共查询到20条相似文献,搜索用时 15 毫秒
1.
Delivery of Plasmid DNA into Mammalian Cell Lines Using pH-Sensitive Liposomes: Comparison with Cationic Liposomes 总被引:9,自引:0,他引:9
We compare the transfection efficiency of plasmid DNA encoding either luciferase or (-galactosidase encapsulated in pH-sensitive liposomes or non-pH-sensitive liposomes or DNA complexed with cationic liposomes composed of dioleoyloxypropyl-trimethylammonium:dioleoylphosphatidyl-ethanolamine (1:1, w/w) (Lipofectin) and delivered into various mammalian cell lines. Cationic liposomes mediate the highest transient transfection level in all cell-lines examined. pH-sensitive liposomes, composed of cholestryl hemisuccinate and dioleoylphosphatidylethanolamine at a 2:1 molar ratio, mediate gene transfer with efficiencies that are 1 to 30% of that obtained with cationic liposomes, while non-pH-sensitive liposome compositions do not induce any detectable transfection. Cationic liposomes mediate a more rapid uptake of plasmid DNA, to about an eightfold greater level than that obtained with pH-sensitive liposomes. The higher uptake of DNA mediated by Lipofectin accounts for part of its high transfection efficiency. Treatment of cells with chloroquine, ammonium chloride, or monensin decreases (threefold) transfection using pH-sensitive liposomes and either has no effect on or enhances cationic liposome-mediated transfection. Therefore plasma membrane fusion is not the only mechanism available to cationic liposomes; in certain cell lines DNA delivery via endocytosis is a possible parallel pathway and could augment the superior transfection efficiency observed with cationic liposomes. 相似文献
2.
Purpose. Genetic immunization using naked plasmid DNA (pDNA) has been shown to elicit broad humoral and cellular immune responses. However, more versatile and perhaps cell-targeted delivery systems are needed. To this end, a novel process to engineer cationic nanoparticles coated with pDNA for genetic immunization was explored.
Methods. Cationic nanoparticles were engineered from warm oil-in-water microemulsion precursors composed of emulsifying wax as the oil phase and cetyltrimethylammonium bromide (CTAB) as the cationic surfactant. Plasmid DNA was coated on the surface of the cationic nanoparticles to produce pDNA-coated nanoparticles. An endosomolytic lipid and/or a dendritic cell-targeting ligand (mannan) were incorporated in or deposited on the nanoparticles to enhance the in vitro cell transfection efficiency and the in vivo immune responses after subcutaneous injection to Balb/C mice. The IgG titer to expressed -galactosidase and the cytokine release from isolated splenocytes after stimulation were determined on 28 days.
Results. Cationic nanoparticles (around 100 nm) were engineered within minutes. The pDNA-coated nanoparticles were stable at 37°C over 30 min in selected biologic fluids. Transmission electron microscopy showed the nanoparticles were spherical. Plasmid DNA-coated nanoparticles, especially those with both an endosomolytic lipid and dendritic cell-targeting ligand, resulted in significant enhancement in both IgG titer (over 16-fold) and T-helper type-1 (Th1-type) cytokine release (up to 300% increase) over naked pDNA.
Conclusion. A novel method to engineer pDNA-coated nanoparticles for enhanced in vitro cell transfection and enhanced in vivo immune responses was reported. 相似文献
3.
Intradermal Injection of Transforming Growth Factor-β1 Gene Enhances Wound Healing in Genetically Diabetic Mice 总被引:2,自引:0,他引:2
Purpose. To evaluate the biologic effect of direct cutaneous TGF-1 gene delivery on impaired wound healing models using genetically diabetic mice.
Methods. Diabetic mice (C57BKS.Cg-m +/+ Leprdb female mice) with 1 cm × 1 cm excisional wounds were intradermally injected with 60 g of plasmid DNA encoding TGF-1 gene. The wound closure was measured up to 14 days postwounding. At days 7 and 14 postwounding, sections of skin were taken for hematoxylin and eosin and Masson's trichome staining to examine the morphology and collagen deposition. The cell proliferation and TGF-1 gene expression were studied using immunohistochemical stainings for 5-bromo-2-deoxy-uridine and for TGF-1.
Results. A higher cell proliferation rate and a denser and more organized new extracellular matrix were observed in the treated wound site. Complete wound closure was detected as early as 7 days for TGF-1-treated group in comparison with 11-14 days for the untreated, control plasmid DNA- and PBS-treated groups.
Conclusion. A single intradermal injection of TGF-1 plasmid DNA was sufficient to enhance wound healing. This approach represents a new strategy that may be applied to the treatment of excisional wounds in human diabetic patients. 相似文献
4.
Purpose. The long-term objective of this research is to understand how the efficiency of iontophoresis depends upon the structural and physicochemical properties of the administered drug. Specifically, the ability of certain drug species to alter the permselective properties of the skin was examined.
Methods. Using conventionalin vitro methodology, the inhibition of electroosmotic flow induced by the iontophoresis of five different -blockers (of varying lipophilicity) was examined. The concomitant electrotransport of the most lipophilic species (propranolol) and the convective movement of solvent in the anode-to-cathode direction were measured. In addition, the possibility that electroosmosis might be augmented by the delivery of anionic drugs was also considered.
Results. Iontophoresis of lipophilic, cationic -blockers caused a concentration-dependent inhibition of conventional electroosmosis. The most hydrophilic analogs elicited no effect. As a result of this charge neutralization phenomenon, the optimal concentration for propranolol iontophoresis was significantly less than the maximum achievable in aqueous solution. Only a very modest improvement in convective solvent flow was induced by the cathodal iontophoresis of anionic compounds.
Conclusions. The permselectivity of the skin can be altered by drugs which are positively charged and which possess a significant, adjacent hydrophobic surface. The latter seems able to 'anchor' the molecule in the skin and the counter charge to the membrane's negative character ensures a tight association. Both lipophilicity and a positive charge are essential—without either, the phenomenon is not observed. The conformational flexibility of the drugs studied to-date, however, prevents unambiguous conclusions about the three-dimensional nature of the putative 'binding site'. 相似文献
5.
Lee Minhyung Nah Jae-Woon Kwon Youngmin Koh Jae Joon Ko Kyung Soo Kim Sung Wan 《Pharmaceutical research》2001,18(4):427-431
Purpose. Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery because of its high positive charges and low cytotoxicity. In this study, low molecular weight chitosan (LMWC, molecular weight of 22 kDa) was characterized and evaluated as a gene carrier.
Methods. Plasmid/LMWC complex was analyzed in 1% agarose gel electrophoresis. To confirm that the LMWC protected plasmids from nuclease, DNase I protection assays were performed. pSV--galactosidase plasmid/LMWC complex was transfected into 293T cells and transfection efficiency was evaluated by -galactosidase assay. Cytotoxicity of LMWC was determined by MTT assay.
Results. Unlike high molecular weight chitosan (HMWC), LMWC is highly water soluble, and can form complex with plasmids in physiological buffer. The plasmid DNA was completely retarded at a weight ratio of 1:2 (plasmid:LMWC) in 1% agarose gel. DNase I protection assay showed that plasmids were protected from DNase I over 60 min. The most efficient transfection was obtained at a weight ratio of 1:3 (plasmid:LMWC). The transfection efficiency of LMWC was significantly higher than naked DNA and higher than poly-L-lysine (PLL). MTT assay showed that LMWC was less cytotoxic than PLL.
Conclusions. LMWC is non-toxic and has higher transfection efficiency than PLL. Therefore, LMWC will be useful in the development of safe gene carriers. 相似文献
6.
Purpose. This study tests the hypothesis that gene transfer efficiency may be improved through the use of transiently stable transfection complexes that degrade within endosomal compartments and promote plasmid escape into the cytosol.
Method. An acid labile cationic lipid, O-(2R-1,2-di-O-(1`Z, 9`Z-octadecadienyl)-glycerol)-3-N-(bis-2-aminoethyl)-carbamate (BCAT), was designed, synthesized, and tested for enhanced gene transfer activity relative to non-labile controls.
Results. The O-alkenyl chains of BCAT were completely hydrolyzed after 4 h incubation in pH 4.5 buffer at 25°C. Addition of BCAT to plasmid DNA in 40%ethanol followed by ethanol evaporation yielded transfection complexes that transfected several cell types in the presence of fetal calf serum and without the need of a helper lipid. Transfection complexes prepared from BCAT displayed higher luciferase expression than the corresponding DCAT complexes (an acid-insensitive derivative of BCAT) for all cell types tested. Uptake studies showed that this increase was not due to a difference in the amount of DNA being delivered. FACS analysis for GFP expression showed that BCAT transfection complexes yielded 1.6 more transfected cells and 20%higher log mean fluorescence than DCAT transfection complexes. In vivo gene transfer was demonstrated in subcutaneous tumor-bearing mice by systemic administration of a 60 g plasmid dose. Expression was observed in the lungs and in the tumor, with the highest activity being observed in the lungs.
Conclusions. Our results show that increased transfection can be obtained by coupling the cationic headgroup to the hydrophobic amphiphilic tails via acid-labile bonds. Acid-catalyzed release of the alkyl chains should facilitate dissociation of the cationic lipid headgroup from the plasmid, thus accelerating one of the rate-limiting steps in cationic lipid mediated transfection. 相似文献
7.
Receptor-Mediated Gene Targeting to Tissues In Vivo Following Intravenous Administration of Pegylated Immunoliposomes 总被引:9,自引:0,他引:9
Purpose. Gene therapy has been limited by the immunogenicity of viral vectors, by the inefficiency of cationic liposomes, and by the rapid degradation in vivofollowing the injection of naked DNA. The present work describes a new approach that enables the non-invasive, non-viral gene therapy of the brain and peripheral organs following an intravenous injection.
Methods. The plasmid DNA encoding -galactosidase is packaged in the interior of neutral liposomes, which are stabilized for in vivo use by surface conjugation with polyethyleglycol (PEG). The tips of about 1% of the PEG strands are attached to a targeting monoclonal antibody (MAb), which acts as a molecular Trojan Horse to ferry the liposome carrying the gene across the biological barriers of the brain and other organs. The MAb targets the transferrin receptor, which is enriched at both the blood-brain barrier (BBB), and in peripheral tissues, such as liver and spleen.
Results. Expression of the exogenous gene in brain, liver, and spleen was demonstrated with -galactosidase histochemistry, which showed persistence of gene expression for at least 6 days after a single intravenous injection of the pegylated immunoliposomes. The persistence of the transgene was confirmed by Southern blot analysis.
Conclusions. Widespread expression of an exogenous gene in brain and peripheral tissues is induced with a single intravenous administration of plasmid DNA packaged in the interior of pegylated im- munoliposomes. The liposomes are formulated to target specific receptor systems that enable receptor-mediated endocytosis of the complex into cells in vivo. This approach allows for non-invasive, non-viral gene therapy of the brain. 相似文献
8.
Purpose. To develop and evaluate a novel artificial lipoprotein delivery system for in vitro gene transfection in human glioma cells.
Method. Nanoemulsion was formulated with similar lipid compositions present in natural lipoproteins. The oil phase of nanoemulsion was composed of triolein (70%), egg phosphatidylcholine (22.7%), lysophosphatidylcholine (2.3%), cholesterol oleate (3.0%), and cholesterol (2.0%). To replace the surface protein as in natural lipoprotein, poly-L-lysine was modified to add palmitoyl chains at a basic condition and was incorporated onto the nanoemulsion particles through hydrophobic interaction. A model plasmid DNA, pSV--Gal containing a reporter gene for -galactosidase was carried by the nanoemulsion/poly-L-lysine particles. The charge variation of so-formed complex was examined by agarose gel electrophoresis and zeta potential measurement. In vitro transfection was conducted on human SF-767 glioma cell line using this new system. After standard X-Gal staining, transfected cells were observed under light microscope. The effect of chloroquine on the transfection was examined and, finally, the cytotoxicity of this new system was evaluated in comparison with commercial Lipofectamine gene transfection system.
Results. The plasmid DNA was effectively carried by this artificial lipoprotein delivery system and the reporter gene was expressed in the glioma cells. Transfection efficiency was significantly increased by the treatment of chloroquine, indicating that endocytosis possibly was the major cellular uptake pathway. Compared to Lipofectamine system, this new delivery system demonstrated similar transfection efficiency but a much lower cytotoxicity. In the experiment, the cell viability showed up to 75% using this system compared to only 24% using Lipofectamine system.
Conclusion. A new artificial lipoprotein delivery system was developed for in vitro gene transfection in tumor cells. The new system showed similar transfection efficiency but a much lower cytotoxicity compared with commercial Lipofectamine system. 相似文献
9.
da Silveira Airton Monza Ponchel Gilles Puisieux Francis Duchêne Dominique 《Pharmaceutical research》1998,15(7):1051-1055
Purpose. The aim of this study was to prepare and characterize nanoparticulate systems constituted of poly(isobutylcyanoacrylate) and cyclodextrins and intended for increasing the loading of the particles with lipophilic substances. Progesterone was used as a model substance.
Methods. Nanoparticles were prepared by polymerization of isobutyl-cyanoacrylate in presence of cyclodextrins or progesterone/ hydroxy-propyl--cyclodextrin complex. Particle size, zeta potential, cyclodextrin and progesterone loading of the particles were determined.
Results. Nanoparticles could be easily prepared in presence of cyclodextrins. An increase in hydroxypropyl--cyclodextrin concentration resulted in small nanoparticles (less than 50 nm). It was found that large amounts of cyclodextrins remained associated to the particles, resulting in a 50 fold increase in progesterone loading compared to nanoparticles prepared in absence of cyclodextrins.
Conclusions. The poly(isobutylcyanoacrylate)—cyclodextrin nanoparticles were characterized by the presence of many lipophilic sites belonging to the cyclodextrins which were firmly anchored to the structure of the particles. Therefore, this new type of nanoparticles offers probably an opportunity for increasing the loading of nanoparticles with various lipophilic drugs. 相似文献
10.
Purpose. The oral buccal mucosa may be an ideal site for mucosal immunization, allowing for the needle-free administration of cost-effective vaccines. A novel mucoadhesive bilayer film was developed to test the feasibility of this route of immunization in rabbits.
Methods. Bilayer films were developed using different ratios of Noveon and Eudragit S-100 as the mucoadhesive layer and a pharmaceutical wax as the impermeable backing layer. Optimal 3/8-inch films were post-loaded with 100 g of plasmid DNA (CMV--gal) or -galactosidase protein. The in vitro release rates and stability of the postloaded antigens were determined. The films were applied to the buccal pouch of rabbits on days 0, 7, and 14, and the humoral and splenocyte proliferative immune responses to -gal were determined through day 28 and compared to those responses after conventional subcutaneous injection of adjuvanted protein.
Results. The weight ratio of Noveon and Eudragit S-100 had a significant effect on adhesion time of bilayer films. Postloaded plasmid DNA and -gal remained stable after being released from bilayer films (release of 60-80% in 2 h for both). Buccal immunization using novel bilayer films (109 ± 6-m thickness) containing plasmid DNA led to comparable antigen-specific IgG titer to that of subcutaneous protein injection. All rabbits immunized with plasmid DNA via the buccal route but none by the subcutaneous route with protein antigen demonstrated splenocyte proliferative immune responses.
Conclusion. The feasibility of buccal (genetic) immunization with these novel bilayer films was demonstrated. 相似文献
11.
Prevention of Autoimmune Insulitis by Delivery of Interleukin-4 Plasmid Using a Soluble and Biodegradable Polymeric Carrier 总被引:4,自引:0,他引:4
Purpose. We delivered interleukin-4 (IL-4) plasmid (pCAGGS-IL-4) using the biodegradable polymer, poly[-(4-aminobutyl)-L-glycolic acid] (PAGA), to prevent autoimmune insulitis in NOD mice.
Methods. The pCAGGS-IL-4/PAGA complex was transfected to 293T cells. The expression level of IL-4 was measured by ELISA. The pCAGGS IL-4/PAGA complex was injected once to NOD mice intravenously at the age of 4 weeks. RT-PCR was performed to evaluate the level of the IL-4 mRNA in the liver. At 6 weeks after the injection, the grade of insulitis of the mice was evaluated by double blind methods.
Results.
In vitro transfecton assays showed that PAGA enhanced the expression of IL-4 in 293T cells. RT-PCR of the liver showed that IL-4 was expressed highest in the complex injected group. In the plasmid/PAGA complex injected group, the prevalence of severe insulitis in NOD mice was markedly improved, suggesting that PAGA enhanced the delivery of IL-4 plasmid.
Conclusion. The pCAGGS-IL-4/PAGA complex is an effective system to prevent autoimmune insulitis in NOD mice and applicable for the prevention of autoimmune diabetes. 相似文献
12.
Biodegradable Polyester,Poly[α-(4-Aminobutyl)-l-Glycolic Acid], as a Non-Toxic Gene Carrier 总被引:3,自引:0,他引:3
Purpose. The aim of this study was to develop a non-toxic polymericgene carrier. For this purpose, biodegradable cationic polymer,poly[-(4-aminobutyl)-l-glycolic acid] (PAGA) was synthesized. PAGA wasdesigned to have ester linkage because polyesters usually showbiodegradability.
Methods. Degradation of PAGA in an aqueous solution was followedby matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS). PAGA/DNA complexes were characterizedby gel electrophoresis, atomic force microscopy (AFM), dynamiclight scattering (DLS). The transfection was measured by using the -galactosidase reporter gene.
Results. PAGA was degraded in aqueous solution very quickly andthe final degradation product was a monomer (l-oxylysine). Formationof self-assembling biodegradable complexes between PAGA and DNAat a charge ratio 1:1 (+/–) was confirmed by gel band shift assay andAFM. In these studies, controlled release of DNA from the complexescould be seen. The complexes showed about 2-fold higher transfectionefficiency than DNA complexes of poly-l-lysine (PLL), a structuralanalogue of PAGA, which is the most commonly used poly-cation forgene delivery. The polymer did not show cytotoxicity, possibly becauseof its degradability and the biocompatibility of the monomer.
Conclusions. The use of the biodegradable poly-cation, PAGA, as aDNA condensing agent will be useful in safe gene delivery. 相似文献
13.
Terplex DNA Delivery System As a Gene Carrier 总被引:1,自引:0,他引:1
Kim Jin-Seok Maruyama Atsushi Akaike Toshihiro Kim Sung Wan 《Pharmaceutical research》1998,15(1):116-121
Purpose. To characterize the physical and biochemical properties of the DNA terplex delivery system, which has previously been shown to deliver and express pSV--gal plasmid efficiently in cultured smooth muscle cells (SMC) (1).
Methods. Atomic force microscopy (AFM), zeta-potential measurement (ZP), gel electrophoresis (GE), circular dichroism (CD), fluorescence quenching and 1H-NMR spectrometry were used.
Results. AFM showed that the plasmid DNA of about 600 nm long in its extended state was condensed to the size of about 100 nm by terplex formation. The DNA condensing effect of the terplex system was as good as unmodified PLL, as shown by an ethidium bromide displacement assay. Zeta-potential measurement showed that the terplex system exerts a slightly positive surface charge (+2 mV) at a 1:1:1 weight ratio of DNA:LDL:stearyl-PLL, which showed the best transfection efficiency on SMC. GE indicated that electrophoretic mobility of the terplex system decreased with increasing amounts of stearyl-PLL, indicating that the surface charge of the terplex system became more positive as more stearyl-PLL was added. Results from CD showed that there was no significant changes in tertiary structure of plasmid DNA from the terplex formation. Presence of strong hydrophobic interaction between stearyl-PLL and LDL was confirmed by 1H-NMR, where about a 30% decrease in -methylene peak of PLL backbone was observed when stearyl-PLL was mixed with LDL, but this phenomenon was not observed when unmodified PLL was used.
Conclusions. Our results indicate that the plasmid DNA, when formulated with the stearyl-PLL and LDL, forms a stable and hydrophobicity / charge balanced terplex system of optimal size for efficient cellular uptake and the DNA is still intact after the terplex formation. This information is expected to be utilized for the development of much improved transfection vectors for in vivogene therapy. 相似文献
14.
Okimoto K Ohike A Ibuki R Aoki O Ohnishi N Irie T Uekama K Rajewski RA Stella VJ 《Pharmaceutical research》1999,16(4):549-554
Purpose. The purpose of this study was to develop a controlled-porosity osmotic pump tablet (OPT) which exhibits pH-independent release profiles for a basic drug using a sulfobutyl ether--cyclodextrin, (SBE)7m--CD, which acts as both a solubilizer and as an osmotic agent.
Methods. Chlorpromazine free base (CLP) was chosen as a model drug for this study. The release of CLP from osmotic pump tablets was studied in vitro. In vivo absorption of CLP from the OPT was evaluated in male beagle dogs.
Results. The CLP release profile from an OPT prepared from a core tablet composed of a 1:10 molar ratio of CLP to (SBE)7m--CD was pH-independent, and was controlled by modulating the membrane thickness of the OPT. Another cyclodextrin, hydroxypropyl--cyclodextrin (HP--CD), and a sugar mixture of lactose and fructose resulted in pH-dependent release at the same molar ratio. An in vivo absorption study in dogs with an OPT containing (SBE)7m--CD correlated very well with the in vitro release profiles using the Japanese Pharmacopoeia dissolution method.
Conclusions. In addition to serving as a solubilizer and osmotic agent, (SBE)7m--CD can also serve as the controlling agent for pH independent release of CLP from OPTs. This system successfully modified the in vivo input rate of CLP without compromising oral bioavailability. 相似文献
15.
Purpose. The main objective of this investigation was to explore thecytotoxic structure-activity relationships of -substituted -aryloxymethyl--methylene--butyrolactones against cancer cells.
Methods. The target compounds were synthesized in two stepscommencing with aryl-OH which was treated with a bromomethyl ketonefollowed by the Reformatsky-type condensation.
Results. Seven types of -methylene--butyrolactones were evaluatedin vitro against 60 human cancer cell lines derived from nine cancercell types. The average values of log G50 indicated that for thearylportion, potencies of these -methylene--butyrolactones are in adecreasing order of quinolin-2(1H)-one (or 2-hydroxyquinoline, 21,–5.89) > quinoline (19, –5.79) > 2-methylquinoline (20, –5.69)> 8-hydroxyquinoline (17, –5.64) > 2-naphthalene (16, –5.59)> benzene (15, –4.90). The same order was obtained for both log TGIand log LC50. However, for the -substituent, the potencies are in adecreasing order of biphenyl (16f–21f) > phenyl and4-substituted phenyl (16b-e–21b-e) > methyl (16a–21a).
Conclusions. Unlike cardiovascular activities of -methylene--butyrolactones in which a -methyl substituent is necessary for vasorelaxingeffect while a phenyl or a halogen-substituted phenyl is prefer for theantiplatelet activities, a -biphenyl substituent proved to be the bestfor their cytotoxicities against various cancer cell lines tested. 相似文献
16.
Purpose. Traditionally, vaccines have been administered by needle injection. Topical immunization through the intact skin with either protein- or DNA-based vaccines has attracted much attention recently. We sought to enhance the immune responses induced by DNA-based vaccines after topical application by developing novel ethanol-in-fluorocarbon (E/F) microemulsion systems to aid in the delivery of plasmid DNA (pDNA).
Methods. Ten different fluorosurfactants were selected or synthesized and screened by pseudo-phase-diagram construction for their ability to form E/F microemulsions. Plasmid DNA was successfully incorporated into E/F microemulsions using several different fluorosurfactants and perfluorooctyl bromide as the continuous fluorocarbon phase. For several reasons, Zonyl® FSN-100 (an ethoxylated nonionic fluorosurfactant) was selected for further studies. In vivo studies were performed in mice to assess pDNA expression in skin and immunologic responses after topical application of this system using a luciferase-encoding plasmid (CMV-luciferase) and a CMV--galactosidase-encoding plasmid, respectively.
Results. Plasmid DNA incorporated into E/F microemulsion using FSN-100 as the surfactant was found to be stable. After topical application of this E/F microemulsion system, significant enhancements in luciferase expression and antibody and T-helper type-1 biased immune responses were observed relative to those of naked pDNA in saline or ethanol. For example, with the E/F microemulsion system, the specific serum IgG and IgA titers were increased by 45-fold and over 1000-fold, respectively.
Conclusion. A novel fluorocarbon-based microemulsion system for potential DNA vaccine delivery was developed. 相似文献
17.
Purpose. To identify factors influencing nonviral vector transfection in differentiated CaCo-2 and mucus-secreting coculture, CaCo-2:Ht29GlucH, cell culture models and to compare these in vitro results with in vivo transfection efficiency in rat intestine.
Methods. A range of nonviral vectors including DOTAP, Lipofectin, Superfect, PEI, and polylysine were investigated. CaCo-2 and a mucus-secreting coculture were used at 21 days. Transfection efficiency was assessed using pCMVluc (firefly luciferase) plasmid, and radiolabeled plasmid was used to determine the binding and internalization of plasmid DNA. The in vivo model used was a ligated rat intestinal loop.
Results. Transfection levels decreased by over 1000-fold in differentiated models relative to nondifferentiated COS-7 cells and were related to reductions in luciferase production by individual cells. Active internalization of DNA by the differentiated cells decreased. Removal of mucus by the mucolytic agent N-acetylcysteine, from the coculture system significantly reduced (p < 0.05) transfection efficiency. In vivo the transfection efficiency of PEI proved superior to DOTAP.
Conclusions. Nonviral gene delivery to the hostile environment of the intestine is possible. Mechanistic studies using differentiated intestinal cell models aid identification of the rate-limiting steps to transfection and represent a more physiologically relevant approach to predict gene delivery to the intestine. 相似文献
18.
Saitoh Hiroshi Fujisaki Hiroko Aungst Bruce J. Miyazaki Katsumi 《Pharmaceutical research》1997,14(5):645-649
Purpose. The purpose of this study was to examine factors limiting the intestinal absorption of orally inactive -lactam antibiotics.
Methods. Permeation behaviors of various -lactam antibiotics across rat intestinal segments were evaluated in vitro using diffusion cells.
Results. Poorly absorbed -lactam antibiotics, like cephaloridine and cefoperazone, commonly exhibit greater serosal-to-mucosal permeation than mucosal-to-serosal permeation, while cephalexin permeation was greater in the mucosal-to-serosal direction. In the absence of D-glucose, secretory-oriented permeation of cephaloridine and cefoperazone disappeared. Addition of sodium azide into an experimental buffer including D-glucose significantly and selectively enhanced mucosal-to-serosal permeation of cephaloridine and cefoperazone. Although benzylpenicillin, ampicillin, and amoxicillin all showed secretory-oriented permeation, the tendency to permeation was greatest with benzylpenicillin and least with amoxicillin. Probenecid stimulated mucosal-to-serosal permeation of cephaloridine, but verapamil and p-aminohippuric acid had no significant effect on it.
Conclusions. It has been suggested that mechanisms which induce secretory-oriented permeation of orally inactive -lactam antibiotics are factors limiting intestinal absorption of such antibiotics. This energy-demanding efflux system was distinct from P-glycoprotein-mediated transport. A free -amino group in the molecule is an important factor for reducing an affinity with the efflux system. 相似文献
19.
Fréville Jean Christophe Dollo Gilles Le Corre Pascal Chevanne François Le Verge Roger 《Pharmaceutical research》1996,13(10):1576-1580
Purpose. To investigate the influence of complexation between bupivacaine and hydroxypropyl--cyclodextrin (HP--CD) on the systemic absorption and on the pharmacodynamic effect of bupivacaine following epidural administration in a rabbit model.
Methods. Bupivacaine and bupivacaine-HP--CD complex were administered according to a randomized and cross-over design in six rabbits chronically instrumented with an epidural catheter. The plasma concentrations of bupivacaine and the duration and intensity of the motor blockade were evaluated.
Results. Complexation with HP--CD led to a decrease in the maximum plasma concentration of bupivacaine. Individual absorption kinetics evaluated by Loo-Riegelman absorption analysis indicated that systemic absorption resulted from two parallel first-order processes. Only the faster absorption phase was slowed by complexation with HP--CD. The duration of the motor blockade was increased almost twice but the intensity was not modified.
Conclusions. Complexation with HP--CD could be a promising drug delivery system to improve the therapeutic index of bupivacaine. 相似文献
20.
Okimoto Kazuto Rajewski Roger A. Uekama Kaneto Jona Janan A. Stella Valentino J. 《Pharmaceutical research》1996,13(2):256-264
Purpose. The objective of this work was to determine the role that charge might play in the interaction of charged and uncharged drugs with neutral (2-hydroxypropyl--cyclodextrin, HP--CD) and anionically charged (SBE7--CD) modified -cyclodextrins. SBE7--CD is a sulfobutyl ether, sodium salt, derivative variably substituted on the 2-, 3- and the 6-positions of -cyclodextrin. The number seven refers to the average degree of substitution.
Methods. The binding of the acidic drugs, indomethacin, naproxen and warfarin and the basic drugs, papaverine, thiabendazole, miconazole and cinnarizine with the two cyclodextrins was determined at 25°C as a function of pH and cyclodextrin concentration by the phase-solubility method.
Results. Except for miconazole and cinnarizine (AP-type diagrams), all other materials studied displayed AL-type diagrams. By comparing the binding constants of both the charged and uncharged forms of the same drugs to both HP--CD and SBE7--CD, the following conclusions could be drawn. The binding constants for the neutral forms of the drugs were always greater with SBE7--CD than with HP--CD. For the anionic agents, the binding constants between SBE7--CD and HP--CD were similar while the binding constants for the cationic agents with SBE7--CD were superior to those of HP--CD, especially when compared with the neutral form of the same drug.
Conclusions. A clear charge effect on complexation, attraction in the case of cationic drugs and perhaps inhibition in the case of anionic drugs, was seen with the SBE7--CD. 相似文献