Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fluorescence microscopy may provide important information regarding interactions between nanoparticulate drugs carriers, such as liposomes and micelles, with target cells as well as their intracellular fate. Current paper describes various applications of fluorescence microscopy to investigate specific targeting of antibody-modified drug carriers to cancer cells. The enhanced antibody-mediated targeting of drug-loaded immunomicelles confirmed by fluorescence microscopy resulted in enhanced cancer cell killing compared to free drug or drug-loaded nontargeted micelles. Fluorescence microscopy was also used to prove the endosomal escape of properly assembled polymeric micelles (based on polyethylene glycol-phosphatidylethanolamine conjugate, PEG-PE) containing various additives destabilizing the endosomal membrane. When loaded with the anticancer drug (paclitaxel or vitamin K3), such micelles demonstrate increased cytotoxicity. Fluorescence microscopy was also applied to investigate the capture of cell-penetrating TAT peptide-modified liposomes by various cells and stability and intracellular trafficking of captured TAT-liposomes inside cells. It was also used to confirm the successful transfection of cells with TAT-liposomes bearing the plasmid encoding for the Green Fluorescent Protein (GFP).
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PMID:Fluorescence microscopy to follow the targeting of liposomes and micelles to cells and their intracellular fate. 1551 23

To develop targeted pharmaceutical carriers additionally capable of responding to certain local stimuli, such as decreased pH values in tumors or infarcts, targeted long-circulating PEGylated liposomes and PEG-phosphatidylethanolamine (PEG-PE)-based micelles have been prepared with several functions. First, they are capable of targeting a specific cell or organ by attaching the monoclonal antimyosin antibody 2G4 to their surface via pNP-PEG-PE moieties. Second, these liposomes and micelles were additionally modified with biotin or TAT peptide (TATp) moieties attached to the surface of the nanocarrier by using biotin-PE or TATp-PE or TATp-short PEG-PE derivatives. PEG-PE used for liposome surface modification or for micelle preparation was made degradable by inserting the pH-sensitive hydrazone bond between PEG and PE (PEG-Hz-PE). Under normal pH values, biotin and TATp functions on the surface of nanocarriers were "shielded" by long protecting PEG chains (pH-degradable PEG(2000)-PE or PEG(5000)-PE) or by even longer pNP-PEG-PE moieties used to attach antibodies to the nanocarrier (non-pH-degradable PEG(3400)-PE or PEG(5000)-PE). At pH 7.4-8.0, both liposomes and micelles demonstrated high specific binding with 2G4 antibody substrate, myosin, but very limited binding on an avidin column (biotin-containing nanocarriers) or internalization by NIH/3T3 or U-87 cells (TATp-containing nanocarriers). However, upon brief incubation (15-30 min) at lower pH values (pH 5.0-6.0), nanocarriers lost their protective PEG shell because of acidic hydrolysis of PEG-Hz-PE and acquired the ability to become strongly retained on an avidin column (biotin-containing nanocarriers) or effectively internalized by cells via TATp moieties (TATp-containing nanocarriers). We consider this result as the first step in the development of multifunctional stimuli-sensitive pharmaceutical nanocarriers.
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PMID:"SMART" drug delivery systems: double-targeted pH-responsive pharmaceutical nanocarriers. 1684 1

The ability to specifically down-regulate gene expression using the RNAi pathway in mammalian cells has tremendous potential in therapy and in basic science. However, delivery systems capable of efficient and biocompatible delivery of siRNA to target cells are not yet satisfactory. Here, we report the synthesis and in vitro characterization of ABC triblock copolymers that self-assemble with siRNA based on electrostatics and with each other by hydrophobic interactions. The ABC triblock copolymer is based on poly(ethylene glycol) (PEG), poly(propylene sulfide) (PPS), and a positively charged peptide (PEG-PPS-peptide). The diblock copolymer PEG(45)-PPS(5,10) was synthesized using anionic polymerization of propylene sulfide upon a PEG macroinitiator, and the peptide domain was coupled to the PPS terminus using a disulfide exchange reaction with an N-terminal cysteine residue on the peptide. The peptides were designed to interact electrostatically with siRNA, selecting the TAT peptide domain of HIV (RKKRRQRRR) and an oligolysine (Lys(9)). The resulting triblock copolymers were able to self-assemble with siRNA as demonstrated by dynamic light scattering and gel electrophoresis. Complex size was found to be dependent on the amount of polymer used (charge ratio) and the length of the hydrophobic PPS block, achieving sizes ranging from 171 nm to 601 nm. Cell internalization and gene expression down-regulation studies showed that the triblock copolymers are able to transport siRNA inside the cell and mediate gene expression down-regulation, with the amount of internalization and gene transfer affected by charge ratio, PPS length, and the presence of serum. The proposed triblock was able to mediate gene expression down-regulation of GAPDH, achieving up to 90.5% +/- 0.02% down-regulation.
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PMID:Synthesis and in vitro characterization of an ABC triblock copolymer for siRNA delivery. 1735 44

A new pH-sensitive micelle delivery system based on TAT cell penetrating peptide and biodegradable sulfonamide grafted disulfide polymer is presented. The system consists of two components: (1) A polymeric micelle made of Poly(L-lactic acid)-b-poly(ethylene glycol) (PLLA-b-PEG) conjugated to TAT (TAT-micelle), (2) A pH-sensitive diblock copolymer (poly(L-cystine bisamide-g-sulfadiazine))-b-PEG (PCBS-b-PEG). The anionic PCBS complexed with cationic TAT of TAT-micelles forms the final carrier. PCBS showed rapid degradation in the presence of cysteine. The TAT-micelles showed increase in particle size between pH 8.0 and 7.0 upon mixing with PCBS-b-PEG indicating complexation. As the pH was further decreased (pH 6.8 to 6.0) two populations were observed, one of normal TAT-micelles and the other of aggregated PCBS-b-PEG. Flow cytometry showed significantly higher uptake of TAT-micelles at pH 6.6 indicating deshielding compared to pH 7.4. The anticancer drug doxorubicin (DOX) was encapsulated into the TAT-micelles, and the in vitro cytotoxicity at different pHs was evaluated. The system was able to distinguish pHs 7.2 and 7.0 in terms of cytotoxicity.
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PMID:A biodegradable pH-sensitive micelle system for targeting acidic solid tumors. 1799 64

To engineer drug carriers capable of spontaneous accumulation in tumors and ischemic areas via the enhanced permeability and retention (EPR) effect and further penetration and drug delivery inside tumor or ischemic cells via the action of the cell-penetrating peptide (CPP), we have prepared liposomes simultaneously bearing on their surface CPP (TAT peptide, TATp) moieties and protective PEG chains. PEG chains were incorporated into the liposome membrane via the PEG-attached phosphatidylethanolamine (PE) residue with PEG and PE being conjugated with the lowered pH-degradable hydrazone bond (PEG-HZ-PE). Under normal conditions, liposome-grafted PEG "shielded" liposome-attached TATp moieties since the PEG spacer for TATp attachment (PEG(1000)) was shorter than protective PEG(2000). PEGylated liposomes are expected to accumulate in targets via the EPR effect, but inside the "acidified" tumor or ischemic tissues lose their PEG coating due to the lowered pH-induced hydrolysis of HZ and penetrate inside cells via the now-exposed TATp moieties. This concept is shown here to work in cell cultures in vitro as well as in ischemic cardiac tissues in the Langendorff perfused rat heart model and in tumors in experimental mice in vivo.
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PMID:"Smart" drug carriers: PEGylated TATp-modified pH-sensitive liposomes. 1802 40

Biologically active polymer core/shell nanoparticles (i.e. micelles) self-assembled from TAT-poly(ethylene glycol) (PEG)-b-cholesterol (TAT-PEG-b-Chol) were fabricated and used as carrier for targeted blood-brain barrier delivery of antibiotics. Ciprofloxacin as a model antibiotic was efficiently loaded into the nanoparticles by a membrane dialysis method. The actual loading level of ciprofloxacin was dependent on initial loading of ciprofloxacin and fabrication temperature. The blank and ciprofloxacin-loaded nanoparticles were characterized using dynamic light scattering and SEM. The nanoparticles were spherical in nature, having an average size lower than 200 nm. The uptake of nanoparticles with TAT by human brain endothelial cells was greater than that of the nanoparticles without TAT. Most importantly, the nanoparticles with TAT were able to cross the blood-brain barrier (BBB), and located around the cell nucleus of neurons. These nanoparticles may provide a promising carrier to deliver antibiotics across the BBB for the treatment of brain infection.
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PMID:Biologically active core/shell nanoparticles self-assembled from cholesterol-terminated PEG-TAT for drug delivery across the blood-brain barrier. 1815 37

Polymeric micelles self-assembled from cholesterol-conjugated poly(ethylene glycol) (PEG) and anchored with transcriptional activator TAT peptide (TAT-PEG-b-Col) were fabricated for delivery of antibiotics across the blood-brain barrier (BBB). Ciprofloxacin, which demonstrated a high bactericidal effect, was efficiently loaded into the micelles by a membrane dialysis method. The ciprofloxacin-loaded micelles were characterized via dynamic light scattering and SEM. The micelles were spherical in nature, having an average diameter of smaller than 180 nm. Sustained release of ciprofloxacin was achieved over 6 h in phosphate-buffered saline (pH 7.4) at 37 degrees C. Confocal laser scanning microscopy reveals that the uptake of Fluorescein 5-isothiocyanate (FITC)-loaded TAT-PEG-b-Col micelles by human astrocytes was much higher than that of free FITC. Animal studies proved that these micelles crossed the BBB and entered the brain. The TAT-conjugated micelles may be used to deliver antibiotics across the BBB for treatment of brain infections.
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PMID:Polymeric micelles anchored with TAT for delivery of antibiotics across the blood-brain barrier. 1841 28

Exon-skipping oligonucleotides (ESOs) with 2'-O-methyl modifications are promising compounds for the treatment of Duchenne muscular dystrophy (DMD). However, the usefulness of these compounds is limited by their poor delivery profile to muscle tissue in vivo. We previously established that copolymers made of poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) enhanced ESO transfection in skeletal muscle of mdx mice, resulting in widespread distribution of dystrophin-positive fibers, but limited dystrophin expression by Western blot. In an attempt to improve ESO delivery and dystrophin expression, a new formulation of PEG-PEI copolymer was used, along with functionalized derivatives containing either the cell-penetrating peptide TAT (trans-activator of transcription), adsorbed colloidal gold (CG), or both TAT and CG. Tibialis anterior muscles were given three intramuscular injections of various PEG-PEI-ESO polyplexes (3 days apart; 5 microg of ESO per injection) and muscles were harvested 3 weeks after the first injection. Surface modifications of PEG-PEI copolymers with TAT showed the highest level of dystrophin recovery, with a 6-fold increase in dystrophin-positive fibers compared with ESO alone and up to 30% of normal dystrophin expression by Western blot. The adsorption of CG to either PEG-PEI or TAT-PEG-PEI copolymers showed no further improvement in dystrophin expression. Our data indicate that TAT-modified PEG-PEI copolymers are effective carriers for delivery of ESOs to skeletal muscle and are promising compounds for the therapeutic treatment of DMD.
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PMID:Functionalized PEG-PEI copolymers complexed to exon-skipping oligonucleotides improve dystrophin expression in mdx mice. 1864 87

Antigen presenting cells (APC) are among the most important cells of the immune system since they link the innate and the adaptative immune responses, directing the type of immune response to be elicited. To modulate the immune response in immune preventing or treating therapies, gene delivery into immunocompetent cells could be used. However, APC are very resistant to transfection. To increase the efficiency of APC transfection, we have used liposome-based lipoplexes additionally modified with cell-penetrating TAT peptide (TATp) for better intracellular delivery of a model plasmid encoding for the enhanced-green fluorescent protein (pEGFP). pEGFP-bearing lipoplexes made of a mixture of PC:Chol:DOTAP (60:30:10 molar ratio) with the addition of 2% mol of polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate (plain-L) or TATp-PEG-PE (TATp-L) were shown to effectively protect the incorporated DNA from degradation. Uptake assays of rhodamine-labeled lipoplexes and transfections with the EGFP reporter gene were performed with APC derived from the mouse spleen. TATp-L-based lipoplexes allowed for significantly enhanced both, the uptake and transfection in APC. Such a tool could be used for the APC transfection as a first step in immune therapy.
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PMID:Improved transfection of spleen-derived antigen-presenting cells in culture using TATp-liposomes. 1905 90

Recently, PEGylation has been extensively employed to increase the circulation time of liposomes and enhance their accumulation in tumor tissue via the enhanced permeability and retention (EPR) effect; however, poly(ethylene glycol) (PEG) is unfavorable for the uptake of liposomes by tumor cells because of its steric hindrance. In this study, thiolytic cleavable PEG modified liposomes were used to solve this dilemma. Before arrival at the tumor tissue, PEG presents on the surface of liposomes, which is useful for passive accumulation in tumor tissue. Upon reaching the tumor tissues, the PEG chain could be removed by a safe cleaving reagent l-cysteine (l-Cys), and thus, the steric hindrance of PEG could be overcome conveniently. To further improve the uptake of liposomes, a "functional molecule" cell-penetrating peptide TAT was attached to the distal end of a shorter PEG spacer anchored to the surface of the liposomes, which could be shielded by cleavable PEG during circulation; upon arriving at tumor tissue, PEG was removed and thus the "functional molecule" TAT was exposed, and then TAT could mediate the uptake of the liposomes with high efficiency. In this study, thiolytic cleavable PEG was synthesized via a disulfide bridge, DOPE-PEG(1600)-TAT was synthesized by sulfhydryl-maleimide reaction, and then Rh-PE labeled liposomes composed of 2% DOPE-PEG(1600)-TAT and various amounts of cleavable PEG(5000) (2%, 4%, and 8%) were prepared, with particle size around 100 nm and slightly negative charge. These liposomes showed good stability in the presence of 10% serum. Their uptake by tumor cells HepG2 in vitro was assessed qualitatively and quantitatively. Liposomes modified with 2% DOPE-PEG(1600)-TAT and 8% DOPE-S-S-mPEG(5000) were regarded as the optimal formulation. In this preparation, nearly no uptake could be observed before addition of l-Cys, which meant undesired uptake during circulation could be avoided, while the uptake upon addition of l-Cys was 4 times as high as that in the absence of l-Cys. For the uptake in vivo, calcein loaded and Rh-PE labeled 8% cleavable PEG + 2% TAT modified liposomes were injected intratumorally into H22 tumor bearing mice. Confocal laser scanning microscopy (CLSM) showed that the uptake of 8% cleavable PEG + 2% TAT modified liposomes was much higher than that of 8% noncleavable PEG + 2% TAT modified liposomes in the presence of l-Cys. Thus, tumor targeted delivery could be achieved efficiently by the liposomal drug delivery system developed here in a controlled manner.
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PMID:Efficient delivery of payload into tumor cells in a controlled manner by TAT and thiolytic cleavable PEG co-modified liposomes. 2070 Dec 88


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