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At least 91 records · Page 5Linked to original sources
PubMed2002

Potassium-dependent folding: a key to intracellular delivery of G-quartet oligonucleotides as HIV inhibitors.

Several groups have demonstrated that G-rich oligonucleotides forming G-quartet structures display activity as potential drugs, such as potent HIV inhibitors. The delivery of G-quartet oligonucleotides to their intracellular targets is a key obstacle to overcome for their clinical success. Here we have developed a novel system to deliver G-rich oligonucleotides into the cell nucleus, e.g., the site of HIV integration. On the basis of the property of potassium-induced formation of G-quartet structure, we explored the difference of K(+) concentrations inside (140 mM) and outside (4 mM) cells to induce the G-rich oligonucleotides to form different structures inside and outside cells. The key steps of this delivery system include the following: (i) First, the G-quartet structure is denatured to form a lipid-DNA complex, so that the molecules can be well delivered into cells. (ii) Then the delivered molecules are induced to form G-quartet structures by potassium inside cells since the G-quartet structure is the primary requirement for inhibition of HIV-1 HIV integrase (IN) activity. The molecules of a novel G-quartet HIV inhibitor, T40214, with the sequence of (GGGC)(4) were successfully delivered into the nuclei of target cells, which significantly decreased HIV-1 replication and increased the probability to target HIV-1 IN in infected cells.

Cell Line↗
PubMed2004

Cell-mediated immune responses in healthy children with a history of subclinical infection with Japanese encephalitis virus: analysis of CD4+ and CD8+ T cell target specificities by intracellular delivery of viral proteins using the human immunodeficiency virus Tat protein transduction domain.

Japanese encephalitis virus (JEV), a single-stranded positive-sense RNA virus of the family Flaviviridae, is the major cause of paediatric encephalitis in Asia. The high incidence of subclinical infections in Japanese encephalitis-endemic areas and subsequent evasion of encephalitis points to the development of immune responses against JEV. Humoral responses play a central role in protection against JEV; however, cell-mediated immune responses contributing to this end are not fully understood. The structural envelope (E) protein, the major inducer of neutralizing antibodies, is a poor target for T cells in natural JEV infections. The extent to which JEV non-structural proteins are targeted by T cells in subclinically infected healthy children would help to elucidate the role of cell-mediated immunity in protection against JEV as well as other flaviviral infections. The property of the Tat peptide of Human immunodeficiency virus to transduce proteins across cell membranes, facilitating intracellular protein delivery following exogenous addition to cultured cells, prompted us to express the four largest proteins of JEV, comprising 71 % of the JEV genome coding sequence, as Tat fusions for enumerating the frequencies of virus-specific CD4(+) and CD8(+) T cells in JEV-immune donors. At least two epitopes recognized by distinct HLA alleles were found on each of the non-structural proteins, with dominant antiviral Th1 T cell responses to the NS3 protein in nearly 96 % of the cohort. The data presented here show that non-structural proteins are frequently targeted by T cells in natural JEV infections and may be efficacious supplements for the predominantly antibody-eliciting E-based JEV vaccines.

Alleles↗
PubMed2002

Intracellular delivery of trehalose into mammalian cells by electropermeabilization.

The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mM trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mM. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues.

Animals↗
PubMed2003

Intracellular delivery of HSP70 using HIV-1 Tat protein transduction domain.

Heat shock protein 70 (HSP70) is an intracellular stress protein that confers cytoprotection to a variety of cellular stressors. Several lines of evidence have suggested that augmentation of the heat shock response by increasing the expression of HSP70 represents a potential therapeutic strategy for the treatment of critically ill patients. The Tat protein of human immunodeficiency virus 1 (HIV-1) has been used previously to deliver functional cargo proteins intracellularly when added exogenously to cultured cells. We generated a Tat-HSP70 fusion protein using recombinant methods and treated HSF -/- cells with either Tat-HSP70 or recombinant HSP70 prior to exposure to hyperoxia or lethal heat shock. We showed that biologically active, exogenous HSP70 can be delivered into cells using the HIV-1 Tat protein, and that the Tat-mediated delivery of HSP70 confers cytoprotection against thermal stress and hyperoxia and may represent a novel approach to augmenting intracellular HSP70 levels.

Animals↗
PubMed2005

Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids.

An analogous series of cationic lipids (1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA) and 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA)) possessing 0, 1, 2 or 3 double bonds per alkyl chain respectively, was synthesized to determine the correlation between lipid saturation, fusogenicity and efficiency of intracellular nucleic acid delivery. 31P-NMR analysis suggests that as saturation increases, from 2 to 0 double bonds, lamellar (L(alpha)) to reversed hexagonal (H(II)) phase transition temperature increases, indicating decreasing fusogenicity. This trend is largely reflected by the efficiency of gene silencing observed in vitro when the lipids are formulated as Stable Nucleic Acid Lipid Particles (SNALPs) encapsulating small inhibitory RNA (siRNA). Uptake experiments suggest that despite their lower gene silencing efficiency, the less fusogenic particles are more readily internalized by cells. Microscopic visualization of fluorescently labelled siRNA uptake was supported by quantitative data acquired using radiolabelled preparations. Since electrostatic binding is a precursor to uptake, the pKa of each cationic lipid was determined. The results support a transfection model in which endosomal release, mediated by fusion with the endosomal membrane, results in cytoplasmic translocation of the nucleic acid payload.

Cations↗
PubMed2005

Intracellular delivery of liposome-encapsulated prolidase in cultured fibroblasts from prolidase-deficient patients.

Prolidase is a cytosolic exopeptidase whose deficiency causes the development of a rare autosomal recessive disorder known as Prolidase Deficiency (PD). The main manifestations of PD are intractable ulcerations of the skin, recurrent infections and mental retardation. At this time only a hazardous and expensive chronic therapy based on blood transfusions is the suggested treatment for PD. The aim of this work was to investigate the capability of utilizing liposomes as enzyme carriers: these vesicular systems have been recently evaluated as protein carriers for their potential in terms of "in vivo" localization, drug release and for protein stabilization in biological fluids. Liposomes were prepared, with a 1:1 PC:Col molar ratio with or without DSPE-PEG, by a thin-film hydration. Ex-vivo experiments were performed, incubating prolidase loaded liposomes with cultured fibroblasts from PD patients and from controls, to determine the amount of active enzyme delivered to cells. Evaluation of liposomes toxicity on cultured skin fibroblasts showed that liposomes did not interfere with cellular growth. Results showed that all the active prolidase encapsulated in the liposomes was completely vehiculated inside fibroblasts after 6 days incubation. SEM analysis suggests that prolidase is vehiculated inside the cell through liposome endocytosis.

Cells, Cultured↗
PubMed2005

Intracellular delivery of proteins into mammalian living cells by polyethylenimine-cationization.

In the post-genomic era, there is pressing need for development of protein manipulation methodology to analyze functions of proteins in living cells. For this purpose, techniques to deliver functional proteins into living cells are currently being evaluated as alternative approaches to the introduction of transcriptionally active DNA. Here, we describe a novel method for efficient protein transduction into living cells in which a protein is simply cationized with polyethylenimine (PEI) by limited chemical conjugation. PEI-cationized proteins appear to adhere to the cell surface by ionic charge interaction and then internalize into cells in a receptor- and transporter-independent fashion. Since PEI is an organic macromolecule with a high cationic-charge density, limited coupling with PEI results in endowment of sufficient cationic charge to proteins without causing serious decline in their fundamental functions. A number of PEI-cationized proteins, such as ribonuclease (RNase), green fluorescent protein (GFP) and immunoglobulin (IgG), efficiently entered cells and functioned in the cytosol. Our results suggest that protein cationization techniques using PEI will be useful for the development of protein transduction technology.

3T3 Cells↗
PubMed2002

Intracellular delivery of monoclonal antibodies.

With some exceptions, antibodies do not have the ability to penetrate cell membranes and act intracellularly. Their usefulness in research and medicine would be considerably enhanced if they had the intrinsic ability to act on intracellular targets. We report here that covalently linking poly-L-arginine (average molecular weight 10750, ca. 68 residues) to the oligosaccharide moiety of the CH2 region of an immunoglobulin makes possible penetration into the cytoplasm and, and in some cases into the nucleus of cells. We demonstrate this with five antibodies and seven cell lines. Retention of specificity is demonstrated by ELISA with an anti-HIV Gag antibody and intracellularly with a monoclonal anti-tubulin antibody. As the antibodies are covalently modified, they have the potential to be used in intact animals.

3T3 Cells↗
PubMed2004

Control of intracellular delivery of oligonucleotides by signal peptides and genetic expression in human cells.

In the present study, membrane permeability and intracellular localization of oligonucleotide (ODN) conjugated with naturally occurring functional peptides or designed peptide were investigated, as well as antisense properties of them to inhibit of telomerase activities. All conjugate antisense ODNs showed higher membrane permeability and nuclease resistance than natural ODN. Intracellular localization of ODN could be precisely controlled by conjugation with functional peptides. Conjugate antisense ODNs indicated thousand fold higher inhibitory effects than natural ODN in cellular extract and 95% suppression in human leukemia cells.

Base Sequence↗
PubMed2003

Intracellular delivery of drugs to macrophages.

Toxic side effects which often complicate successful therapy in a number of diseases possibly arise due to the fact that at therapeutically effective concentrations the non-target cells in the body are also exposed to the cytotoxic effects of the drugs. Minimization of such adverse reactions might be feasible through drug delivery modalities that would reduce the uptake of the drugs by non-target cells and selectively deliver the drug only to the target cells (and/or intracellular sites) at relatively low extracellular concentrations. The current generic approach to site-specific drug delivery consists of attaching the therapeutic agent to a carrier recognized only by the cells where the pharmacological action is desired. Two types of recognition elements on the surface of target cells are being exploited for this purpose, viz., (i) antigens capable of generating specific, non-cross reactive antibodies, and (ii) receptors on the cell surface capable of efficient transport of the ligands. In general, incomplete specificity for the target cells and poor internalization of antibody-drug conjugates still limit the usefulness of antibodies for site-specific drug delivery applications necessitating exploration of alternatives. The alternate possibility is to exploit the exquisite cell type specificity and high efficiency of endocytosis of macromolecules mediated by specific receptors present on the surface of target cells for delivering drugs. A large number of infectious, metabolic, and neoplastic diseases are associated with macrophages leading to morbidities and mortalities to millions of people worldwide, thus an appropriate design of a drug delivery system to macrophages will be of tremendous help.

Drug Carriers↗
PubMed2002

Targetability and intracellular delivery of anti-BCG antibody-modified, pH-sensitive fusogenic immunoliposomes to tumor cells.

We prepared tumor-specific immunoliposomes by coupling anti-BCG monoclonal antibodies to pH-sensitive fusogenic liposomes modified with succinylated polyglycidol (sucPG), in order to obtain efficient binding to, and endocytotic internalization into, the tumor cells. Mouse colon carcinoma 26 cells, which are known to share a common antigen with BCG, were used in in vitro experiments. BCG-sucPG immunoliposomes showed fusion ability under acidic conditions. Fluorescence microscopic observation indicated that BCG-sucPG immunoliposomes bound to colon 26 tumor cells and induced receptor-mediated endocytosis at 37 degrees C. Fusion assay by resonance energy transfer using N-(7-nitro-2-1,3-benzoxadiazol-4-yl) diacyl phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl) diacyl phosphatidylethanolamine suggested that fusion between BCG-sucPG immunoliposomes and endosomal and/or lysozomal membrane did occur. These results imply that the BCG-sucPG immunoliposomes transfer their content into the cytoplasm by fusing with the endosomal and/or lysozomal membrane after recognition of target cells and subsequent internalization into the cells by endocytosis.

Animals↗
PubMedDate not supplied

Intracellular delivery of nanometric DNA particles via the folate receptor.

The size of condensed DNA particles is a key determinant for both diffusion to target cells in vivo and intracellular trafficking. The smallest complexes are obtained when each DNA molecule collapses individually. This was achieved using a designed cationic thiol-detergent, tetradecyl-cysteinyl-ornithine (C(14)COrn). The resulting particles were subsequently stabilized by air-induced dimerization of the detergent into a disulfide lipid on the DNA template. Particles are anionic (zeta potential = -45 mV), and their size (30 nm) corresponds to the volume of a single plasmid DNA molecule. The electrophoretic mobility of the condensed DNA, though quasi-neutralized, was found higher than that of the extended DNA. Moreover, the dimerized (C(14)COrn)(2) lipid was found to be an efficient transfection reagent for various cell lines. In an attempt to achieve extended circulation times and to target tumors by systemic delivery, we have coated the particles with PEG-folate residues. Plasmid DNA was condensed into monomolecular particles as described above and coated by simple mixing with DPPE-PEG-folate. Physicochemical measurements showed particles coated with 2% of DPPE-PEG(3400)-folate remain monomolecular and are stable in the cell-culture medium. Caveolae-mediated cell entry was demonstrated by ligand-dependence, by competition with excess folic acid as well as by confocal microscopy.

3T3 Cells↗
PubMedDate not supplied

Efficient intracellular delivery of oligonucleotides formulated in folate receptor-targeted lipid vesicles.

In this study, a novel lipid vector has been developed for targeted delivery of oligodeoxynucleotides (ODN) to tumors that overexpress folate receptor. This is based on a method developed by Semple et al. (1), which utilizes an ionizable aminolipid (1,2-dioleoyl-3-(dimethylammonio)propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities of polyanionic ODN in lipid vesicles. Folate is incorporated into the lipid vesicles via a distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG) spacer. These vesicles are around 100-200 nm in diameter with an ODN entrapment efficiency of 60-80%. Folate mediated efficient delivery of ODN to KB cells that overexpress folate receptor. Uptake of folate-targeted lipidic ODN by KB cells is about 8-10-fold more efficient than that of lipidic ODN without a ligand or free ODN. This formulation is resistant to serum. Thus, targeted delivery of ODN via this novel lipid vector may have potential in treating tumors that overexpress folate receptors.

Base Sequence↗
PubMed1994

Specific inhibition of a human papillomavirus E2 trans-activator by intracellular delivery of its repressor.

Papillomaviruses are the causative agents of benign and malignant epithelial tumors of the skin and mucosa. They encode a DNA-binding protein, E2, that regulates viral transcription and replication, making it an important therapeutic target. By deleting the amino-terminal trans-activation domain of human papillomavirus type 16 (HPV-16) E2 while retaining its carboxy-terminal DNA binding and dimerization domain, an E2 repressor (E2R) that efficiently inhibits transcriptional activation by full-length HPV E2 was generated. To deliver this repressor protein into animal cells, we have utilized the human immunodeficiency virus type 1 (HIV-1) Tat protein which itself is taken up efficiently into intact cells. Chimeras of E2R and the cellular uptake domain of Tat specifically inhibited E2-dependent reporter gene expression in COS-7 cells. Treatment of cervical intraepithelial neoplasia cells having episomally replicating HPV-31 DNA with this Tat-E2R protein led to a dose-dependent loss of HPV DNA copies and inhibition of cell growth. Tat-mediated delivery can be a valuable tool for assessing protein function and may allow the development of novel therapeutic proteins having intracellular targets.

Amino Acid Sequence↗
PubMedDate not supplied

[Approaches to the targeted intracellular delivery of photosensitizers in order to enhance their efficacy and cell specificity].

The main physicochemical properties of photosensitizers used in the photodynamic therapy of cancer and their subcellular distribution after in vitro and in vivo administration were analyzed. It was shown that the effect of photosensitizers is realized at very short distances from the sites of their intracellular localization, and the sensitivities of different cellular compartments to the photocytotoxic action of photosensitizers are different. The necessity of intranuclear delivery of photosensitizers into the nuclei of target cells in order to enhance their efficacy and cell specificity was shown and the available approaches to the targeted delivery of photosensitizers were analyzed. The mechanisms of nucleocytoplasmic transport through the nuclear pore complex, which can be used for the delivery of photosensitizers inward the nucleus, are reviewed. Different modular transporters for photosensitizers comprising (i) a ligand module, which binds to an internalizable receptor overexpressed on the target cells, (ii) an intracellular localization signal, (iii) a carrier module, and (iv) an endosomolytic module were characterized. All these modules were shown to be fully functional within the chimeric polypeptide and the polypeptide as a whole. A significant enhancement of photocytotoxicity and cell specificity of photosensitizers delivered by these transporters were demonstrated. The transporters described represent a new generation of pharmaceuticals which can be widely used for targeted drug delivery.

Animals↗
PubMed2001

Synthesis, analysis, purification, and intracellular delivery of peptide nucleic acids.

Peptide nucleic acids (PNAs) are nonionic DNA mimics. Their novel chemical properties may facilitate the development of selective and potent antisense and antigene strategies for regulating intracellular processes. Described herein are procedures for the synthesis, purification, handling, and characterization of PNAs. A simple protocol for the lipid-mediated introduction of PNAs into in vitro cultures of mammalian cells is provided.

Animals↗
PubMed2000

A weak electrical current enhances intracellular delivery of therapeutic substances.

Cell membranes have electrical properties which can be measured and modified. Administering a weak electrical signal across a tissue is a technique which can be used to determine the quantity of water in the intracellular and extracellular compartments. Using this real-time method of bioelectrical impedance, it was found that a variety of stimuli (including an electrical current) can enhance the passage of water and other substances into the cell. We propose an inexpensive and safe technique which could be utilized to enhance delivery of a wide variety of therapeutic agents into cells for the enhanced delivery of antibiotics, chemotherapy or other therapeutic agents.

Animals↗