RESUMO
While persistence of fear memories is essential for survival, a failure to inhibit fear in response to harmless stimuli is a feature of anxiety disorders. Extinction training only temporarily suppresses fear memory recovery in adults, but it is highly effective in juvenile rodents. Maturation of GABAergic circuits, in particular of parvalbumin-positive (PV+) cells, restricts plasticity in the adult brain, thus reducing PV+ cell maturation could promote the suppression of fear memories following extinction training in adults. Epigenetic modifications such as histone acetylation control gene accessibility for transcription and help couple synaptic activity to changes in gene expression. Histone deacetylase 2 (Hdac2), in particular, restrains both structural and functional synaptic plasticity. However, whether and how Hdac2 controls the maturation of postnatal PV+ cells is not well understood. Here, we show that PV+- cell specific Hdac2 deletion limits spontaneous fear memory recovery in adult mice, while enhancing PV+ cell bouton remodeling and reducing perineuronal net aggregation around PV+ cells in prefrontal cortex and basolateral amygdala. Prefrontal cortex PV+ cells lacking Hdac2, show reduced expression of Acan, a critical perineuronal net component, which is rescued by Hdac2 re-expression. Pharmacological inhibition of Hdac2 before extinction training is sufficient to reduce both spontaneous fear memory recovery and Acan expression in wild-type adult mice, while these effects are occluded in PV+-cell specific Hdac2 conditional knockout mice. Finally, a brief knock-down of Acan expression mediated by intravenous siRNA delivery before extinction training but after fear memory acquisition is sufficient to reduce spontaneous fear recovery in wild-type mice. Altogether, these data suggest that controlled manipulation of PV+ cells by targeting Hdac2 activity, or the expression of its downstream effector Acan, promotes the long-term efficacy of extinction training in adults.
Assuntos
Condicionamento Psicológico , Parvalbuminas , Camundongos , Animais , Parvalbuminas/metabolismo , Regulação para Baixo , Condicionamento Psicológico/fisiologia , Memória/fisiologia , Medo/fisiologia , Camundongos Knockout , Extinção Psicológica/fisiologiaRESUMO
Systemic delivery of nucleic acids to the central nervous system (CNS) is a major challenge for the development of RNA interference-based therapeutics due to lack of stability, target specificity, non-permeability to the blood-brain barrier (BBB), and lack of suitable carriers. Using a designed bi-functional fusion protein TARBP-BTP in a complex with siRNA, we earlier demonstrated knockdown of target genes in the brain of both AßPP-PS1 (Alzheimer's disease, AD) and wild-type C57BL/6 mice. In this report, we further substantiate the approach through an extended use in AßPP-PS1 mice, which upon treatment with seven doses of ß-secretase AßPP cleaving Enzyme 1 (BACE1) TARBP-BTP:siRNA, led to target-specific effect in the mouse brain. Concomitant gene silencing of BACE1, and consequent reduction in plaque load in the cerebral cortex and hippocampus (greater than 60%) in mice treated with TARBP-BTP:siRNA complex, led to improvement in spatial learning and memory. The study validates the efficiency of TARBP-BTP fusion protein as an efficient mediator of RNAi, giving considerable scope for future intervention in neurodegenerative disorders through the use of short nucleic acids as gene specific inhibitors.
Assuntos
Doença de Alzheimer/terapia , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Proteínas de Ligação a RNA/genética , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/administração & dosagem , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/administração & dosagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Inativação Gênica , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Oligopeptídeos/genética , Placa Amiloide/genética , Placa Amiloide/metabolismo , Interferência de RNA , Proteínas de Ligação a RNA/administração & dosagemRESUMO
Tocopherol-based lipids are widely used for nucleic acid delivery. Using tocopherol molecules, we designed and synthesized 5-HT functionalized lipids by tethering 5-hydroxytryptamine (5-HT), a small molecule ligand as the head group to a natural amphiphilic molecule namely α-tocopherol (Vitamin E). This is with the aim of delivering nucleic acids specifically into cells expressing the serotonin receptors (5-hydroxytryptamine[5-HT]) which are abundant in the central nervous system. In order to achieve target recognition, we adopted an approach wherein two structurally different lipid molecules having serotonin as the head group was conjugated to tocopherol via different linkers thus generating lipids with either free -NH2 or -OH moiety. The corresponding lipids designated as Lipid A (Tocopheryl carbonate serotonin-NH2) and Lipid B (Tocopheryl 2-hydroxy propyl ammonium serotonin-OH), were formulated with co-lipids 1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-sn-3-phosphatidylcholine (DOPC) and evaluated for their ability to deliver plasmid DNA through reporter gene expression assays in vitro. Furthermore, the physicochemical characteristics and cellular interactions of the formulations were examined using serotonin-receptor enriched cells in order to distinguish the structural and functional attributes of both lipids. Cell-based gene expression studies reveal that in comparison to Lipid A, a formulation of Lipid B prepared with DOPE as the co-lipid, contributes to efficient uptake leading to significant enhancement in transfection. Specific interactions explored by molecular docking studies suggests the role of the hydroxyl moiety and the enantiospecific significance of serotonin- conjugated tocopherol lipids in recognizing these receptors thus signifying a promising lipid-based approach to target the serotonin receptors in the central nervous system.
Assuntos
DNA/administração & dosagem , Lipídeos/química , Serotonina/administração & dosagem , Tocoferóis/administração & dosagem , Animais , Linhagem Celular , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter/genética , Humanos , Lipossomos , Simulação de Acoplamento Molecular , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Plasmídeos/administração & dosagem , Receptores de Serotonina/metabolismo , Serotonina/química , Relação Estrutura-Atividade , Tocoferóis/química , TransfecçãoRESUMO
Targeted gene delivery of wild type tumor suppressor gene p53 is a promising approach to inhibit the progression of ovarian cancer. Although several gene delivery vehicles have been reported earlier, there is paucity for targeted delivery of wild type p53 to ovarian cancer using gold nanoparticles. As it is well-known that EGFR (epidermal growth factor receptor) is overexpressed in ovarian cancer, in this study we hypothesized that the FDA approved monoclonal antibody C225 (cetuximab) that targets EGFR could be used for targeted delivery of wild type p53 gene. With this impetus, we devised an approach wherein cationic gold nanoparticles (AuNPs) were employed to generate gold nanoparticle-based drug delivery system (DDS, Au-C225-p53DNA where p53DNA is pCMVp53 plasmid) that was formulated and characterized by biochemical and biophysical methods. The nanoconjugate complexed with DNA (Au-C225-p53DNA) is serum-stable and protects the bound DNA from digestion by DNase-I. Additionally, in vitro reporter gene expression assays demonstrated efficient and specific gene transfection in EGFR overexpressing SK-OV-3 cells. Further, the intraperitoneal administration of Au-C225-p53DNA in SK-OV-3 xenograft mouse model displayed significant tumor targeting and tumor regression. Altogether, these studies indicated a promising nanoparticle-based approach for targeting ovarian cancers caused by mutated p53.
RESUMO
Designed recombinant proteins comprising functional domains offer selective targeting of cancer cells for the efficient delivery of therapeutic agents. The efficacy of these carriers can be further enhanced by conjugating engineered proteins to nanoparticle surfaces. However, recombinant protein-loaded nanoparticle-based drug delivery systems are not well addressed for ovarian cancer therapy. In the present study, using a combinatorial approach, we designed and fabricated a drug delivery system by combining gold nanoparticles (AuNPs) with an engineered bi-functional recombinant fusion protein TRAF(C) (TR), loaded with an anticancer drug, namely doxorubicin (DX), and erbB2-siRNA (si), to mediate target specific delivery into SK-OV-3, a model human ovarian cancer cell line over expressing HER2 receptors (i.e. human epidermal growth factor receptor-2). The nanoparticle-based targeted drug delivery system, designated as TDDS (Au-TR-DX-si), was found to be stable and homogenous as revealed by physicochemical and biochemical studies in vitro. In addition, TDDS was functional upon evaluation in vivo. Intraperitoneal administration of TDDS at 2.5 mg kg-1 of DX and 0.25 mg kg-1 of erbB2 siRNA into SK-OV-3 xenograft nude mice, revealed target specific uptake and consequent gene silencing resulting in significant tumor suppression. We attribute these results to specific co-delivery of erbB2 siRNA and DX mediated by TDDS into SK-OV-3 cells via HER2 receptors. Additionally, the biodistribution of TDDS, as quantitated by ICP-OES, confirmed tumor-specific accumulation of AuNPs primarily in tumor tissues, which firmly establishes the efficacy of the nanomedicine-based combinatorial approach for the treatment of ovarian cancer in a non-toxic manner. Based on these findings, we strongly believe that the nanomedicine-based combinatorial approach can be developed as a universal strategy for treatment of HER2+ ovarian cancers.
RESUMO
Gene therapy, a promising strategy for the delivery of therapeutic nucleic acids, is greatly dependent on the development of efficient vectors. In this study, we designed and synthesized several tocopherol-based lipids varying in the head group region. Here, we present the structure-activity relationship of stable aqueous suspensions of lipids that were synthetically prepared and formulated with 1,2-dioleoyl phosphatidyl ethanolamine (DOPE) as the co-lipid. The physicochemical properties such as the hydrodynamic size, zeta potential, stability and morphology of these formulations were investigated. Interaction with plasmid DNA was clearly demonstrated through gel binding and EtBr displacement assays. Further, the transfection potential was examined in mouse neuroblastoma Neuro-2a, hepatocarcinoma HepG2, human embryonic kidney and Chinese hamster ovarian cell lines, all of different origins. Cell-uptake assays with N-methylpiperidinium, N-methylmorpholinium, N-methylimidazolium and N,N-dimethylaminopyridinium head group containing formulations evidently depicted efficient cell uptake as observed by particulate cytoplasmic fluorescence. Trafficking of lipoplexes using an endocytic marker and rhodamine-labeled phospholipid DHPE indicated that the lipoplexes were not sequestered in the lysosomes. Importantly, lipoplexes were non-toxic and mediated good transfection efficiency as analyzed by ß-Gal and GFP reporter gene expression assays which established the superior activity of lipids whose structures correlate strongly with the transfection efficiency.
Assuntos
DNA/administração & dosagem , Lipídeos/química , Plasmídeos/administração & dosagem , Tocoferóis/química , Transfecção/métodos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cricetulus , DNA/genética , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Células Hep G2 , Humanos , Camundongos , Fosfatidiletanolaminas/química , Plasmídeos/genética , Relação Estrutura-AtividadeRESUMO
We have developed active targeting liposomes to deliver anticancer agents to ASGPR which will contribute to effective treatment of hepatocellular carcinoma. Active targeting is achieved through polymeric ligands on the liposome surface. The liposomes were prepared using reverse phase evaporation method and doxorubicin hydrocholoride, a model drug, was loaded using the ammonium sulphate gradient method. Liposomes loaded with DOX were found to have a particle size of 200nm with more than 90% entrapment efficiency. Systems were observed to release the drug in a sustained manner in acidic pH in vitro. Liposomes containing targeting ligands possessed greater and selective toxicity to ASGPR positive HepG2 cell lines due to specific ligand receptor interaction. Bio-distribution studies revealed that liposomes were concentrated in the liver even after 3h of administration, thus providing conclusive evidence of targeting potential for formulated nanosystems. Tumor regression studies indicated greater tumor suppression with targeted liposomes thereby establishing superiority of the liposomal system. In this work, we used a novel methodology to guide the determination of the optimal composition of the targeting liposomes: molecular dynamics (MD) simulation that aided our understanding of the behaviour of the ligand within the bilayer. This can be seen as a demonstration of the utility of this methodology as a rational design tool for active targeting liposome formulation.
Assuntos
Antineoplásicos/administração & dosagem , Receptor de Asialoglicoproteína/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Colesterol/química , Galactanos/química , Lipossomos/química , Neoplasias Hepáticas/tratamento farmacológico , Antineoplásicos/química , Antineoplásicos/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Doxorrubicina/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Células Hep G2 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Tamanho da Partícula , Polietilenoglicóis/química , Distribuição TecidualRESUMO
RNA interference represents a novel therapeutic approach to modulate several neurodegenerative disease-related genes. However, exogenous delivery of siRNA restricts their transport into different tissues and specifically into the brain mainly due to its large size and the presence of the blood-brain barrier (BBB). To overcome these challenges, we developed here a strategy wherein a peptide known to target specific gangliosides was fused to a double-stranded RNA binding protein to deliver siRNA to the brain parenchyma. The designed fusion protein designated as TARBP-BTP consists of a double-stranded RNA-binding domain (dsRBD) of human Trans Activation response element (TAR) RNA Binding Protein (TARBP2) fused to a brain targeting peptide that binds to monosialoganglioside GM1. Conformation-specific binding of TARBP2 domain to siRNA led to the formation of homogenous serum-stable complex with targeting potential. Further, uptake of the complex in Neuro-2a, IMR32 and HepG2 cells analyzed by confocal microscopy and fluorescence activated cell sorting, revealed selective requirement of GM1 for entry. Remarkably, systemic delivery of the fluorescently labeled complex (TARBP-BTP:siRNA) in ΑßPP-PS1 mouse model of Alzheimer's disease (AD) led to distinctive localization in the cerebral hemisphere. Further, the delivery of siRNA mediated by TARBP-BTP led to significant knockdown of BACE1 in the brain, in both ΑßPP-PS1 mice and wild type C57BL/6. The study establishes the growing importance of fusion proteins in delivering therapeutic siRNA to brain tissues.
Assuntos
Doença de Alzheimer/terapia , Encéfalo/metabolismo , Técnicas de Transferência de Genes , Peptídeos/metabolismo , RNA Interferente Pequeno/administração & dosagem , Proteínas de Ligação a RNA/metabolismo , Terapêutica com RNAi , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Barreira Hematoencefálica/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos , Gangliosídeo G(M1)/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/uso terapêutico , Proteínas de Ligação a RNA/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Efficiency of systemically delivered siRNA in gene silencing is compromised due to lack of target-specific delivery and rapid clearance of siRNA by in vivo elimination pathways. We designed a fusion protein consisting of a dsRNA binding domain of transactivation response RNA binding protein (TRBP2) fused to ErbB2 binding affibody (AF) for target specific delivery of siRNA. Designated as TRAF, the fusion protein is stable and binds efficiently and specifically to siRNA, forming homogenous non-aggregated and nuclease-resistant particles that efficiently and selectively transport siRNA into HER-2 overexpressing cancer cells and tissues. Administration of siRNA by TRAF into cells resulted in significant silencing of chosen genes involved in cell proliferation viz. AURKB and ErbB2. Noticeably, intravenous administration of TRAF:siRNA against these genes resulted in remarkable tumor suppression in the SK-OV-3 xenograft mouse model. Our results establish the potential of engineered proteins for specific and systemic delivery of siRNA for cancer therapy. FROM THE CLINICAL EDITOR: The use of siRNA in one of many novel treatments in cancer therapy. However, a major challenge for using siRNA is the lack of specificity and rapid RNA clearance. In this article, the authors designed a tumor targeting fusion protein, which can deliver siRNA specifically. In the experimental xenograft model, it was shown that intravenous administration of this resulted in significant tumor suppression. The results seem to hold promise in future clinical studies.
Assuntos
RNA Interferente Pequeno/administração & dosagem , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Animais , Aurora Quinase B/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Inativação Gênica , Genes erbB-2 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , RNA Interferente Pequeno/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lipid-based nanoparticles are considered as promising candidates for delivering siRNA into the cytoplasm of targeted cells. However, in vivo efficiency of these nanoparticles is critically dependent on formulation strategies of lipid-siRNA complexes. Adsorption of serum proteins to lipid-siRNA complexes and its charge determine siRNA degradation and serum half-life, thus significantly altering the bioavailability of siRNA. To address these challenges, we developed a formulation comprising dihydroxy cationic lipid, N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium chloride (DHDEAC), cholesterol, and varying concentrations of 1,2-distearoryl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol-2000)] (DSPE-PEG 2000). Using an ethanol dilution method, addition of these lipids to siRNA solution leads to formation of stable and homogeneous population of siRNA-encapsulated vesicles (SEVs). Biodistribution of these SEVs, containing 5 mol % of DSPE-PEG 2000 in xenograft mice, as monitored by live animal imaging and fluorescence microscopy, revealed selective accumulation in the tumor. Remarkably, four intravenous injections of the modified vesicles with equimolar amounts of siRNA targeting ErbB2 and AURKB genes led to significant gene silencing and concomitant tumor suppression in the SK-OV-3 xenograft mouse model. Safety parameters as evaluated by various markers of hepatocellular injury indicated the nontoxic nature of this formulation. These results highlight improved pharmacokinetics and effective in vivo delivery of siRNA by DHDEAC-based vesicles.
Assuntos
Lipídeos/química , RNA Interferente Pequeno/química , Animais , Colesterol/química , Inativação Gênica , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , RNA Interferente Pequeno/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Present study investigates the potential of chemically modified (Shah et al., 2013) palmitoylated arabinogalactan (PAG) in guiding liposomal delivery system and targeting asialoglycoprotein receptors (ASGPR) which are expressed in hepatocellular carcinoma (HCC). PAG was incorporated in liposomes during preparation and doxorubicin hydrochloride was actively loaded in preformed liposomes with and without PAG. The liposomal systems with or without PAG were evaluated for in vitro release, in vitro cytotoxicity, in vitro cell uptake on ASGPR(+) cells, in vivo pharmacokinetic study, in vivo biodistribution study, and in vivo efficacy study in immunocompromised mice. The particle size for all the liposomal systems was below 200 nm with a negative zeta potential. Doxorubicin loaded PAG liposomes released significantly higher amount of doxorubicin at pH 5.5 as compared to pH 7.4, providing advantage for targeted tumor therapy. Doxorubicin in PAG liposomes showed superior cytotoxicity on ASGPR(+) HepG2 cells as compared to ASGPR(-), MCF7, A549, and HT29 cells. Superior uptake of doxorubicin loaded PAG liposomes as compared to doxorubicin loaded conventional liposomes was evident in confocal microscopy studies. Higher AUC in pharmacokinetic study and higher deposition in liver was observed for PAG liposomes compared to conventional liposomes. Significantly higher tumor suppression was noted in immunocompromised mice for mice treated with PAG liposomes as compared to the conventional liposomes. Targeting ability and superior activity of PAG liposomes is established pre-clinically suggesting potential of targeted delivery system for improved treatment of HCC.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Doxorrubicina/administração & dosagem , Galactanos/química , Neoplasias Hepáticas/tratamento farmacológico , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacologia , Carcinoma Hepatocelular/patologia , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Feminino , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Lipossomos , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos SCID , Microscopia Confocal , Tamanho da Partícula , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
This study evaluates the efficiency of novel non-viral vectors consisting of super paramagnetic iron oxide nanoparticles functionalized with the chemically tunable cationic polymer for in vitro gene magnetofection. The cationic polymer, poly(vinyl pyridinium alkyl halide), with a reactive alkoxysilyl group at one terminal of the polymer (VPCmn, m = length of the side chain and n = polymerization degree), was grafted onto the surface of iron oxide nanoparticles through a silane coupling reaction. The VPCmn grafted-magnetic nanoparticles (Mag-VPCmn) were quaternized with various alkyl halides such as methyl iodide (m = 1), ethyl bromide (m = 2), butyl bromide (m = 4), hexyl bromide (m = 6) and octyl bromide (m = 8). Mag-VPCmn quaternized with a shorter alkyl chain (m = 1, 2, 4 and 6) were water dispersible, but that quaternized with a longer alkyl chain (m = 8) was precipitated in water. The surface of water dispersible Mag-VPCmns was positively charged in pH ranging from 2 to 11, and is stable for more than one month in this pH range. The complexes of Mag-VPCmns and nucleoside molecules with various N/P ratios were evaluated using gel electrophoresis, surface charge (ζ-potential) measurement, and particle size measurement. In vitro transfection experiments were assayed in human embryonic kidney 293 cells (HEK293 cells) using pmaxGFP plasmid as a reporter gene. Gene expression was found to be strongly influenced by the length of the side alkyl chains. Higher transfection efficiencies were observed with longer alkyl chains (C6 > C4 > C2 ≥ C1), indicating that hydrophobic side chains were effective in increasing the transfection efficiency.
RESUMO
Non-viral gene therapy approaches have strongly established the utility of peptides as integral constituents of delivery platforms devised for efficient transfer of therapeutic molecules into cells. Among these, cell-penetrating peptides (CPPs), encompassing a family of short peptide sequences and their chimeric derivatives, have gained versatility through the addition of de novo peptide ligands primarily to facilitate cell-specific nucleic acid delivery in vitro and in vivo. The review illustrates the structural requirements of a noteworthy peptide TAT-PTD and other derivatives chiefly to exemplify their implication in gene therapy. An overview of the emerging concept and recent explorations will be presented through unique examples which form a facet in nanotechnology-based cancer therapy. Finally the basis for the utility of CPPs in plants will be discussed in view of its biotechnological potential.
Assuntos
Peptídeos Penetradores de Células , Portadores de Fármacos/administração & dosagem , Produtos do Gene tat , Ácidos Nucleicos/administração & dosagem , Animais , Humanos , Nanotecnologia , Plantas/genéticaRESUMO
In this study, a combination of magnetic nanoparticles (MNPs) together with cationic lipid N,N-di-n-hexadecyl-N,N-dihydroxyethylammonium chloride formulated with colipid cholesterol, upon magnetofection, enhanced DNA uptake into human glioblastoma-astrocytoma, epithelial-like cell line U-87 MG, hepatocellular carcinoma Hep G2, cervical cancer HeLa and breast cancer MDA-MB-231 cells. Having confirmed this, we monitored uptake of plasmid DNA mediated by ternary magnetoplexes by fluorescence microscopy, flow cytometry and reporter gene expression assays in the presence and absence of a magnetic field. Our observations clearly indicate enhanced transfection efficiency in vitro, upon magnetofection, in the presence of serum as seen from ß-Gal reporter gene expression. The observed activity in serum suggests the suitability of MNPs for in vivo applications. Further, we measured the transverse relaxation time (T2) and obtained T2-weighted MRI images of treated U-87 MG cells. T2 determined for MNP-VP-Me22 and MNP-VP-Et22 corresponds to 22.6±0.8 ms and 36.0±2.1 ms, respectively, as compared to 47±1.7 ms for control, suggesting their applicability in molecular imaging. Our results collectively highlight the potential of lipid-based approach to augment magnetic-field guided-gene delivery using MNPs and additionally towards developing intracellular molecular probes for magnetic resonance imaging.
Assuntos
DNA/administração & dosagem , Etanolaminas/química , Nanopartículas/química , Compostos de Amônio Quaternário/química , Transfecção/métodos , Animais , Linhagem Celular Tumoral , DNA/química , Humanos , Lipossomos , Fenômenos Magnéticos , CamundongosRESUMO
Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMVß-gal, resulted in enhanced ß-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of ß-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P<0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.
Assuntos
Materiais Biocompatíveis/farmacologia , Peptídeos Penetradores de Células/farmacologia , Terapia de Alvo Molecular/métodos , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Desoxirribonuclease I/metabolismo , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Técnicas de Transferência de Genes , Humanos , Ligantes , Camundongos , Camundongos SCID , Plasmídeos/metabolismo , Protaminas , Estabilidade Proteica/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Receptor ErbB-2/metabolismo , Soro/metabolismo , Tromboplastina/metabolismo , TransfecçãoRESUMO
In the design of new cationic lipids for gene transfection, the chemistry of linkers is widely investigated from the viewpoint of biodegradation and less from their contribution to the biophysical properties. We synthesized two dodecyl lipids with glutamide as the backbone and two lysines to provide the cationic headgroup. Lipid 1 differs from Lipid 2 by the presence of an amide linkage instead of an ester linkage that characterizes Lipid 2. The transfection efficiency of lipoplexes with cholesterol as colipid was found to be very high with Lipid 1 on Chinese Hamster Ovary (CHO) and HepG2 cell lines, whereas Lipid 2 has shown partial transfection efficiency on HepG2 cells. Lipid 1 was found to be stable in the presence of serum when tested in HepG2 and CHO cells albeit with lower activity. Fluorescence-based dye-binding and agarose gel-based assays indicated that Lipid 1 binds to DNA more efficiently than Lipid 2 at charge ratios of >1:1. The uptake of oligonucleotides with Lipid 1 was higher than Lipid 2 as revealed by confocal microscopy. Transmission electron microscopy (TEM) images reveal distinct formation of liposomes and lipoplexes with Lipid 1 but fragmented and unordered structures with Lipid 2. Fusion of Lipids 1 and 2 with anionic vesicles, with composition similar to plasma membrane, suggests that fusion of Lipid 2 was very rapid and unlike a fusion event, whereas the fusion kinetics of Lipid 1 vesicles was more defined. Differential scanning calorimetry (DSC) revealed a high T(m) for Lipid 1 (65.4 °C) while Lipid 2 had a T(m) of 23.5 °C. Surface area-pressure isotherms of Lipid 1 was less compressible compared to Lipid 2. However, microviscosity measured using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed identical values for vesicles made with either of the lipids. The presence of amide linker apparently resulted in stable vesicle formation, higher melting temperature, and low compressibility, while retaining the membrane fluid properties suggesting that the intermolecular hydrogen bonds of Lipid 1 yielded stable lipoplexes of high transfection efficiency.
Assuntos
Cátions/química , Lipídeos/química , Oligopeptídeos/química , Transfecção/métodos , Animais , Anisotropia , Células CHO , Varredura Diferencial de Calorimetria , Cricetinae , Cricetulus , Técnicas de Transferência de Genes , Células Hep G2 , Humanos , Fusão de Membrana , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Difração de Raios XRESUMO
Cell targeted delivery of drugs, including nucleic acids, is known to enhance the therapeutic potential of free drugs. We used serotonin (5-HT) as the targeting ligand to deliver plasmid DNA to cells specifically expressing 5-HT receptor. Our liposomal formulation includes the 5-HT conjugated targeting lipid, a cationic lipid and cholesterol. DNA-binding studies indicate that the targeting 5-HT-lipid binds DNA efficiently. The formulation was tested and found to efficiently deliver DNA into CHO cells stably expressing the human serotonin(1A) receptor (CHO-5-HT(1A)R) compared to control CHO cells. Liposomes without the 5-HT moiety were less efficient in both cell lines. Similar enhancement in transfection efficiency was also observed in human neuroblastoma IMR32 and hepatocellular carcinoma (HepG2) cells. Cell uptake studies using CHO-5-HT(1A)R cells by flow cytometry and confocal microscopy clearly indicated that the targeting liposomes through 5-HT moiety may have a direct role in increasing the cellular uptake of DNA-lipid complexes. To our knowledge this is the first report that demonstrates receptor-targeted nucleic acid delivery into cells expressing 5-HT receptor.
Assuntos
DNA/administração & dosagem , Receptor 5-HT1A de Serotonina/metabolismo , Serotonina/administração & dosagem , Animais , Células CHO , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Citometria de Fluxo , Técnicas de Transferência de Genes , Células Hep G2 , Humanos , Ligantes , Lipossomos , Neoplasias Hepáticas/metabolismo , Microscopia Confocal , Neuroblastoma/metabolismo , Plasmídeos , Serotonina/metabolismo , TransfecçãoRESUMO
A major rate-limiting step in nonviral gene delivery is the entry of nucleic acids across various membrane barriers and eventually into the nucleus where it must be transcribed. Cell-penetrating peptides and proteins are employed to generate formulations that overcome these challenges to facilitate DNA delivery into cells efficiently. However, these are limited by their inability to deliver nucleic acids selectively due to lack of specificity because they deliver to both cancer and normal cells. In this study, through modular design, we generated a recombinant fusion protein designated as Her-nuclear localization sequence (Her-NLS), where heregulin-α (Her), a targeting moiety, was cloned in frame with cationic NLS peptide to obtain a cell-specific targeting biomolecule for nucleic acid delivery. The heregulin-α(1) isoform possesses the epidermal growth factor-like domain and binds to HER2/3 heterodimers which are overexpressed in certain breast cancers. Purified recombinant Her-NLS fusion protein binds plasmid DNA and specifically transfects MDA-MB-453 cells overexpressing the epidermal growth factor receptors HER2/3 in vitro. The approach described would also permit replacement of heregulin ligand with other targeting moieties that would be suited to cell-specific nucleic acid delivery mediated via receptor-ligand interactions.
Assuntos
Plasmídeos/administração & dosagem , Receptor ErbB-2/administração & dosagem , Sequência de Aminoácidos , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Primers do DNA/genética , Sistemas de Liberação de Medicamentos/métodos , Feminino , Terapia Genética/métodos , Proteínas de Fluorescência Verde/genética , Humanos , Dados de Sequência Molecular , Nanomedicina , Sinais de Localização Nuclear/administração & dosagem , Sinais de Localização Nuclear/genética , Plasmídeos/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , TransfecçãoRESUMO
Cell-penetrating peptides comprising cloned epitopes that contribute to membrane transduction, DNA-binding and cell targeting functions are known to facilitate nucleic acid delivery. Using the ITASSER software, we predicted the 3-D structure of a well characterized and efficient transfecting cell-penetrating peptide, namely TAT-Mu and its derivative TAT-Mu-AF protein that harbors a targeting ligand, the HER2-binding affibody. Our model predicts TAT-Mu-AF fusion protein as primarily comprising α-helices. The affibody in TAT-Mu-AF is predicted as a 3-helical domain that is distinct from the TAT-Mu domain. Its positioning in three-dimensional structure is oriented in a manner that possibly favors interactions with receptor and facilitates transport to the target site. The linker region between TAT-Mu and the affibody is also predicted as a helix that is likely to stabilize the overall fold of the TAT-Mu-AF complex. Further, the evaluation of secondary structure of the designed TAT-Mu-AF fusion protein by circular dichroism is in support of our predictions.
RESUMO
Protein-based nucleic acid carriers offer attractive possibilities to enhance in vitro and in vivo gene delivery to combat diseases. A multi-domain fusion protein, namely TAT-NLS-Mu, designated as TNM, has been designed, cloned, heterologously expressed in E. coli and purified to homogeneity by affinity chromatography. The recombinant chimera TNM harbors three epitopes, a cell-penetrating (TAT) domain, a nuclear localization domain comprising of three nuclear localization sequence (NLS) motifs in tandem and a DNA-binding (Mu) domain. Complexes prepared by combining plasmid DNA with TNM (DP) transfect MCF-7, COS, CHO and HepG2 cells. Ternary complexes prepared with DNA, protein and cationic lipid (DPL) resulted in ~5-7 fold enhancement in reporter gene expression over the DP alone. Treatment of cells with chloroquine during transfection, with DP complexes, resulted in remarkable increases in reporter gene expression suggesting the involvement of endosomal compartments in the uptake process. Interestingly, DPL prepared with Lipofectin or 1, 2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP) exhibited enhanced transfection in the presence of serum in MCF-7 and HepG2 cells. Microinjection of DP complexes, with and without NLS sequence, into the cytoplasm and nucleus of smooth muscle cells (SMC) indicated that the presence of NLS sequence in protein carrier significantly enhanced transgene expression. Together the data suggest that modular design of proteins is a promising method to develop gene delivery carriers and also the role of NLS epitopes in mediating nuclear transfer of DNA complexes into various cell types.