DNA condensing agent, poly-L-lysine could be replaced by polyethylenimine (PEI) as the DNA condensing agent in the formulations. For the lipidic components, CHEMS could be replaced by other anioniclipids including oleic acid, dicetylphosphate and phosphatidylserine, but DOPE, a fusogenic helper lipid, could not be replaced by dioleolyphosphatidylcholine. LPDII formulation showed significantly less cytotoxicity compared to the commonly used cationic lipsomes or PEI mediated transfection and several cell lines were transfected with high efficiency. LPDII vectors avoid the use of toxic cationic lipids and may have potential application in gene therapy.     

Cationic Liposomes Enhance the Rate of Transduction by a Recombinant Retroviral Vector In Vitro and In Vivo

Cationic liposomes enhanced the rate of transduction of target cells with retroviral vectors. The greatest effect was seen with the formulation DC-Chol/DOPE, which gave a 20-fold increase in initial transduction rate. This allowed an efficiency of transduction after brief exposure of target cells to virus plus liposome that could be achieved only after extensive exposure to virus alone. Enhancement with DC-Chol/DOPE was optimal when stable virion-liposome complexes were preformed. The transduction rate for complexed virus, as for virus used alone or with the polycation Polybrene, showed first-order dependence on virus concentration. Cationic liposomes, but not Polybrene, were able to mediate envelope-independent transduction, but optimal efficiency required envelope-receptor interaction. When virus complexed with DC-Chol/DOPE was used to transduce human mesothelioma xenografts, transduction was enhanced four- to fivefold compared to that for virus alone. Since the efficacy of gene therapy is dependent on the number of cells modified, which is in turn dependent upon the balance between transduction and biological clearance of the vector, the ability of cationic liposomes to form stable complexes with retroviral vectors and enhance their rate of infection is likely to be important for in vivo application.     

Symmetrical cationic triglycerides: an efficient synthesis and application to gene transfer

Some cationic triglycerides 1Aa-1Cb which have a symmetrical structure were effectively synthesized and formulated into cationic liposomes with the co-lipid dioleoylphosphatidylethanolamine (DOPE) and/or dilauroylphosphatidylcholine (DLPC). The plasmid encoding a luciferase was delivered into CHO cells by using these cationic liposomes. Our symmetrical cationic triglycerides showed high transfection activity when DOPE was used as a co-lipid. Among the symmetrical cationic triglycerides synthesized here, 1Ab and 1Ac, which have an oleoyl group at the 1- and 3-position in the glycerol backbone and also have a relatively long linker connecting the 2-hydroxy group in glycerol with the quaternary ammonium head group, were found to be the most suitable for gene delivery into cells. The transfection activity of the symmetrical cationic triglyceride 1Ab was comparable with that of its asymmetrical congener 6 and several times higher than that of Lipofectin.     

Delivery and pathway in MCF7 cells of DNA vectorized by cationic liposomes derived from cholesterol

We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.     

Gene transfer mediated by YKS-220 cationic particles: convenient and efficient gene delivery reagent.

A monocationic lipid, YKS-220, with a symmetrical and biodegradable structure can be used as an effective gene transfer vector in a cationic particle form (not a cationic liposome form), and is obtained by diluting an ethanol solution of YKS-220 and DOPE (1:5, molar ratio) with an aqueous medium. This preparation method is more convenient than that for cationic liposomes. YKS-220 cationic particles showed a heterogeneous large mean diameter of 4.4 microm. An obvious size change was not observed when plasmid DNA was added. The transfection activity of YKS-220 cationic particles was comparable to those of YKS-220 liposomes and DOSPA liposomes (LipofectAMINE), and even higher than that of DOGS (TRNSFECTAM). Interestingly, the YKS-220 cationic particle/DNA complexes were resistant to the neutralizing effect of serum. All of these findings indicate that YKS-220 cationic particles are a convenient and efficient gene delivery reagent.     

Cationic lipid gene transfer of an IL-2 transgene leads to activation of natural killer cells in a SCID mouse human tumor xenograft

Natural killer (NK) cells play an important role in combating infectious and malignant diseases and interleukin-2 (IL-2) has been shown to promote proliferation and activation of NK cells in vitro and in vivo. Here we investigate the effects of local cationic lipid-mediated IL-2 gene transfer on intratumoral accumulation and activation of NK cells in a SCID mouse tumor model. UM449 human melanoma tumors in SCID mice received intratumoral injections of DMRIE/DOPE admixed with VR1103, a DNA plasmid encoding the gene for human IL-2. Dissagregated tumor cells were tested for IL-2 secretion and were characterized using antibodies to asGM1, MAC-1, and F4/80 antigens. Granzyme A, a proteolytic serine esterase, was also measured in tumor cell lysates. IL-2 secretion from tumors injected with VR1103:DMRIE/DOPE peaked at 48 h after injection and fell to baseline levels on day 8. Intratumoral granzyme A activity was significantly increased in tumors injected with IL-2 plasmid:DMRIE/DOPE complexes, but not by an irrelevant plasmid DNA:DMRIE/DOPE control. Importantly, the growth of UM449 tumors was slowed in VR1103:DMRIE/DOPE-injected tumors. These results indicate that local cationic lipid-mediated gene transfer of IL-2 induces activation of intratumoral NK cells and slows tumor growth.     

Synthesis and evaluation of vitamin D-based cationic lipids for gene delivery in vitro

A new panel of steroidal cationic lipids has been synthesized for gene delivery. Using commercially available vitamin D2 (calciferol) or vitamin D3 (cholecalciferol) as hydrophobic motifs and a variety of cationic head groups as binding sites for negatively charged phosphate groups in DNA, we demonstrated that the transfection activity of the synthetic vitamin D-based cationic lipids 1d, 2d formulated with dioleoylphosphatidylethanolamine (DOPE) as a co-lipid is comparable to that of 3-(-[N-N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol). These synthetic lipids are effective in transfecting a variety of cell lines. These results suggest that vitamin D-based cationic lipids are useful transfection reagents for in vitro gene transfer studies.     

Using disaccharides to enhance in vitro and in vivo transgene expression mediated by a lipid-based gene delivery system

BACKGROUND: Lipid-based vectors have been widely applied to in vivo and in vitro gene delivery. Disaccharides can effectively stabilize lipid membranes. This study examined whether disaccharides could enhance the transgene expression mediated by lipid-based vectors. METHODS: Different disaccharides were incorporated into the vectors prepared with DOTAP/protamine/DNA (LPD) or with DNA/cationic liposomes containing DOTAP, DOTAP/Chol, DOTAP/DOPE, or DC-Chol/DOPE. The levels of transgene expression and internalized plasmid of CHO cells were represented by the percentages of GFP-positive cells and the fluorescence intensity of ethidium-monoazide covalently labeled plasmid, respectively. The vectors containing either cellobiose or trehalose were also intravenously injected into mouse tail vein to investigate the potentials of in vivo applications. RESULTS: For enhancing the transgene expression, cellobiose was found to be effective for all the vectors whereas maltose decreased the effectiveness of DOTAP/Chol liposomes and LPD. For the internalization of plasmid, most disaccharides were able to increase the cellular delivery of DOTAP, DOTAP/Chol, and DOTAP/DOPE liposomes, but caused decreases in the cellular entry of DC-Chol/DOPE liposomes. An approximately linear correlation between the internalized plasmid and the transgene expression was observed for all the treatments in this study. When the vectors were administered to mouse by intravenous injection, 10-fold and 3-fold increases in the luciferase expression of lung were observed for DOTAP liposomes containing 330 mM cellobiose and trehalose, respectively. CONCLUSIONS: This study showed that using trehalose and cellobiose with a lipid-based delivery system provides a straightforward approach to effectively enhance both in vitro and in vivo transgene expression.     

Synthesis and gene transfer activities of novel serum compatible cholesterol-based gemini lipids possessing oxyethylene-type spacers

Four novel cholesterol-based gemini cationic lipids differing in the length of oxyethylene-type spacers [-CH2-(CH2-O-CH2)n-CH2-] between each ammonium headgroup have been synthesized. These formed stable suspensions in aqueous media. Cationic liposomes were prepared from each of these lipids individually and as mixtures of cationic lipid and DOPE. These were used as nonviral gene delivery agents. All the cholesterol-based gemini lipids induced better transfection activity than their monomeric counterpart. Inclusion of DOPE in co-liposomal formulation of the cationic gemini lipid potentiates their gene transfer activity significantly. A major characteristic feature of these oxyethylene spacer based cholesterol gemini lipids was that serum does not inhibit the transfection activity of these gemini lipids, whereas the transfection activity of their monomeric counterpart decreased drastically in the presence of serum. One of the cholesterol-based gemini lipids 2a possessing a -CH2-CH2-O-CH2-CH2- spacer showed the highest transfection activity.     

Effect of human interferon β gene transfer upon human glioma, transplanted into nude mouse brain, involves induced natural killer cells

We investigated the immunological responses induced by human interferon β (IFNβ) gene transfer in human gliomas produced in the brains of nude mice. A suspension of human glioma U251-SP cells was injected into the brains of nude mice. The IFNβ gene was transferred by intratumoral injection with cationic liposomes or cationic liposomes associated with anti-glioma monoclonal antibody (immunoliposomes). When intratumoral injection of liposomes or immunoliposomes containing the human IFNβ gene was performed every second day for a total of six injections, starting 7 days after tumor transplantation, complete disappearance of the tumor was observed in six of seven mice that had received liposomes and in all seven mice receiving immunoliposomes. In addition, experimental gliomas injected with immunoliposomes were much smaller than those injected with ordinary liposomes following delayed injections beginning 14 days after transplantation. An immunohistochemical study of the treated nude mouse brains revealed a remarkable induction of natural killer (NK) cells expressing asialoGM1 antigen. To investigate the significance of NK cells in the antitumor effect, we injected liposomes or immunoliposomes containing the human IFNβ gene into tumors in nude mice depleted of NK cells by irradiation and anti-asialoGM1 antibody administration. The antitumor effect of the liposomes or immunoliposomes was abolished. Subsequent subcutaneous glioma challenge of the nude mice after intracerebral tumor implantation and gene transfer resulted in no subcutaneous tumor growth. These results suggest that the induction of NK cells is important in the cytocidal effect of liposomes or immunoliposomes containing the human IFNβ gene upon experimental gliomas. Received: 10 February 1998 / Accepted: 1 September 1998     

脂质体DC-Chol/DOPE对HEK293FT细胞的高效转染及其在腺病毒制备中的应用

重组病毒载体系统因为具有高效的基因转移能力得到了广泛应用,而病毒包装细胞的转染是重组病毒制备过程中的关键步骤。优化了脂质体DC-Chol/DOPE介导的转染常用的病毒包装细胞系HEK293FT的实验条件,比较了DC-Chol/DOPE、Lipofectamine2000和磷酸钙共沉淀法转染细胞的效率,并且比较了用DC-Chol/DOPE和磷酸钙共沉淀法转染293FT细胞制备重组腺病毒的结果,发现DC-Chol/DOPE对293FT细胞的转染效率以及最终收获的病毒滴度都远高于磷酸钙共沉淀法转染。所以,利用DC-Chol/DOPE转染293FT细胞制备重组病毒是一种简单、高效、成本低廉的方法。     

Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity.

Four different cationic derivatives of cholesterol were synthesized which contain either a tertiary or a quaternary amino head group, with and without a succinyl spacer-arm. Their ability to inhibit protein kinase C (PKC) activity was measured in a detergent mixed micellar solution. Derivatives containing a quaternary amino head group were effective inhibitors (Ki approx. 12 and 59 microM) of PKC and derivatives containing a tertiary amino head group were approx. 4-20-fold less inhibitory. Liposomes containing an equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and a cationic cholesterol derivative were tested for the DNA-mediated transfection activity in mouse L929 cells. Highest activity was found with the derivative with low PKC inhibitory activity and with a succinyl spacer-arm. The transfection activity of this tertiary amine derivative, N,N-dimethylethylenediaminyl succinyl cholesterol was dependent on DOPE as a helper lipid; liposomes containing dioleoylphosphatidylcholine and this derivative had little activity. The transfection protocol of this new cationic liposome reagent was optimized with respect to the ratio of liposome/DNA, dose of the complex and time of incubation with cells. Several adherent cell lines could be efficiently transfected with this liposome reagent without any apparent cytotoxicity. However, the transfection activity was strongly inhibited by the presence of serum components.