RESUMO
BACKGROUND: Gene therapy remains a significant challenge due to lots of barriers limiting the genetic manipulation technologies. As for non-viral delivery vectors, they often suffer insufficient performance due to inadequate cellular uptake and gene degradation in endosome or lysosome. The importance of overcoming these conserved intracellular barriers is increasing as the delivery of genetic cargo. RESULTS: A surface-functionalized non-viral vector involving the biomimetic mannitol moiety is initiated, which can control the cellular uptake and promote the caveolae-mediated pathway and intracellular trafficking, thus avoiding acidic and enzymatic lysosomal degradation of loaded gene internalized by clathrin-mediated pathway. Different degrees of mannitol moiety are anchored onto the surface of the nanoparticles to form bio-inspired non-viral vectors and CaP-MA-40 exhibits remarkably high stability, negligible toxicity, and significantly enhanced transgene expression both in vitro and in vivo. CONCLUSIONS: This strategy highlights a paradigmatic approach to construct vectors that need precise intracellular delivery for innovative applications.
Assuntos
Fosfatos de Cálcio/química , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Nanopartículas/química , Transgenes , Cavéolas/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Clatrina/metabolismo , Endossomos/metabolismo , Regulação Viral da Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Lisossomos/metabolismo , Manitol/metabolismo , Transdução de Sinais , Propriedades de Superfície , Transdução Genética , TransfecçãoRESUMO
Stimuli-responsive materials are promising paradigm applied to construct diagnostic and therapeutic intracellular controlled release vectors, while highlighting many challenges and opportunities. In this paper, six α-cyclodextrin-based supramolecular nanovectors were constructed and the efficacy of amine groups, stimuli-responsive profiles and endocytic mechanisms were investigated. The results indicated that the designed supermolecules can compact DNA to form stable complexes and display low cytotoxicity. Among them, PRPEI-2 with suitable PEI amine group exhibited enhanced transfecting performance, high dilution stability, nice serum compatibility, and good acid-responsive profiles to enable endosome escape, significantly higher than commercially available transfecting agent PEI25000, the most effective vector studied to date. The endocytic uptake mechanisms involved in the transfection was mainly through clathrin-mediated pathway, which is closely associated with and can be improved by endosome escape. Moreover, PRPEI-2/DNA polyplex can be effectively expressed in vivo even after 48 h via only single tail-vein injection, and the gene expression and main tissue distribution appeared in the testis, liver, brain and spleen. These excellent characteristics demonstrated that the supramolecular PRPEI-2 represents an excellent prospect as stimuli-responsive nanovectors for gene diagnosis and therapy.
Assuntos
Técnicas de Transferência de Genes , DNA , Endossomos , Polietilenoimina , Transfecção , alfa-CiclodextrinasRESUMO
Non-viral nanovectors have attracted much attention owing to their ability to condense genetic materials and their ease of modification. However, their poor stability, low biocompatibility and gene degradation in endosomes or lysosomes has significantly hampered their application in vivo and in the clinic. In an attempt to overcome these difficulties a series of bovine serum albumin (BSA)-calcium phosphate (CaP) nanoparticles were constructed. The CaP condenses with DNA to form nanocomplexes coated with a biomimetic corona of BSA. Such complexes may retain the inherent endocytosis profile of BSA, with improved biocompatibility. In particular the transgene performance may be enhanced by stimulating the cellular uptake pathway via caveolae-mediated endocytosis. Two methods were employed to construct and optimize the formulation of BSA-CaP nanomaterials. The optimized BSA-CaP-50-M2 nanoparticles prepared by our second method exhibited good stability, negligible cytotoxicity and enhanced transgene performance with long-term expression for 72 h in vivo even with a single dose. Determination of the cellular uptake pathway and Western blot revealed that cellular uptake of the designed BSA-CaP-50-M2 nanoparticles was mainly via caveolae-mediated endocytosis in a non-degradative pathway in which the biomimetic uptake profile of BSA was retained.
Assuntos
Fosfatos de Cálcio/química , Cavéolas/metabolismo , Endocitose , Nanoestruturas/química , Coroa de Proteína/metabolismo , Soroalbumina Bovina/química , Transgenes , Animais , Bovinos , Caveolina 1/metabolismo , Morte Celular , DNA/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos Nus , Tamanho da Partícula , Fosforilação , Distribuição TecidualRESUMO
The nano self-assembly profiles of amphiphilic gene delivery vectors could improve the density of local cationic head groups to promote their DNA condensation capability and enhance the interaction between cell membrane and hydrophobic tails, thus increasing cellular uptake and gene transfection. In this paper, two series of cationic amphiphilic ß-cyclodextrin (ß-CD) derivatives were designed and synthesized by using 6-mono-OTs-ß-CD (1) as the precursor to construct amphiphilic gene vectors with different building blocks in a selective and controlled manner. The effect of different type and degree of cationic head groups on transfection and the endocytic mechanism of ß-CD derivatives/DNA nanocomplexes were also investigated. The results demonstrated that the designed ß-cyclodextrin derivatives were able to compact DNA to form stable nanocomplexes and exhibited low cytotoxicity. Among them, PEI-1 with PEI head group showed enhanced transfection activity, significantly higher than commercially available agent PEI25000 especially in the presence of serum, showing potential application prospects in clinical trials. Moreover, the endocytic uptake mechanism involved in the gene transfection of PEI-1 was mainly through caveolae-mediated endocytosis, which could avoid the lysosomal degradation of loaded gene, and had great importance for improving gene transfection activity.
Assuntos
Vetores Genéticos , Nanopartículas/química , Transfecção/métodos , beta-Ciclodextrinas , DNA/química , DNA/farmacologia , Vetores Genéticos/química , Vetores Genéticos/farmacologia , Células HEK293 , Humanos , Iminas/química , Iminas/farmacologia , Polietilenos/química , Polietilenos/farmacologia , beta-Ciclodextrinas/química , beta-Ciclodextrinas/farmacologiaRESUMO
Polyethylenimine (PEI), a commercially available gene transfection reagent, is a promising nonviral vector due to its inherent ability to efficiently condense genetic materials and its successful transfection performance in vitro. However, its low transfection efficiency in vivo, along with its high cytotoxicity, limit any further applications in gene therapy. To enhance the gene transfection performance and reduce the cytotoxicity of linear polyethylenimine, pseudopolyrotaxane PEI25k/CD and the polyrotaxanes PEI25k/CD-PA and PEI25k/CD-PB were prepared and their transfection efficiencies were then evaluated. The pseudopolyrotaxane PEI25k/CD exhibited better transfection efficiency and lower cytotoxicity than the transfection reagent linear PEI25k, even in the presence of serum. It also showed a remarkably higher cell viability, similar DNA protecting capability, and better DNA decondensation and release ability, and could be useful for the development of novel and safe nonviral gene delivery vectors for gene therapy.
Assuntos
Materiais Biocompatíveis/química , Ciclodextrinas/química , Poloxâmero/química , Polietilenoimina/química , Rotaxanos/química , Transfecção/métodos , alfa-Ciclodextrinas/química , Sobrevivência Celular , DNA/metabolismo , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Tamanho da Partícula , Soro/metabolismo , Eletricidade EstáticaRESUMO
The covalently cross-linked chitosan-poly(ethylene glycol)1540 derivatives have been developed as a controlled release system with potential for the delivery of protein drug. The swelling characteristics of the hydrogels based on these derivatives as the function of different PEG content and the release profiles of a model protein (bovine serum albumin, BSA) from the hydrogels were evaluated in simulated gastric fluid with or without enzyme in order to simulate the gastrointestinal tract conditions. The derivatives cross-linked with difunctional PEG1540-dialdehyde via reductive amination can swell in alkaline pH and remain insoluble in acidic medium. The cumulative release amount of BSA was relatively low in the initial 2h and increased significantly at pH 7.4 with intestinal lysozyme for additional 12h. The results proved that the release-and-hold behavior of the cross-linked CS-PEG1540H-CS hydrogel provided a swell and intestinal enzyme controlled release carrier system, which is suitable for oral protein drug delivery.
Assuntos
Aldeídos/química , Aminas/química , Quitosana/química , Portadores de Fármacos , Polietilenoglicóis/química , Soroalbumina Bovina/administração & dosagem , Materiais Biocompatíveis , Células CACO-2 , Sequência de Carboidratos , Humanos , Oxirredução , Espectroscopia de Prótons por Ressonância Magnética , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Cholesterol derivatives M1-M6 as synthetic cationic lipids were designed and the biological evaluation of the cationic liposomes based on them as non-viral gene delivery vectors were described. Plasmid pEGFP-N1, used as model gene, was transferred into 293T cells by cationic liposomes formed with M1-M6 and transfection efficiency and GFP expression were tested. Cationic liposomes prepared with cationic lipids M1-M6 exhibited good transfection activity, and the transfection activity was parallel (M2 and M4) or superior (M1 and M6) to that of DC-Chol derived from the same backbone. Among them, the transfection efficiency of cationic lipid M6 was parallel to that of the commercially available Lipofectamine2000. The optimal formulation of M1 and M6 were found to be at a mol ratio of 1:0.5 for cationic lipid/DOPE, and at a N/P charge mol ratio of 3:1 for liposome/DNA. Under optimized conditions, the efficiency of M1 and M6 is greater than that of all the tested commercial liposomes DC-Chol and Lipofectamine2000, even in the presence of serum. The results indicated that M1 and M6 exhibited low cytotoxicity, good serum compatibility and efficient transfection performance, having the potential of being excellent non-viral vectors for gene delivery.
Assuntos
Colesterol/química , Terapia Genética/métodos , Lipídeos/química , Lipossomos/química , Relação Estrutura-Atividade , Colesterol/análogos & derivados , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lipídeos/síntese química , Tamanho da Partícula , Fosfatidiletanolaminas/química , Soro/química , Transfecção/métodosRESUMO
In this work two novel cationic lipids using natural tartaric acid as linking backbone were synthesized. These cationic lipids were simply constructed by tartaric acid backbone using head group 6-aminocaproic acid and saturated hydrocarbon chains dodecanol (T-C12-AH) or hexadecanol (T-C16-AH). The physicochemical properties, gel electrophoresis, transfection activities, and cytotoxicity of cationic liposomes were tested. The optimum formulation for T-C12-AH and T-C16-AH was at cationic lipid/dioleoylphosphatidylethanolamine (DOPE) molar ratio of 1 : 0.5 and 1 : 2, respectively, and N/P charge molar ratio of 1 : 1 and 1 : 1, respectively. Under optimized conditions, T-C12-AH and T-C16-AH showed effective gene transfection capabilities, superior or comparable to that of commercially available transfecting reagent 3ß-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol) and N-[2,3-dioleoyloxypropyl]-N,N,N-trimethylammonium chloride (DOTAP). The results demonstrated that the two novel tartaric acid-based cationic lipids exhibited low toxicity and efficient transfection performance, offering an excellent prospect as nonviral vectors for gene delivery.
Assuntos
Técnicas de Transferência de Genes , Lipídeos/química , Tartaratos/química , Ácido Aminocaproico , Sobrevivência Celular/efeitos dos fármacos , DNA/administração & dosagem , DNA/química , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células HeLa , Humanos , Lipossomos/química , Lipossomos/farmacologiaRESUMO
OBJECTIVES: This paper aims to investigate the influence of single-nucleotide polymorphisms (SNPs) in the receptor of activator of nuclear factor kappaB ligand (RANKL) gene (TNFSF11) and osteoprotegerin (OPG) gene (TNFRSF11B) on bone and joint injury in patients with rheumatoid arthritis (RA). METHODS: Two hundred RA patients and 201 matched controls were analysed by case-control design, and their samples were genotyped. Bone mineral density (BMD) and serum OPG and RANKL levels were measured. Clinical and laboratory parameters were recorded, and the radiographic changes in both hands of RA were evaluated by Sharp's method. RESULTS: Our results showed no significant differences in the distribution frequency of the alleles and genotypes of TNFRSF11B (rs2073618 and rs3102735) and TNFSF11 (rs2277438) between the RA group and controls (p>0.05). Compared to patients with TNFSF11 (rs2277438) AA or GG genotype, RA with TNFSF11 (rs2277438) AG genotype had significantly decreased BMD values at lumbar spine 3, lumbar spine 4, lumbar spine 2-4 (p<0.05-0.01), and apparently elevated Sharp scores (p<0.05), respectively. The RA group showed significantly higher serum levels of RANKL, RANKL/OPG ratio and a lower serum level of OPG than that of the controls (p<0.05-0.0001). RA patients with RANKL-rs2277438 heterozygotic genotype (AG) had significantly increased serum levels of RANKL (p<0.05), compared to homozygotic genotype (AA or GG). CONCLUSIONS: These results indicate that SNP of TNFRSF11B (rs2073618 and rs3102735) and TNFSF11 (rs2277438) may not be susceptibility factors for RA in Chinese Han population. SNP of TNFSF11 (rs2277438) may have an important influence on bone and joint injury in RA.