RESUMO
The design of modified oligonucleotides that combine in one molecule several therapeutically beneficial properties still poses a major challenge. Recently a new type of modified mesyl phosphoramidate (or µ-) oligonucleotide was described that demonstrates high affinity to RNA, exceptional nuclease resistance, efficient recruitment of RNase H, and potent inhibition of key carcinogenesis processes in vitro. Herein, using a xenograft mouse tumor model, it was demonstrated that microRNA miR-21-targeted µ-oligonucleotides administered in complex with folate-containing liposomes dramatically inhibit primary tumor growth via long-term down-regulation of miR-21 in tumors and increase in biosynthesis of miR-21-regulated tumor suppressor proteins. This antitumoral effect is superior to the effect of the corresponding phosphorothioate. Peritumoral administration of µ-oligonucleotide results in its rapid distribution and efficient accumulation in the tumor. Blood biochemistry and morphometric studies of internal organs revealed no pronounced toxicity of µ-oligonucleotides. This new oligonucleotide class provides a powerful tool for antisense technology.
Assuntos
Amidas/química , Antineoplásicos/farmacologia , MicroRNAs/genética , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Ácidos Fosfóricos/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Masculino , Melanoma/genética , Melanoma/patologia , Camundongos SCID , Terapia de Alvo Molecular , Oligonucleotídeos Antissenso/farmacocinética , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Unsymmetric lipophilic polyamine derivatives are considered as potential antitumor agents. Here, a series of novel symmetric lipophilic polyamines (LPAs) based on norspermine and triethylenetetramine (TETA) backbones bearing alkyl substituents with different lengths (from decyl to octadecyl) at C(1) atom of glycerol were synthesized. Performed screening of the cytotoxicity of novel compounds on the panel of tumor cell lines (MCF-7, KB-3-1, B16) and non-malignant fibroblasts hFF3 in vitro revealed a correlation between the length of the aliphatic moieties in LPAs and their toxic effects - LPAs with the shortest decyl substituent were found to exhibit the highest cytotoxicity. Furthermore, norspermine-based LPAs displayed somewhat more pronounced cytotoxicity compared with their TETA-based counterparts. Further mechanistic studies demonstrated that hit LPAs containing the norspermine backbone and tetradecyl or decyl substituents efficiently induced apoptosis in KB-3-1 cells. Moreover, decyl-bearing LPA inhibited motility and enhanced adhesiveness of murine B16 melanoma cells in vitro, showing promising antimetastatic potential. Thus, developed novel symmetric norspermine-based LPAs can be considered as promising anticancer chemotherapeutic candidates.
Assuntos
Poliaminas , Animais , Camundongos , Poliaminas/farmacologiaRESUMO
Upconverting nanoparticles have unique spectral and photophysical properties that make them suitable for development of theranostics for imaging and treating large and deep-seated tumors. Nanoparticles based on NaYF4 crystals doped with lanthanides Yb3+ and Er3+ were obtained by the high-temperature decomposition of trifluoroacetates in oleic acid and 1-octadecene. Such particles have pronounced hydrophobic properties. Therefore, to obtain stable dispersions in aqueous media for the study of their properties in vivo and in vitro, the polyethylene glycol (PEG)-glycerolipids of various structures were obtained. To increase the circulation time of PEG-lipid coated nanoparticles in the bloodstream, long-chain substituents are needed to be attached to the glycerol backbone using ether bonds. To prevent nanoparticle aggregation, an L-cysteine-derived negatively charged carboxy group should be included in the lipid molecule.
Assuntos
Nanopartículas , Polietilenoglicóis , Cisteína , Fluoretos/química , Nanopartículas/química , Ácido Oleico , Polietilenoglicóis/química , Ítrio/químicaRESUMO
Photo-controlled or photo-regulated molecules, especially biologically active and operating in physiological conditions, are in steady demand. Herein, furocoumaric and furocoumarinic acids being (Z/E)-isomers relative to each other were obtained in two stages starting from psoralen: the alkaline solvolysis of psoralen led to furocoumaric acid, which was further Z â E photoisomerized (365 nm) to furocoumarinic acid. The kinetics of Z â E photoisomerization was monitored by HPLC and UV-vis spectrophotometry. Photophysical characteristics in the aqueous phase for both acids, as well as the reversibility of (Z/E) photoisomerization process, were also assessed. Furocoumarinic acid was found to be visibly fluorescent at pH 2.0-12.0, with the maxima of fluorescence emission spectra being pH-dependent. The reverse E â Z photoisomerization predicted by quantum chemistry calculations as energetically favorable for the monoanionic form of furocoumarinic acid was proved in the experiment while being complicated by pyrone ring closure back to psoralen in acidic and neutral conditions. The preparative synthesis of furocoumarinic acid outlined in this work is particularly valuable in view of a wide range of pharmacological effects previously predicted for this compound.
Assuntos
Furocumarinas/química , Furocumarinas/efeitos da radiação , Luz , Ficusina/química , Fluorescência , Concentração de Íons de Hidrogênio , Isomerismo , Conformação Molecular , Pironas/química , Espectrofotometria UltravioletaRESUMO
Three novel polycationic gemini amphiphiles with different spacers were developed and evaluated in terms of their physiochemical properties and transfection efficiencies. Cationic liposomes formed by these amphiphiles and the helper lipid DOPE were able to successfully condense DNA, as shown by gel mobility shift and ethidium bromide intercalation assays. Transfection activity of the liposomes was superior to Lipofectamine® 2000 and was dependent on spacer structure, hydrophobicity, and nucleic acid type (pDNA or siRNA). We demonstrated that the cationic liposomes 2X6/DOPE and 2X7/DOPE are potential non-toxic vehicles for gene delivery.
Assuntos
DNA/administração & dosagem , Plasmídeos/administração & dosagem , Poliaminas/química , RNA Interferente Pequeno/administração & dosagem , Tensoativos/química , Transfecção/métodos , DNA/genética , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Lipossomos/química , Fosfatidiletanolaminas/química , Plasmídeos/genética , Polieletrólitos , RNA Interferente Pequeno/genéticaRESUMO
A novel redox-sensitive polycationic amphiphile (2S3) with disulphide linkers for nucleic acid delivery was developed. Cationic liposomes formed by 2S3 and the helper lipid DOPE demonstrated effective DNA delivery into HEK293 cells with a maximal transfection activity that is superior than both nonredox-sensitive cationic liposomes and Lipofectamine® 2000 at an N/P ratio of 6/1. Redox-sensitivity was tested by experiments with extracellular glutathione which shown the ability of disulphide linker degradation. Our results suggest that polycationic amphiphile 2S3 is a promising candidate for nucleic acid delivery.
Assuntos
DNA/genética , Desenho de Fármacos , Técnicas de Transferência de Genes , Poliaminas/química , Tensoativos/química , DNA/metabolismo , Células HEK293 , Humanos , Estrutura Molecular , Oxirredução , Poliaminas/síntese química , Polieletrólitos , Tensoativos/síntese química , TransfecçãoRESUMO
Over the past decade, mRNA-based therapy has displayed significant promise in a wide range of clinical applications. The most striking example of the leap in the development of mRNA technologies was the mass vaccination against COVID-19 during the pandemic. The emergence of large-scale technology and positive experience of mRNA immunization sparked the development of antiviral and anti-cancer mRNA vaccines as well as therapeutic mRNA agents for genetic and other diseases. To facilitate mRNA delivery, lipid nanoparticles (LNPs) have been successfully employed. However, the diverse use of mRNA therapeutic approaches requires the development of adaptable LNP delivery systems that can control the kinetics of mRNA uptake and expression in target cells. Here, we report effective mRNA delivery into cultured mammalian cells (HEK293T, HeLa, DC2.4) and living mouse muscle tissues by liposomes containing either 1,26-bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) or the newly applied 1,30-bis(cholest-5-en-3ß-yloxycarbonylamino)-9,13,18,22-tetraaza-3,6,25,28-tetraoxatriacontane tetrahydrochloride (2X7) cationic lipids. Using end-point and real-time monitoring of Fluc mRNA expression, we showed that these LNPs exhibited an unusually delayed (of over 10 h in the case of the 2X7-based system) but had highly efficient and prolonged reporter activity in cells. Accordingly, both LNP formulations decorated with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) provided efficient luciferase production in mice, peaking on day 3 after intramuscular injection. Notably, the bioluminescence was observed only at the site of injection in caudal thigh muscles, thereby demonstrating local expression of the model gene of interest. The developed mRNA delivery systems hold promise for prophylactic applications, where sustained synthesis of defensive proteins is required, and open doors to new possibilities in mRNA-based therapies.
RESUMO
Cationic liposomes are promising candidates for the delivery of various therapeutic nucleic acids. Here, we report a convenient synthesis of carbamate-type cationic lipids with various hydrophobic domains (tetradecanol, dialkylglycerol, cholesterol) and positively charged head-groups (pyridinium, N-methylimidazolium, N-methylmorpholinium) and data on the structure-transfection activity relationships. It was found that single-chain lipids possess high surface activity, which correlates with high cytotoxicity due to their ability to disrupt the cellular membrane by combined hydrophobic and electrostatic interactions. Liposomes containing these lipids also display high cytotoxicity with respect to all cell lines. Irrespective of chemical structures, all cationic lipids form liposomes with similar sizes and surface potentials. The characteristics of complexes composed of cationic liposomes and nucleic acids depend mostly on the type of nucleic acid and P/N ratios. In the case of oligodeoxyribonucleotide delivery, the transfection activity depends on the type of cationic head-group regardless of the type of hydrophobic domain: all types of cationic liposomes mediate efficient oligonucleotide transfer into 80-90% of the eukaryotic cells, and liposomes based on lipids with N-methylmorpholinium cationic head-group display the highest transfection activity. In the case of plasmid DNA and siRNA, the type of hydrophobic domain determines the transfection activity: liposomes composed of cholesterol-based lipids were the most efficient in DNA transfer, while liposomes containing glycerol-based lipids exhibited reasonable activity in siRNA delivery under serum-free conditions.
Assuntos
Antineoplásicos/farmacologia , DNA/administração & dosagem , Portadores de Fármacos/farmacologia , Compostos Heterocíclicos/farmacologia , Lipídeos/farmacologia , Lipossomos/farmacologia , RNA Interferente Pequeno/administração & dosagem , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Carbamatos/química , Cátions/administração & dosagem , Cátions/química , Cátions/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA/genética , DNA/metabolismo , Relação Dose-Resposta a Droga , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Células HeLa , Compostos Heterocíclicos/administração & dosagem , Compostos Heterocíclicos/química , Humanos , Lipídeos/administração & dosagem , Lipídeos/química , Lipossomos/administração & dosagem , Lipossomos/química , Estrutura Molecular , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/química , Oligodesoxirribonucleotídeos Antissenso/genética , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
In this study, the impact of different delivery systems on the cytokine-inducing, antiproliferative, and antitumor activities of short immunostimulatory double-stranded RNA (isRNA) was investigated. The delivery systems, consisting of the polycationic amphiphile 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20 tetraazahexacosan tetrahydrochloride (2X3), and the lipid-helper dioleoylphosphatidylethanolamine (DOPE), were equipped with polyethylene glycol lipoconjugates differing in molecular weight and structure. The main findings of this work are as follows: (i) significant activation of MCP-1 and INF-α, ß, and γ production in CBA mice occurs under the action of isRNA complexes with liposomes containing lipoconjugates with long PEG chains, while activation of MCP-1 and INF-γ, but not INF-α or ß, was observed under the action of isRNA lipoplexes containing lipoconjugates with short PEG chains; (ii) a pronounced antiproliferative effect on B16 melanoma cells in vitro, as well as an antitumor and hepatoprotective effect in vivo, was induced by isRNA pre-complexes with non-pegylated liposomes, while complexes containing lipoconjugates with long-chain liposomes were inactive; (iii) the antitumor activity of isRNA correlated with the efficiency of its accumulation in the cells and did not explicitly depend on the activation of cytokine and interferon production. Thus, the structure of the delivery system plays a vital role in determining the response to isRNA and allows for the choice of a delivery system depending on the desired effect.
RESUMO
Cell-free antitumor vaccines represent a promising approach to immunotherapy of cancer. Here, we compare the antitumor potential of cell-free vaccines based on microvesicles derived from dendritic cells (DCs) with DC- and cationic-liposome-based vaccines using a murine model of drug-resistant lymphosarcoma RLS40 in vivo. The vaccines were the following: microvesicle vaccinescytochalasin B-induced membrane vesicles (CIMVs) obtained from DCs loaded with total tumor RNA using cholesterol/spermine-containing cationic liposomes L or mannosylated liposomes ML; DC vaccinesmurine DCs loaded with total tumor-derived RNA using the same liposomes; and liposomal vaccineslipoplexes of total tumor-derived RNA with liposomes L or ML. Being non-hepatotoxic, CIMV- and DC-based vaccines administered subcutaneously exhibited comparable potential to stimulate highly efficient antitumor CTLs in vivo, whereas liposomal vaccines were 25% weaker CTL inducers. Nevertheless, the antitumor efficiencies of the different types of the vaccines were similar: sizes of tumor nodes and the number of liver metastases were significantly decreased, regardless of the vaccine type. Notably, the booster vaccination did not improve the overall antitumor efficacy of the vaccines under the study. CIMV- and DC- based vaccines more efficiently than liposome-based ones decreased mitotic activity of tumor cells and induced their apoptosis, stimulated accumulation of neutrophil inflammatory infiltration in tumor tissue, and had a more pronounced immunomodulatory activity toward the spleen and thymus. Administration of CIMV-, DC-, and liposome-based vaccines resulted in activation of Th1/Th17 cells as well as the induction of positive immune checkpoint 4-1BBL and downregulation of suppressive immune checkpoints in a raw PD-1 >>> TIGIT > CTLA4 > TIM3. We demonstrated that cell-free CIMV-based vaccines exhibited superior antitumor and antimetastatic activity in a tumor model in vivo. The obtained results can be considered as the basis for developing novel strategies for oncoimmunotherapy.
RESUMO
In this study, we examined the in vivo toxicity of the liposomes F consisting of 1,26-bis(cholest-5-en-3-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride, lipid-helper 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and folate lipoconjugate (O-{2-[rac-2,3-di(tetradecyloxy)prop-1-yloxycarbonyl]aminoethyl}-O'-[2-(pteroyl-L-glutam-5-yl)aminoethyl]octadecaethyleneglycol) and investigated the antitumor effect of combined antitumor therapy consisting of MDR1-targeted siMDR/F complexes and conventional polychemotherapy using tumor xenograft initiated in immunodeficient mice. Detailed analysis of acute and chronic toxicity of this liposomal formulation in healthy C57BL/6J mice demonstrated that formulation F and parent formulation L (without folate lipoconjugate) have no acute and chronic toxicity in mice. The study of the biodistribution of siMDR/F lipoplexes in SCID mice with xenograft tumors formed by tumor cells differing in the expression level of folate receptors showed that the accumulation in various types of tumors strongly depends on the abandons of folate receptors in tumor cells and effective accumulation occurs only in tumors formed by cells with the highest FR levels. Investigating the effects of combined therapy including anti-MDR1 siRNA/F complexes and polychemotherapy on a multidrug-resistant KB-8-5 tumor xenograft in SCID mice demonstrated that siMDR/F increases the efficiency of polychemotherapy: the treatment leads to pronounced inhibition of tumor growth, reduced necrosis and inflammation, and stimulates apoptosis in KB-8-5 tumor tissue. At the same time, it does not induce liver toxicity in tumor-bearing mice. These data confirm that folate-containing liposome F mediated the extremely efficient delivery of siRNA in FR-expressing tumors in vivo and ensured the safety and effectiveness of its action.
RESUMO
Folate receptors (FR) are cellular markers highly expressed in various cancer cells. Here, we report on the synthesis of a novel folate-containing lipoconjugate (FC) built of 1,2-di-O-ditetradecyl-rac-glycerol and folic acid connected via a PEG spacer, and the evaluation of the FC as a targeting component of liposomal formulations for nucleic acid (NA) delivery into FR expressing tumor cells. FR-targeting liposomes, based on polycationic lipid 1,26-bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride (2X3), lipid helper dioleoylphosphatidylethanolamine (DOPE) and novel FC, formed small compact particles in solution with diameters of 60⯱â¯22â¯nm, and were not toxic to cells. Complexes of NAs with the liposomes were prepared at various nitrogen to phosphate ratios (N/P) to optimize liposome/cell interactions. We showed that FR-mediated delivery of different nucleic acids mediated by 2X3-DOPE/FC liposomes occurs in vitro at low N/P (1/1 and 2/1); under these conditions FC-containing liposomes display 3-4-fold higher transfection efficiency in comparison with conventional formulation. Lipoplexes formed at N/P 1/1 by targeted liposomes and cargo (Cy7-labeled siRNA targeting MDR1 mRNA) in vivo efficiently accumulate in tumor (â¼15-18% of total amount), and kidneys (71%), and were retained there for more than 24â¯h, causing efficient downregulation of p-glycoprotein expression (to 40% of control) in tumors. Thus, FC containing liposomes provide effective targeted delivery of nucleic acids into tumor cells in vitro and in xenograft tumors in vivo.
Assuntos
Ácido Fólico/química , Xenoenxertos/efeitos dos fármacos , Lipossomos/química , Neoplasias/tratamento farmacológico , Ácidos Nucleicos/administração & dosagem , Ácidos Nucleicos/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Feminino , Transportadores de Ácido Fólico/metabolismo , Células HEK293 , Xenoenxertos/metabolismo , Humanos , Rim/efeitos dos fármacos , Camundongos , Camundongos SCID , Neoplasias/metabolismo , Tamanho da Partícula , Fosfatidiletanolaminas/química , Transfecção/métodosRESUMO
Here we demonstrate the ability of mannosylated liposomes (ML) targeted to mannose receptors (MR) to perform the targeted delivery of model plasmid DNA encoding EGFP and total tumour RNA into murine bone-marrow-derived dendritic cells (DCs) and enhance the efficiency of anti-tumour response triggered by these DCs in murine melanoma model. ML consist of cationic lipid 2X3 (1,26-Bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and 2.5, 5 or 10% mol. of novel mannosylated lipoconjugates. In the structure of lipoconjugates D-mannose was attached to ditetradecylglycerol residue via succinyl (lipoconjugate 1) or diethylsquarate (lipoconjugate 2) linker groups. ML spontaneously form complexes with plasmid DNA and RNA due to electrostatic interaction between positively charged lipid amino group and negatively charged phosphate of nucleic acids. ML demonstrated the benefit in transfection efficiency (TE) of pDNA into DC progenitors and immature DCs in comparison with the control liposomes at low N/P (nitrogen to phosphate) ratios (1/1 and 2/1) but not at high N/P ratios where the TE was comparable with control liposomes. Moreover, ML at low N/P were more effective in RNA delivery into immature DCs in comparison with DC progenitors. At high N/P ratios liposomal formulations containing 5 and 10% mol. of mannosylated lipoconjugate 2 with diethylsquarate linker were the most effective (up to 50% of transfected cells). DCs transfected ex vivo with ML/melanoma B16 RNA complexes after i.v. injection into mice caused five- to six-fold inhibition of melanoma lung metastasis number. Moreover, the i.v. injection of ML/melanoma B16 RNA complexes into mice induced generation of the melanoma B16-specific cytotoxic T-lymphocytes, which were two-fold more efficient in B16 cell killing than those from control liposome group.
Assuntos
Células Dendríticas/transplante , Lipossomos/química , Manose/química , Melanoma Experimental/terapia , RNA Neoplásico/administração & dosagem , Animais , Linhagem Celular Tumoral , DNA/administração & dosagem , DNA/genética , DNA/uso terapêutico , Células Dendríticas/metabolismo , Terapia Genética , Proteínas de Fluorescência Verde/genética , Lectinas Tipo C/metabolismo , Lipossomos/metabolismo , Masculino , Manose/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Neoplásico/genética , RNA Neoplásico/uso terapêutico , Receptores de Superfície Celular/metabolismo , TransfecçãoRESUMO
New polycationic lipids corresponding to the two different classes of amphiphiles ("head-tail" and "gemini") were designed and used as components of non-viral gene delivery systems. The hydrophobic domain of lipids is based on the cholesterol residue and the hydrophilic one--on the naturally occurring polyamine--spermine. Ester and carbamate linker groups as well as oligomethylene spacers of various lengths were used to connect cholesterol and spermine motifs in order to estimate the structure-activity relationships of novel polycationic lipids and to determine an effective and safe transfectant suitable for the delivery of different nucleic acids. The cationic liposomes composed of the synthesized polycationic lipids and DOPE provided delivery of FITC-labeled oligonucleotide, plasmid DNA and siRNA into HEK293 cells with an efficiency significantly higher than that of Lipofectamine 2000. We found that the transfection activity of polycationic lipids is influenced by a linker type, a spacer length and the amount of cholesterol residues. The lipid containing two cholesterol units, carbamate linker and spacer of six methylene groups demonstrated the best in vitro transfection results among other analogues tested and was defined as a promising candidate for further transfection studies to be hold in vivo.
Assuntos
Colesterol/química , DNA/administração & dosagem , Portadores de Fármacos/química , Técnicas de Transferência de Genes , RNA Interferente Pequeno/administração & dosagem , Espermina/química , Animais , Sobrevivência Celular , Colesterol/análogos & derivados , Cricetinae , DNA/genética , Portadores de Fármacos/síntese química , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células HeLa , Humanos , Lipossomos , Estrutura Molecular , Tamanho da Partícula , Fosfatidiletanolaminas/química , Plasmídeos , RNA Interferente Pequeno/genética , Espermina/análogos & derivados , Relação Estrutura-Atividade , Propriedades de Superfície , Tensoativos/química , TransfecçãoRESUMO
Here we report on the application of cationic liposomes formed by new cationic lipids and the lipid-helper DOPE (dioleoylphosphatidylethanolamine) for the transfection of plasmid DNA and mRNA into dendritic cells (DCs) progenitors and immature DCs of bone-marrow origin in vitro and the use of these DCs to induce the suppression of B16 melanoma metastases in vivo. The cationic lipids contain one (X2, S1, S2 and S3) or two (2X3) cholesterol residues or long-chain hydrocarbon substituent (2D3) linked with spermine. Data show that liposomes 2X3-DOPE, 2D3-DOPE, X2-DOPE and S2-DOPE display high transfection efficiency in respect to DNA (30-47% of DC progenitors and up to 57% of immature DC were transfected) and RNA (up to 57% of cells were transfected). The studied lipids exhibited an efficiency of DNA and RNA delivery in DCs several times higher in comparison with Lipofectamine 2000. Observed ex vivo the higher transfection efficiencies of DCs with mRNAs encoding of a set of tumor-associated antigens provided by cationic liposomes 2X3-DOPE and 2X2-DOPE corresponded to a 3-5 fold suppression of metastasis number in a model of murine B16 melanoma in vivo. The injection into mice of these pulsed DCs resulted in a slight pro-inflammatory response which was balanced by the positive effect of the antitumor cytokine production induced by the DCs. The obtained data show that the novel spermine-based polycationic lipids can be applied in the preparation of antitumor DC-based vaccine.
Assuntos
Células da Medula Óssea/metabolismo , DNA/administração & dosagem , Células Dendríticas/metabolismo , Portadores de Fármacos/química , RNA Mensageiro/administração & dosagem , Células-Tronco/metabolismo , Animais , Células da Medula Óssea/imunologia , Linhagem Celular Tumoral , Citocinas/sangue , DNA/genética , Células Dendríticas/imunologia , Proteínas de Fluorescência Verde/genética , Lipossomos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/secundário , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Transplante de Neoplasias , Fosfatidiletanolaminas/química , Plasmídeos , Poliaminas/química , Polieletrólitos , RNA Mensageiro/genética , Células-Tronco/imunologia , TransfecçãoRESUMO
Two types of complexes were prepared from a cationic cholesterol derivative, dioleoylphos-phatidylcholine and DNA. Depending on the preparation procedure complexes were either dense snarls of lipid covered DNA (type A) or multilayer liposomes with DNA between layers (type B). The transfection efficiency of the snarl-shaped complexes was low but positive. The transfection efficiency of the liposome-shaped complexes was zero, while DNA release upon their interaction with anionic liposomes was 1.7 times higher. The differences in transfection efficacy and DNA release could not be ascribed to the difference in resistance of complexes to decomposition upon interaction with anionic liposomes or intracellular environment since the lipid composition of complexes is the same. Instead the complexes in which lipoplex phase is more continuous (type A) should require more anionic lipids or more time within a cell for complete decomposition. Prolonged life time should lead to the higher probability of DNA expression.
Assuntos
DNA/química , Lipídeos/química , Transfecção/métodos , Linhagem Celular Tumoral , Humanos , Lipossomos/químicaRESUMO
Cholesterol amphiphiles containing positively charged groups (pyridinium, N-methylimidazolium, N-methylmorpholinium, and N-methylpiperidinium) linked via ß-glucosyl spacer were prepared by alkylation of the corresponding bases with 6-Ð-mesyl-ß-D-cholesteryl glucopyranoside. IC(50) values were in the range 20-35µM for the series of compounds and liposomal formulations with DOPE (1:1) were significantly less toxic. The liposomal formulations provided the accumulation of FITC-labeled oligonucleotide in cells, and the efficiency of this process was comparable to that of Lipofectamine 2000. Cationic liposomes were able to deliver siRNA into the cells, and the liposomal formulation 7d/DOPE provided the most pronounced down-regulation of EGFP expression both in the presence and in the absence of serum (up to 30%).
Assuntos
Colesterol/análogos & derivados , Portadores de Fármacos/síntese química , Portadores de Fármacos/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Fenômenos Químicos , Química Farmacêutica , Colesterol/síntese química , Colesterol/química , Colesterol/metabolismo , Colesterol/toxicidade , Portadores de Fármacos/química , Portadores de Fármacos/toxicidade , Fluoresceína-5-Isotiocianato/química , Compostos Heterocíclicos/química , Lipossomos/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
Gene therapy based on gene delivery is a promising strategy for the treatment of human disease. Here we present data on structure/biological activity of new biodegradable cholesterol-based cationic lipids with various heterocyclic cationic head groups and linker types. Enhanced accumulation of nucleic acids in the cells mediated by the lipids was demonstrated by fluorescent microscopy and flow cytometry. Light scattering and atomic force microscopy were used to find structure/transfection activity correlations for the lipids. We found that the ability of the lipids to stimulate intracellular accumulation of the oligodeoxyribonucleotides and plasmid DNA correlates well with their ability to form in solution lipid/NA complexes of sizes that do not exceed 100 nm. Screening of the lipids revealed the most promising transfection agents both in terms of low toxicity and efficient delivery: cholesterol-based lipids with positively charged pyridine and methyl imidazole head groups and either the ester or carbamate linker.