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1.
Methods Mol Biol ; 991: 275-80, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23546677

RESUMO

The medical applications of protein-based therapeutics are hampered by low bioavailability associated with inefficient intracellular delivery. Various delivery materials have been developed and tested to interact with protein cargos in a manner of stabilizing proteins extracellularly and facilitating cellular uptake of proteins, thus enhancing delivery efficiency. Peptides that can form stable complexes with proteins through non-covalent interaction appear to be a promising tool to improve intracellular delivery of proteins. Here we describe the preparation of complexes formed between ß-galactosidase and peptide-based carrier, protein transfer of the complexes, and the methods to evaluate delivery efficiency qualitatively and quantitatively.


Assuntos
Portadores de Fármacos , Peptídeos/administração & dosagem , Proteínas/administração & dosagem
2.
Biochem Biophys Res Commun ; 434(1): 110-6, 2013 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-23537655

RESUMO

Imaging of transduced cells and tissues is valuable in developing gene transfer vectors and evaluating gene therapy efficacy. We report here a simple method to use bright and photostable quantum dots to label baculovirus, an emerging gene therapy vector. The labeling was achieved through the non-covalent interaction of glutathione-capped CdTe quantum dots with the virus envelope, without the use of chemical conjugation. The quantum dot labeling was nondestructive to viral transduction function and enabled the identification of baculoviral vector-transduced, living cells based on red fluorescence. When the labeled baculoviral vectors were injected intravenously or intraventricularly for in vivo delivery of a transgene into mice, quantum dot fluorescence signals allow us monitor whether or not the injected tissues were transduced. More importantly, using a dual-color whole-body imaging technology, we demonstrated that in vivo viral transduction could be evaluated in a real-time manner in living mice. Thus, our method of labeling a read-to-use gene delivery vector with quantum dots could be useful towards the improvement of vector design and will have the potential to assess baculovirus-based gene therapy protocols in future.


Assuntos
Vetores Genéticos , Nucleopoliedrovírus/genética , Imagem Óptica , Pontos Quânticos , Transdução Genética , Animais , Linhagem Celular Tumoral , Feminino , Terapia Genética , Vetores Genéticos/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nucleopoliedrovírus/ultraestrutura , Ligação Proteica/genética , Transdução Genética/métodos
3.
Nanoscale ; 5(6): 2322-7, 2013 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-23392168

RESUMO

Highly emissive and air-stable AgInS2-ZnS quantum dots (ZAIS QDs) with quantum yields of up to 20% have been successfully synthesized directly in aqueous media in the presence of polyacrylic acid (PAA) and mercaptoacetic acid (MAA) as stabilizing and reactivity-controlling agents. The as-prepared water-dispersible ZAIS QDs are around 3 nm in size, possess the tetragonal chalcopyrite crystal structure, and exhibit long fluorescence lifetimes (>100 ns). In addition, these ZAIS QDs are found to exhibit excellent optical and colloidal stability in physiologically relevant pH values as well as very low cytotoxicity, which render them particularly suitable for biological applications. Their potential use in biological labelling of baculoviral vectors is demonstrated.


Assuntos
Índio/química , Pontos Quânticos , Compostos de Prata/química , Sulfetos/química , Compostos de Zinco/química , Resinas Acrílicas/química , Baculoviridae/química , Células Hep G2 , Humanos , Índio/farmacologia , Compostos de Prata/farmacologia , Coloração e Rotulagem/métodos , Sulfetos/farmacologia , Tioglicolatos/química , Compostos de Zinco/farmacologia
4.
Adv Mater ; 24(24): 3280-4, 2012 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-22628070

RESUMO

Self-assembled amphiphilic peptide units and supercoiled, circular double-stranded plasmid DNA are used as building blocks to form peptide/DNA fibers for virus encapsulation. Since the fiber formation process takes place under ambient conditions and is aqueous-based without the use of denaturing organic solvents, the bioactivity of viruses is well preserved.


Assuntos
DNA/química , Portadores de Fármacos/química , Terapia Genética , Vetores Genéticos/genética , Glioma/terapia , Microtecnologia/métodos , Peptídeos/química , Animais , Baculoviridae/genética , Cápsulas , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Portadores de Fármacos/metabolismo , Vetores Genéticos/metabolismo , Glioma/genética , Glioma/patologia , Humanos , Camundongos , Transdução Genética
5.
Biochem Biophys Res Commun ; 421(3): 616-20, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22538367

RESUMO

The advance in stem cell research relies largely on the efficiency and biocompatibility of technologies used to manipulate stem cells. In our previous study, we had designed an amphipathic peptide RV24 that can deliver proteins into cancer cell lines efficiently without significant side effects. Encouraged by this observation, we moved forward to test whether RV24 could be used to deliver proteins into human embryonic stem cells and human induced pluripotent stem cells. RV24 successfully mediated protein delivery into these pluripotent stem cells, as well as their derivatives including neural stem cells and dendritic cells. Based on NMR studies and particle surface charge measurements, we proposed that hydrophobic domain of RV24 interacts with ß-sheet structures of the proteins, followed by formation of "peptide cage" to facilitate delivery across cellular membrane. These findings suggest the feasibility of using amphipathic peptide to deliver functional proteins intracellularly for stem cell research.


Assuntos
Peptídeos/química , Células-Tronco Pluripotentes/metabolismo , Proteínas/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Proteínas/metabolismo
6.
Biochem Biophys Res Commun ; 419(2): 170-4, 2012 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-22326265

RESUMO

Intracellular delivery of proteins offers an alternative to genetic modification or siRNA transfection for functional studies of proteins in live cells, especially for studies in cancer cells for therapeutics development. However, lack of efficient and biocompatible delivery system has limited the use of protein for in vitro cancer research. In this study, we design and evaluate an amphipathic peptide RV24, composing of a hydrophobic domain for protein binding, a flexible linker, and a hydrophilic domain to facilitate cell penetration. When using ß-galactosidase as a cargo protein for comparison with commercially available peptide- and lipid-based carriers, RV24 peptide provides up to 5-fold increase in quantity delivered into 3 different cancer cell lines. Green fluorescent protein could also be delivered rapidly within 4h and transduced up to 83% of tested cancer cell lines. Although having a cell penetrating domain, RV24 peptide did not compromise cell viability, morphology and granularity significantly. These findings suggest the feasibility of using biocompatible amphipathic peptide to efficiently deliver protein-based molecules intracellularly for in vitro cancer research.


Assuntos
Portadores de Fármacos/metabolismo , Neoplasias/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Portadores de Fármacos/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Peptídeos/química , Transporte Proteico , beta-Galactosidase/metabolismo
7.
Biomaterials ; 31(4): 769-78, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19819012

RESUMO

Nonviral magnetofection facilitates gene transfer by using a magnetic field to concentrate magnetic nanoparticle-associated plasmid delivery vectors onto target cells. In light of the well-established effects of the Tat peptide, a cationic cell-penetrating peptide, that enhances the cytoplasmic delivery of a variety of cargos, we tested whether the combined use of magnetofection and Tat-mediated intracellular delivery would improve transfection efficiency. Through electrostatic interaction, gene transfer complexes were generated by mixing polyethylenimine-coated cationic magnetic iron beads with plasmid DNA, followed by addition of a bis(cysteinyl) histidine-rich Tat peptide. These ternary magnetofection complexes provided a 4-fold improvement in transgene expression at a dose of 1 microg of plasmid DNA per 20,000 cells over the binary complexes without the Tat peptide and transfected up to 60% of cells in vitro. The enhanced transfection efficiency was also observed in vivo in the rat spinal cord after lumbar intrathecal injection. Moreover, the injected ternary magnetofection complexes in the cerebrospinal fluid responded to a moving magnetic filed by shifting away from the injection site and mediating transgene expression in a remote region. Thus, our approach could potentially be useful for effective gene therapy treatments of localized diseases.


Assuntos
Técnicas de Transferência de Genes , Magnetismo , Nanopartículas Metálicas/química , Fragmentos de Peptídeos/química , Plasmídeos/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Ratos , Ratos Wistar , Medula Espinal/metabolismo
8.
Macromol Rapid Commun ; 31(13): 1134-41, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21590866

RESUMO

Successful molecular therapy using protein-based therapeutic agents for intracellular targets depends on the development of efficient and safe protein delivery systems that are able to overcome the problem of poor permeability of cell membrane to proteins. Here, we summarize recent studies elucidating how one particular class of peptide-based carriers, amphipathic peptide, has been designed and utilized for intracellular protein delivery in a simple yet effective manner. The unique feature of these delivery systems lies in the noncovalent binding of amphipathic peptides to protein cargos, mainly through hydrophobic interactions. At least five different types of amphipathic peptides have been developed and demonstrated to be able to deliver various biologically active proteins into a variety of cell types without the use of chemical conjugation. In view of their efficiency and presumably low toxicity, we anticipate that amphipathic peptides will continue to be developed as powerful carriers for intracellular delivery of protein therapeutics.

9.
Biomaterials ; 30(29): 5767-74, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19577293

RESUMO

Recombinant baculoviral vectors efficiently transduce many types of mammalian cells. However, their in vivo applications are hampered by the sensitivity of the virus to complement-mediated inactivation. Based on our observation that the surface charge of baculovirus is negative at neutral pH, we developed a procedure to coat baculoviral vectors with positively charged polyethylenimine 25 kDa, a commonly tested non-viral gene delivery vector, through electrostatic interaction. This coating was effective in protecting baculoviral vectors against human and rat serum-mediated inactivation in vitro, providing transduction efficiencies comparable with that generated by the control virus used under a serum-free condition. Enhanced in vivo gene expression in the liver and spleen was observed after tail vein injection of the coated viruses into mice. When injected directly into human tumor xenografts in nude mice, the coated viruses suppressed tumor development more effectively than uncoated viral vectors. These findings demonstrated the usefulness of using a simple coating method to circumvent a major obstacle to in vivo application of baculoviral vectors. The method may also serve as a flexible platform technology for improved use of the vectors, for example introducing a targeting ligand and minimizing immune responses.


Assuntos
Baculoviridae/química , Baculoviridae/genética , DNA Viral/administração & dosagem , DNA Viral/farmacocinética , Portadores de Fármacos/síntese química , Iminas/química , Polietilenos/química , Soro/química , Transfecção/métodos , Animais , Materiais Revestidos Biocompatíveis/química , Vetores Genéticos/genética , Humanos , Teste de Materiais , Camundongos , Camundongos Nus , Ratos
10.
J Control Release ; 130(2): 140-5, 2008 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-18632177

RESUMO

Aiming at identification of novel peptides that can be employed for effective targeting of malignant gliomas, we used a 12-mer peptide phage display library and cultured human malignant glioma cells for phage selection. Several common phage clones emerged after 4 rounds of biopanning against the U87MG glioblastoma cell line. The most abundant phage clone VTW, expressing a sequence of VTWTPQAWFQWV, bound to U87MG cells 700-fold more efficiently than the original unselected library. The VTW phage also bound strongly to other human glioma cell lines, including H4, SW1088 and SW1783, but very weakly to normal human astrocytes and SV40-immortalized human astroglial cells. When compared to other non-glial tumor cells, the phage showed 400- to 1400-fold higher binding efficiency for U87MG cells. After linked to positively charged lysine peptides, the VTW peptide became water soluble and was able to deliver biologically active, hydrophilic beta-galactosidase into U87MG cells, with up to 90% of the cells being stained intensively blue. This peptide carrier did not show obvious protein delivery activities in the human astrocytes. Our results provide a proof of principle to the concept that peptides identified through phage display technology can be used to develop protein carriers that are capable of mediating intracellular delivery of hydrophilic macromolecules in a tumor cell-specific manner.


Assuntos
Portadores de Fármacos/química , Neuroglia/metabolismo , Biblioteca de Peptídeos , Proteínas/administração & dosagem , Sequência de Aminoácidos , Astrócitos/metabolismo , Carbocianinas/química , Linhagem Celular Tumoral , Endocitose , Glioblastoma , Humanos , Dados de Sequência Molecular , Ligação Proteica , Proteínas/genética , Receptores de Superfície Celular/metabolismo , Transfecção , beta-Galactosidase/administração & dosagem , beta-Galactosidase/genética
11.
Biomaterials ; 29(15): 2408-14, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18295328

RESUMO

Peptides as functional biomaterials offer the possibility of incorporating various biological activities required for different biomedical applications. Here, we take advantage of this property of peptide materials and design a DNA delivery vector equipped with multiple functions critical to efficient gene transfection. The Tat peptide, a cationic cell-penetrating peptide, is known to enhance the cellular uptake of a large variety of molecules such as drugs and proteins. However, the application of the Tat peptide in DNA delivery is limited by the inability to release DNA in endosomes and the instability of peptide/DNA complexes. We incorporate in the Tat sequence histidine and cysteine residues that are able to promote endosomal escape of DNA and protect DNA in the extracellular environment. We observe up to 7000-fold improvement in gene transfection efficiency by a modified Tat peptide covalently fused with 10 histidine residues (Tat-10H) over the original Tat peptide. After incorporating two cysteine residues into the Tat-10H design, the resulting bis(cysteinyl) histidine-rich peptide is more effective than the Tat-10H peptide, because interpeptide disulfide bonds form by air oxidation upon binding to DNA, leading to enhanced stability of peptide/DNA complexes. These findings demonstrate the feasibility of using multi-functional peptide materials to extend the applications of the Tat vector to efficient gene delivery.


Assuntos
Cisteína/química , Histidina/química , Transfecção/métodos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Sistema Nervoso Central/metabolismo , DNA/química , DNA/genética , DNA Circular/química , Endossomos/metabolismo , Expressão Gênica/efeitos dos fármacos , Técnicas de Transferência de Genes , Vetores Genéticos/química , Vetores Genéticos/genética , Humanos , Luciferases/genética , Luciferases/metabolismo , Macrolídeos/farmacologia , Masculino , Microscopia de Força Atômica , Oxirredução , Tamanho da Partícula , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ratos , Ratos Wistar , Eletricidade Estática
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