Syringe loading introduces macromolecules into living mammalian cell cytosol.
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We describe a simple, efficient, gentle and inexpensive technique for the introduction of normally impermeant macromolecules into the cytosol of living mammalian cells growing in suspension or attached to the culturing substratum. Loading is achieved by the production of transient, survivable plasma membrane disruptions as cells are passed back and forth through a standard syringe needle or similar narrow orifice. The loading volume required, which contains cells and the macromolecule to be loaded, can be as little as 5 microliters, thus minimizing the use of valuable reagents. In addition, we report that the surfactant molecule, Pluronic F-68, is capable of altering the physical properties of the plasma membrane in such a way as to increase loading efficiency and the long-term survivability of cells loaded by this and other mechanically based cell-loading techniques. (+info)
Effect of pluronic P85 on ATPase activity of drug efflux transporters.
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PURPOSE: Pluronic block copolymers are potent sensitizers of multi-drug resistant (MDR) cancer cells. The sensitization effect by Pluronics is a result of two processes acting in concert: i) intracellular ATP depletion, and ii) inhibition of ATPase activity of drug efflux proteins. This work characterizes effects of Pluronic P85 on ATPase activities of Pgp, MRP1, and MRP2 drug efflux transport proteins and interaction of these proteins with their substrates, vinblastine, and leucotriene C4. METHODS: Using membranes overexpressing Pgp, MRP1, and MRP2, the current study evaluates effects of Pluronic P85 (P85) on the kinetic parameters (Vmax, Km, Vmax/Km) of ATP hydrolysis by these ATPases. RESULTS: The decreases in the maximal reaction rates (Vmax) and increases in apparent Michaelis constants (Km) for these transporters in the presence of various concentrations of P85 were observed. The mechanism of these effects may involve i) conformational changes of the transporter due to membrane fluidization and/or ii) nonspecific steric hindrance of the drug-binding sites by P85 chains embedded into cellular membranes. The extent of these alterations was increased in the row MRP1 < MRP2 << Pgp. CONCLUSIONS: These data suggest that there are unifying pathways for the inhibition of Pgp and MRPs by the block copolymer. However, the effect of P85 on Pgp ATPase activity is considerably greater compared with the effects on MRP1 and MRP2 ATPases. This may be a reason for greater inhibitory effects of Pluronic in Pgp- compared with MRP-overexpressing cells. (+info)
Effect of intestinal chylomicron secretory blockade on apolipoprotein synthesis in the newborn piglet.
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Pluronic L-81 is a hydrophobic surfactant which blocks intestinal chylomicron secretion at the pre-Golgi level without affecting triacylglycerol uptake and re-esterification. To study the effects of such blockade on apolipoprotein synthesis, newborn female piglets received 24 h intraduodenal infusions of low-triacylglycerol, or high-triacylglycerol with or without Pluronic L-81, diets, followed by determination of apolipoprotein (apo) B-48, A-I and A-IV synthesis and content and apo B and A-IV mRNA levels in the small intestine. Jejunal apo B-48 content, synthesis and mRNA levels were down-regulated below basal levels by the addition of Pluronic to the high-triacylglycerol infusion. The normal increase in apo A-I synthesis induced by triacylglycerol absorption was ablated in both jejunum and ileum, even though the expected increase in apo A-I content in jejunum still occurred. Although attenuated, the expected increase in jejunal apo A-IV synthesis and mRNA levels with triacylglycerol absorption was still present with Pluronic treatment. These results suggest very different mechanisms of cellular regulation and trafficking for the various apolipoproteins incorporated into nascent intestinal chylomicrons. Apo B may be specifically down-regulated by the chylomicron secretory blockade induced by Pluronic L-81. (+info)
The polyoxyethylene/polyoxypropylene block co-polymer poloxamer-407 selectively redirects intravenously injected microspheres to sinusoidal endothelial cells of rabbit bone marrow.
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Small colloidal particulates (150 nm and below, in diameter) can be redirected specifically to the rabbit bone marrow following intravenous administration by coating their surface with the block co-polymer poloxamer-407, a non-ionic surfactant. The coated colloids are sequestered by the sinusoidal endothelial cells of the bone marrow and are accumulated in dense bodies within these cells. The uptake of poloxamer-407-coated colloids by marrow endothelial cells suggests that the steric repulsive barrier, imposed by the polyoxyethylene segment of the polymer, to particle-cell interaction can apparently be overcome by a specific interaction mechanism(s) with the cell surface. Such a dramatic uptake cannot be achieved with other block co-polymers of similar structure to poloxamer-407. The application of the current model for the site-specific targeting of drug carriers to bone marrow and the prevention of the adherence of metastases of tumours which selectively colonize the bone marrow endothelium is discussed. (+info)
Alteration of genomic responses to doxorubicin and prevention of MDR in breast cancer cells by a polymer excipient: pluronic P85.
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Polymer therapeutics has emerged as a new clinical option for the treatment of human diseases. However, little is known about pharmacogenetic responses to drugs formulated with polymers. In this study, we demonstrate that a formulation containing the block copolymer Pluronic P85 and antineoplastic drug doxorubicin (Dox) prevents the development of multidrug resistance in the human breast carcinoma cell line, MCF7. Specifically, MCF7 cells cultured in the presence of Pluronic were unable to stably grow in concentrations of Dox that exceeded 10 ng of Dox/mL of culture medium. In sharp contrast, MCF7 cells cultured in the absence of the block copolymer resulted in the selection and stable growth of cells that tolerated a 1000 times higher concentration of the drug (10 000 ng of Dox/mL of culture medium). Detailed characterization of the isolated sublines demonstrated that those cells selected in the polymer-drug formulation did not show amplification of the MDR1 gene, likely resulting in their high sensitivity to the drug. Conversely, cells selected with Dox alone showed an elevated level in the expression of the MDR1 gene along with a corresponding increase in the expression level of the drug efflux transporter, Pgp, and likely contributing to the high resistance of the cells to Dox. Global analysis of the expression profiles of 20K genes by DNA microarray revealed that the use of Pluronic in combination with Dox drastically changed the direction and magnitude of the genetic response of the tumor cells to Dox and may potentially enhance therapeutic outcomes. Overall, this study reinforces the need for a thorough assessment of pharmacogenomic effects of polymer therapeutics. (+info)
Comparison of nanogel drug carriers and their formulations with nucleoside 5'-triphosphates.
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PURPOSE: The aim of the study is to synthesize and characterize nanogel carriers composed of amphiphilic polymers and cationic polyethylenimine for encapsulation and delivery of cytotoxic nucleoside analogs 5'-triphosphates (NTPs) into cancer cells. METHODS: Nanogels were synthesized by a novel micellar approach and compared with carriers prepared by the emulsification/evaporation method. Complexes of nanogels with NTP were prepared; particle size and in vitro drug release were characterized. Resistance of the nanogel-encapsulated NTP to enzymatic hydrolysis was analyzed by ion-pair high-performance liquid chromatography. Binding to isolated cellular membranes, cellular accumulation and cytotoxicity were compared using breast carcinoma cell lines CL-66, MCF-7, and MDA-MB-231. In vivo biodistribution of the 3H-labeled NTP encapsulated in different types of nanogels was evaluated in comparison to the injected NTP alone. RESULTS: Nanogels with a particle size of 100-300 nm in the unloaded form and less than 140 nm in the NTP-loaded form were prepared. An in vitro release of NTP was >50% during the first 24 h. Nanogel formulations ensured increased NTP drug stability against enzymatic hydrolysis as compared to the drug alone. Pluronic-based nanogels NG(F68), NG(F127), NG(P85), and NGM(P123) demonstrated 2-2.5 times enhanced interaction with cellular membranes and association with various cancer cells compared to NG(PEG). Among them, NG(F68) and NG(F127) exhibited the lowest cytotoxicity. Injection of nanogel-formulated NTP significantly modulated the drug accumulation in different mouse organs. CONCLUSIONS: Nanogels composed of Pluronic F68 and P123 were shown to display certain advanced properties compared to NG(PEG) as a drug delivery system for NTP analogs. Formulations of nucleoside analogs in active NTP form with these nanogels will improve the delivery of these cytotoxic drugs to cancer cells and the therapeutic potential of this anticancer chemotherapy. (+info)
Pharmacokinetics and biodistribution of polymeric micelles of paclitaxel with Pluronic P123.
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AIM: To investigate the preparation, in vitro release, in vivo pharmacokinetics and tissue distribution of a novel polymeric micellar formulation of paclitaxel (PTX) with Pluronic P123. METHODS: The polymeric micelles of paclitaxel with Pluronic P123 were prepared by a solid dispersion method. The characteristics of micelles including particle size distribution, morphology and in vitro release of PTX from micelles were carried out. PTX-loaded micellar solutions were administered through the tail vein to healthy Sprague-Dawley rats and Kunming strain mice to assess the pharmacokinetics and tissue distribution of PTX, respectively. Taxol, the commercially available intravenous formulation of PTX, was also administered as control. RESULTS: By using a dynamic light scattering sizer and a transmission electron microscopy, it was shown that the PTX-loaded micelles had a mean size of approximately 25 nm with narrow size distribution and a spherical shape. PTX was continuously released from Pluronic P123 micelles in release medium containing 1 mol/L sodium salicylate for 24 h at 37 centigrade degree. In the pharmacokinetic assessment, t(1/2beta) and AUC of micelle formulation were 2.3 and 2.9-fold higher than that of Taxol injection. And the PTX-loaded micelles increased the uptake of PTX in the plasma, ovary and uterus, lung, and kidney, but decreased uptake in the liver and brain in the biodistribution study. CONCLUSION: Polymeric micelles using Pluronic P123 can effectively solubilize PTX, prolong blood circulation time and modify the biodistribution of PTX. (+info)
Novel delivery system enhances efficacy of antiretroviral therapy in animal model for HIV-1 encephalitis.
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Most potent antiretroviral drugs (e.g., HIV-1 protease inhibitors) poorly penetrate the blood-brain barrier. Brain distribution can be limited by the efflux transporter, P-glycoprotein (P-gp). The ability of a novel drug delivery system (block co-polymer P85) that inhibits P-gp, to increase the efficacy of antiretroviral drugs in brain was examined using a severe combined immunodeficiency (SCID) mouse model of HIV-1 encephalitis (HIVE). Severe combined immunodeficiency mice inoculated with HIV-1 infected human monocyte-derived macrophages (MDM) into the basal ganglia were treated with P85, antiretroviral therapy (ART) (zidovudine, lamivudine and nelfinavir (NEL)), or P85 and ART. Mice were killed on days 7 and 14, and brains were evaluated for levels of viral infection. Antiviral effects of NEL, P85, or their combination were evaluated in vitro using HIV-1 infected MDM and showed antiretroviral effects of P85 alone. In SCID mice injected with virus-infected MDM, the combination of ART-P85 and ART alone showed a significant decrease of HIV-1 p24 expressing MDM (25% and 33% of controls, respectively) at day 7 while P85 alone group was not different from control. At day 14, all treatment groups showed a significant decrease in percentage of HIV-1 infected MDM as compared with control. P85 alone and combined ART-P85 groups showed the most significant reduction in percentage of HIV-1 p24 expressing MDM (8% to 22% of control) that were superior to the ART alone group (38% of control). Our findings indicate major antiretroviral effects of P85 and enhanced in vivo efficacy of antiretroviral drugs when combined with P85 in a SCID mouse model of HIVE. (+info)