RESUMO
Gene therapy is a promising treatment option for cancers generated by mutation of oncogenes or tumor suppressor genes. The transcriptional process is activated by doxorubicin (DXR), and gene expression efficiency followed by gene transfection can be enhanced by the combination-use of DXR. Therefore, co-encapsulation of plasmid DNA (pDNA) and DXR into non-viral gene carriers can enhance gene expression. Here, we prepared DXR-loaded liposome/pDNA complexes (DXR-loaded PEGylated lipoplexes) by co-encapsulating pDNA and DXR into liposomes. Gene expression was enhanced by DXR encapsulation into lipoplexes in colon-26 cells and cultured mouse macrophages, and this gene expression level was significantly higher than that obtained by the combination of PEGylated lipoplexes and free DXR. Moreover, the activation profiles of transcriptional factors induced by DXR-loaded lipoplexes were different from those induced by free DXR; therefore, co-encapsulation of pDNA and DXR into gene carriers might be contributed to effective enhancement of gene expression. These findings provide a new approach for achieving effective gene transfection using PEGylated lipoplexes.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacologia , DNA/administração & dosagem , DNA/genética , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Expressão Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Análise de Variância , Animais , Linhagem Celular , Corantes , DNA/biossíntese , Portadores de Fármacos , Feminino , Lipossomos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/genética , Tamanho da Partícula , Polietilenoglicóis , Reação em Cadeia da Polimerase em Tempo Real , Sais de Tetrazólio , Tiazóis , Fator de Transcrição AP-1/genéticaRESUMO
Ablation of expanded polytetrafluoroethylene without disruption of the fine porous structure is demonstrated using an intense femtosecond-pulse laser. As a result of laser-matter interactions near ablation threshold fluence, high-energy ions are emitted, which cannot be produced by thermal dissociation of the molecules. The ion energy is produced by Coulomb explosion of the elements of (-CF(2)-CF(2)-)(n) and the energy spectra of the ions show contributions from the Coulomb explosions of the ions rather than those of thermal expansion to generate high-energy ions. The dependence of ion energy on the laser fluence of a 180-fs pulse, compared with that of a 400-ps pulse, also suggests that the high-energy ions are accelerated by Coulomb explosion.
Assuntos
Lasers , Óptica e Fotônica , Politetrafluoretileno/química , Carbono/química , Desenho de Equipamento , Íons , Espectrometria de Massas/métodos , Microscopia Eletrônica de Varredura/métodos , Modelos Estatísticos , Distribuição Normal , Física/métodos , Polímeros/química , PorosidadeRESUMO
After intravenous administration of plasmid DNA (pDNA)/cationic liposome complexes, the gene expression is predominantly observed in the lung due to the physicochemical properties of the liposome complexes and the physiology of the lung. To determine the physicochemical properties and distribution behavior of cationic liposomes for lung-selective drug and/or gene delivery systems, N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA)/cholesterol and 1,2-dioleoyl-3-trimethyl-ammoniopropane (DOTAP)/cholesterol liposomes were studied. The particle sizes of DOTMA/cholesterol and DOTAP/cholesterol liposomes were very similar: 126 and 128 nm, respectively. Furthermore, the zeta potentials of these two liposomes were 51 and 66 mV, respectively. After intravenous injection into mice, both cationic liposomes were rapidly eliminated from the blood circulation and preferentially recovered in the lung. Interestingly, the highest lung accumulation was observed at 1 min, and then, decreased gradually. The distribution characteristics of DOTMA/cholesterol and DOTAP/cholesterol liposomes were almost identical due to the similarities in their physicochemical properties. These results demonstrated that DOTMA/cholesterol and DOTAP/cholesterol liposomes, which possess a positive charge, are promising carriers for lung-selective drug and/or gene delivery systems.
Assuntos
Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Lipossomos/química , Lipossomos/farmacocinética , Animais , Área Sob a Curva , Fenômenos Químicos , Físico-Química , Colesterol/química , Sistemas de Liberação de Medicamentos , Eletroquímica , Ácidos Graxos Monoinsaturados , Feminino , Camundongos , Camundongos Endogâmicos ICR , Tamanho da Partícula , Compostos de Amônio QuaternárioRESUMO
NFkappaB decoy, double stranded oligonucleotides containing NFkappaB binding sequences, inhibits NFkappaB-mediated production of inflammatory cytokines, and therefore NFkappaB decoy has been applied to several diseases. However, naked NFkappaB decoy, which is quickly cleared from the circulation in mice after intravenous injection, is readily absorbed into the systemic circulation. In order to deliver enough NFkappaB decoy for a therapeutic effect, it is necessary to develop a carrier, which enables much more NFkappaKB decoy to transfer to the target cells. In this study, using N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA)/cholesterol (1 :1) liposomes, the therapeutic effect of NFkappaB decoy was investigated in an LPS induced acute hepatitis model mice. The mean diameter of the cationic liposomes/NFkappaB decoy complex was about 70.9 nm and the zeta potential of complex was about 37.4 mV. Tissue distribution was determined by measuring the radioactivity of a cationic liposomes/ [32P] NFkappaB decoy complex after intravenous injection. The cationic liposomes/[32P] NFkappaB decoy complex was rapidly accumulated in the lung and gradually moved to the liver. The therapeutic effect was determined by the serum concentration of TNFalpha in LPS treated mice. The production of TNFalpha was significantly inhibited by cationic liposomes/NFkappaB decoy complex but not by cationic liposomes/random decoy complex or naked NFkappaB decoy. These results suggested that NFkappaB decoy therapy could be achieved using cationic liposomes. This information is of great value for the design of NFkappaB decoy carrier systems.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/terapia , Terapia Genética , Lipopolissacarídeos/toxicidade , NF-kappa B/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Animais , Cátions , Fenômenos Químicos , Físico-Química , Citocinas/metabolismo , DNA/administração & dosagem , DNA/genética , Sistemas de Liberação de Medicamentos , Injeções Intravenosas , Lipossomos , Camundongos , Mimetismo Molecular , Oligonucleotídeos/uso terapêutico , Tamanho da Partícula , Fosforilação , TransfecçãoRESUMO
Absorption and lymphatic transfer of a polymeric prodrug of mitomycin C (MMC), mitomycin C-dextran conjugate (MMC-D), following i.m. injection were studied in rats in order to assess the feasibility of a macromolecular prodrug as a lymphotropic delivery system. Three types of MMC-D, conjugates with dextran with molecular weights of 10,000, 70,000 and 500,000, were synthesized, and the disposition of MMC was determined by bioassay. Following i.m. injection of MMC-D, MMC was retained at the injection site for a long period in a conjugated form while MMC administered as a free form disappeared rapidly. The disappearance was markedly influenced by the size of carrier dextran, because the remaining amount of MMC increased with an increase of molecular size. The lymphatic uptake of the drug was evaluated by determining the concentration in the regional lymph nodes and thoracic lymph fluid. In contrast to a slight lymphatic uptake following i.v. and i.m. injection of free MMC, MMC-D exhibited remarkable accumulation in the regional lymph nodes after i.m. injection which persisted up to 48 hr. MMC-D (Mr 10,000) appeared in the thoracic lymph as both the conjugated and the free form. Larger MMC-D gave a persistent supply of free MMC in thoracic lymph, suggesting that it was accumulated in the lymph node and supplying MMC continuously. These MMC-Ds suppressed the lymph node metastases introduced by a s.c. inoculation of L1210 leukemia cells. The usefulness of MMC-D as a lymphotropic delivery system for preventing lymphatic metastasis of cancer was suggested.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Dextranos/administração & dosagem , Linfa/fisiologia , Mitomicinas/administração & dosagem , Animais , Transporte Biológico , Dextranos/metabolismo , Cinética , Linfonodos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Camundongos Endogâmicos , Mitomicina , Mitomicinas/metabolismo , Peso Molecular , Ratos , Ratos EndogâmicosRESUMO
Cellular interaction and in vitro antitumor activity of a polymeric prodrug of mitomycin C (MMC), mitomycin C-dextran conjugate (MMC-D), were studied in relation to its physicochemical characteristics. MMC-D with cationic and anionic charges were examined. The cationic MMC-D was synthesized using a spacer, epsilon-aminocaproic acid and dextrans with molecular weights of 10,000, 70,000, or 500,000 [MMC(C6)Dcat]. The anionic MMC-D was synthesized using 6-bromohexanoic acid as a spacer and dextran with a molecular weight of 70,000 [MMC(C6)Dan]. Cellular adsorption was determined by measuring the concentration of the drug in the medium after incubation with three tumor cell lines, Ehrlich ascites carcinoma, L1210 leukemia, and AH66 ascites hepatoma cells. MMC(C6)Dcat was adsorbed more readily than MMC or MMC(C6)Dan on the tumor cell surface by an electrostatic force. The percentage of adsorption remained almost constant during the course of incubation and no significant difference was observed between the incubation at 4 degrees C and that at 37 degrees C. A corresponding increase in the amounts of MMC(C6)Dcat adsorbed on with higher molecular weights was noted, which conformed to Langmuir's adsorption isotherm. In vitro antitumor activity was evaluated using L1210 and EAC cell culture systems and human tumor colony forming assay. MMC(C6)Dcat showed growth inhibition essentially equal to that of MMC in continuous drug exposure experiments. In a 1-h drug exposure experiment, MMC(C6)Dcat with a molecular weight of 70,000 or 500,000 was more active than MMC, and a good correlation was observed between the effects of MMC(C6)Dcat and the extent of cellular interaction. These results show that cellular interaction played an important role in the manifestation of the antitumor effect of MMC-D and that these phenomena are governed by the physicochemical properties of macromolecular prodrugs, such as electric charge and molecular weight.
Assuntos
Mitomicinas/administração & dosagem , Neoplasias Experimentais/tratamento farmacológico , Adsorção , Animais , Transporte Biológico , Compartimento Celular , Ciclo Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Meios de Cultura , Dextranos/administração & dosagem , Inibidores do Crescimento/administração & dosagem , Ponto Isoelétrico , Camundongos , Mitomicina , Peso Molecular , Ratos , Ácidos Siálicos/fisiologia , Relação Estrutura-Atividade , Fatores de TempoRESUMO
Disposition characteristics of the macromolecular prodrug of mitomycin C (MMC), mitomycin C-dextran conjugate (MMC-D), in normal and tumor (VX2 carcinoma)-bearing rabbit thigh muscles were studied using the in situ vascular perfusion technique. Three types of cationic MMC-D (MMC-Dcat) and two types of anionic MMC-D (MMC-Dan) with different carrier molecular weights were used. After bolus arterial injection in normal muscles, 83-96% of injected MMC-D was recovered in the venous outflow regardless of the carrier size or charge, whereas less than 60% of MMC was recovered in the same system. By applying statistical moment analysis to the outflow pattern of these drugs, pharmacokinetic parameters representing their disposition characteristics were obtained. Smaller intrinsic clearance (CLint) and distribution volume (V) were noted for MMC-D than for MMC, indicating low extravascular diffusion of MMC-D. In the tumor-bearing muscle, blood contamination from other parts of the body increased and a shortage of flow recovery due to the neovascularization of the tumors occurred. The disposition parameters of MMC-Dcat with a molecular weight of 500,000 (T-500) indicated some tissue distribution and sequestration in the tumor preparation. After constant infusion of [14C]MMC-D (T-500) for 4 h, tissue radioactivity concentrations were determined in various tissues. A higher radioactivity was observed in the viable region of the tumor and the lymph node compared with the normal muscle tissue and the necrotic region of the tumors. 131I-Labeled human serum albumin also gave similar results. In conclusion, higher tumor localization of antitumor agents may be made possible by the application of macromolecular prodrugs.
Assuntos
Dextranos/farmacocinética , Mitomicinas/farmacocinética , Músculos/metabolismo , Neoplasias Experimentais/metabolismo , Animais , Masculino , Mitomicina , Coelhos , Albumina Sérica/farmacocinéticaRESUMO
Disposition characteristics of various anticancer drugs in a tissue-isolated tumor preparation were studied in Walker 256 carcinosarcoma-bearing rats using an in situ single-pass vascular perfusion technique. Three anticancer drugs, 5-fluorouracil, mitomycin C, and Adriamycin, and two lipophilic prodrugs of mitomycin C were tested in the tumor preparation perfused with Tyrode's solution containing 4.7% bovine serum albumin. After bolus arterial injection of test drugs, their outflow concentration-time curves were analyzed based on statistical moment theory. In each tumor preparation, the injection of drug was paired with that of vascular reference substance, Evans' blue-labeled bovine serum albumin, and disposition parameters of the drug were corrected with those of vascular reference substance. From the mean transit time values of vascular reference substance, the average vascular volume of the tumor preparation was calculated to be 0.063 ml/g, which decreased with tumor growth. All drugs showed significant extraction by the tumor tissue, depending on their physicochemical properties. Distribution volumes of tested drugs were from 1.53 to 3.33 times larger than the vascular volume. Calculated intrinsic clearance values for the protein-unbound fractions increased as the lipophilicity of the drug increased. The potential increase in tumor uptake was observed in lipophilic prodrugs of mitomycin C. The present experimental system is thus suggested to be useful for analyzing drug disposition in tumor tissue.
Assuntos
Carcinoma 256 de Walker/metabolismo , Doxorrubicina/farmacocinética , Fluoruracila/farmacocinética , Mitomicinas/farmacocinética , Pró-Fármacos/farmacocinética , Animais , Carcinoma 256 de Walker/irrigação sanguínea , Doxorrubicina/administração & dosagem , Feminino , Fluoruracila/administração & dosagem , Infusões Intra-Arteriais , Mitomicina , Mitomicinas/administração & dosagem , Pró-Fármacos/administração & dosagem , Ratos , Ratos EndogâmicosRESUMO
The pharmacokinetic properties and gene expression of naked plasmid DNA and its cationic liposome complexes were studied after direct intratumoral injection. Using a Walker 256 tissue-isolated tumor perfusion system, we quantified the recovery of naked plasmid DNA and cationic liposome complexes in the tumor, leakage from the tumor surface, and the venous outflow after intratumoral injection. Approximately 50% of naked plasmid DNA had been eliminated from the tumor 2 h after injection, whereas more than 90% of plasmid DNA was retained in the tumor when it was complexed with cationic liposomes. However, the distribution of these complexes in the tumor was restricted to the tissue surrounding the injection site. Pharmacokinetic analysis of the venous outflow profiles suggested that the rate-limiting process that determines the retention of plasmid DNA in the tumor is transferred from the injection site in the tumor tissue and that complexation with cationic liposomes may retard this process. Furthermore, we examined the gene expression of chloramphenicol acetyltransferase DNA constructs (naked pCMV-CAT) and the corresponding cationic liposome [3-beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol] complexes. A similar level of gene expression was observed in vivo after direct intratumoral injection of naked DNA and its cationic liposome complexes. In both cases, a great variation was observed between tumors, and localization of gene-transduced cells in the tumor tissue was limited to the area in the vicinity of the injection site. Thus, these pharmacokinetic and gene expression studies have demonstrated that cationic liposomes can enhance the retention of injected DNA in the tumor model, whereas cationic liposome complex does not necessarily improve gene expression because of its poor dissemination in this tumor. The present study also suggested that there is a need to control the behavior of the injected naked plasmid DNA and its cationic liposome complexes to ensure better distribution throughout the tumor.
Assuntos
Carcinoma 256 de Walker/genética , DNA Complementar/farmacocinética , Técnicas de Transferência de Genes , Lipossomos/farmacocinética , Plasmídeos/metabolismo , Plasmídeos/farmacocinética , Animais , Carcinoma 256 de Walker/metabolismo , Carcinoma 256 de Walker/terapia , Cátions , Cloranfenicol O-Acetiltransferase/metabolismo , DNA Complementar/administração & dosagem , DNA Complementar/metabolismo , Feminino , Expressão Gênica , Injeções Intralesionais , Lipossomos/metabolismo , Plasmídeos/administração & dosagem , Ratos , Ratos WistarRESUMO
Disposition characteristics of model macromolecules with different physicochemical characteristics and macromolecular prodrugs of mitomycin C, namely mitomycin C-dextran conjugates, were studied in tissue-isolated tumor preparations of Walker 256 carcinoma with the use of a single-pass vascular perfusion technique. In constant infusion experiments, all radiolabeled macromolecules accumulated in the tumor tissue, but the degree and pattern of distribution greatly varied, depending on their electric charges. Positively charged macromolecules were markedly accumulated compared with those that were neutral or negatively charged. In addition, their concentrations were significantly higher in viable than in necrotic regions, while neutral and negative compounds were distributed in necrotic rather than in viable regions. Pharmacokinetic analysis of tissue concentration-time courses of positively charged diethylaminoethyl and neutral dextrans showed that their movement occurred by convective fluid flow, and that high tissue accumulation of positively charged macromolecules could be explained by strong binding due to electrostatic interaction. For neutral and anionic macromolecules with negligible affinity to the tissue, it was suggested that the final concentration gradient between the viable and necrotic regions was decided by their tissue fluid content. Thus, the present study revealed the basic disposition characteristics of macromolecules in tumor tissue relative to their physicochemical properties.
Assuntos
Carcinoma 256 de Walker/metabolismo , Dextranos/farmacocinética , Espaço Extracelular/metabolismo , Mitomicina/farmacocinética , Pró-Fármacos/farmacocinética , Animais , Permeabilidade Capilar , Carcinoma 256 de Walker/irrigação sanguínea , Dextranos/administração & dosagem , Dextranos/química , Feminino , Mitomicina/administração & dosagem , Mitomicina/química , Perfusão , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Ratos , Ratos EndogâmicosRESUMO
PurposeTo investigate the relationship between the background of preoperative cataract patients and bacterial conjunctival flora.MethodsA total of 990 cataract patients who had completed preoperative examinations in 2007 and 2008 were included. Patients using topical antibiotics at the preoperative examination or having a history of intraocular surgery were excluded. Conjunctival cultures had been preoperatively obtained. Patient characteristics were investigated via medical records. Risk factors for conjunctival flora of seven typical bacteria were analyzed by univariate and multivariate analyses.ResultsThe detection rate of alpha-hemolytic streptococci and Enterococcus faecalis increased with age (P=0.044 and P=0.002, respectively). The detection rate of Gram-negative bacilli was higher among patients with oral steroid use or lacrimal duct obstruction (P=0.038 and P=0.002, respectively). The detection rate of Corynebacterium species was higher among older patients and men, and lower among patients with glaucoma eye drop use (P<0.001, P=0.012 and P=0.001, respectively). The detection rate of methicillin-susceptible coagulase-negative Staphylococci was higher among men and lower among patients with a surgical history in other departments (P=0.003 and P=0.046, respectively). The detection rate of methicillin-resistant coagulase-negative Staphylococci (MR-CNS) was higher among patients with oral steroid use, a visit history to ophthalmic facilities, or a surgical history in other departments (P=0.002, P=0.037 and P<0.001, respectively).ConclusionsElderly patients, men, patients with lacrimal duct obstruction or immunosuppressed patients are more likely to be colonized by pathogens that cause postoperative endophthalmitis. Moreover, MR-CNS colonization was associated with healthcare-associated infection.
Assuntos
Bactérias Aeróbias/isolamento & purificação , Túnica Conjuntiva/microbiologia , Facoemulsificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas Bacteriológicas , Endoftalmite/microbiologia , Endoftalmite/prevenção & controle , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Período Pré-Operatório , Fatores de RiscoRESUMO
The pharmacokinetics and disposition characteristics of recombinant decorin after intravenous administration were investigated in mice. Following bolus injection of 111In-labeled decorin at doses of 0.02 and 0.1 mg/kg, radioactivity rapidly disappeared from the circulation and approximately 70% of the dose accumulated in liver within 10 min. 111In-labeled decorin was preferentially localized in hepatic nonparenchymal cells. At a higher dose of 1 mg/kg, clearance from the circulation and hepatic uptake of [111In]decorin were slower than at lower doses. Both the accumulation in other tissues and urinary excretion of [111In]decorin were 5% or less. Pharmacokinetic analysis demonstrated that hepatic uptake clearance was large and accounted almost completely for total body clearance; in addition the clearance values decreased as the dose increased, suggesting that the hepatic uptake of decorin is mediated by a specific mechanism which becomes saturated at higher doses. In competitive inhibition experiments, hepatic uptake of 111In-labeled decorin was partially inhibited (about 20-30%) by several sulfated glycans such as glycosaminoglycans and dextran sulfate and by mannosylated bovine serum albumin (BSA), mannan and mannose to a lesser extent (about 10%). On the other hand, polyinosinic acid, polycytidylic acid and succinylated BSA were ineffective, suggesting that the scavenger receptor for polyanions in the liver is not involved in the hepatic uptake of decorin. A basic protein, protamine, and a ligand of the apoE receptor, lactoferrin, also had no effect. Taken together, the present results have demonstrated that recombinant decorin is rapidly eliminated from the blood circulation through extensive uptake by the liver, primarily by the nonparenchymal cells, following systemic administration. The sugar structure and mannose residue in decorin have also been suggested to play an important role in the hepatic uptake of decorin. These findings provide useful information for the development of decorin as a therapeutic agent.
Assuntos
Proteoglicanas/farmacocinética , Animais , Decorina , Proteínas da Matriz Extracelular , Radioisótopos de Índio , Injeções Intravenosas , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Proteoglicanas/administração & dosagem , Proteínas Recombinantes/farmacocinética , Distribuição Tecidual , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
To elucidate the mechanisms underlying the dysfunctions of intestinal absorption induced by antitumor drugs, the effect of pretreatment with mitomycin C on sodium gradient-dependent D-glucose and L-alanine transports was studied in rat brush-border membrane vesicles. 24, 48, 96, or 120 h following a single intravenous injection of mitomycin C, brush-border membrane vesicles were prepared from rat small-intestines. The uptake of D-glucose and L-alanine was shown to be Na+ gradient-dependent even in the case of vesicles obtained from mitomycin C-treated rats, but uptake rates measured at 15 s and magnitude of overshooting effect in uptake of both solutes were decreased in vesicles maximally from 48 h mitomycin C-treated rats. The rate of D-glucose uptake calculated at 15 s recovered to the control level in vesicles prepared at 96 h and 120 h after mitomycin C-treatment, indicating that the effect of mitomycin C on Na+ gradient-dependent D-glucose transport would be fully reversible. Tracer exchange experiments under Na+ and D-glucose equilibrated conditions indicated that the Na+/D-glucose transporters were similarly operative in the vesicles from control and 48 h mitomycin C-treated rats. Rates of 22Na+ uptake measured at 15 s in vesicles from 48 h mitomycin C-treated rats, however, were increased. The increased permeability to Na+ might bring about a more rapid dissipation of the Na+ gradient in these vesicles and this would secondarily cause the decrease in Na+-dependent D-glucose uptake in vesicles from mitomycin C-treated rats.
Assuntos
Alanina/metabolismo , Glucose/metabolismo , Intestino Delgado/metabolismo , Mitomicinas/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Permeabilidade da Membrana Celular , Polarização de Fluorescência , Intestino Delgado/efeitos dos fármacos , Cinética , Lipossomos/metabolismo , Masculino , Fluidez de Membrana/efeitos dos fármacos , Microvilosidades/efeitos dos fármacos , Microvilosidades/metabolismo , Mitomicina , Proteínas de Transporte de Monossacarídeos/metabolismo , Ratos , Ratos Endogâmicos , Sódio/metabolismoRESUMO
The effect of molecular mass and surface density of galactose residues on hepatic uptake and subsequent biliary excretion of galactosylated proteins was investigated in rats. Several proteins with different molecular weights (15-70 kDa) and different numbers of galactose units were synthesized and radiolabeled with 111In. Galactosylated proteins were administered i.v. to anaesthetized rats and samples of plasma and bile were collected for 3 h. Liver was harvested at the end of the experiments and the radioactivity of all samples was measured. Galactosylated proteins accumulated primarily in the liver and 2-10% of the administered dose appeared in the bile, mainly in undegraded form. The hepatic uptake clearance (Cl liver) and biliary excretion rate constant (kbile) of galactosylated proteins were calculated. No direct effect of molecular weight was observed, however, on increasing the galactose density, Cl liver increased from about 4 to 400 ml/h whereas kbile gradually decreased from about 0.057 to 0.007 (h-1). In conclusion, both hepatic uptake and biliary excretion of galactosylated proteins were found to be affected by the extent of galactosylation.
Assuntos
Bile/metabolismo , Proteínas de Ligação ao Cálcio , Galactose/metabolismo , Fígado/metabolismo , Proteínas de Transporte de Monossacarídeos/farmacocinética , Proteínas Periplásmicas de Ligação , Animais , Bile/química , Galactose/análise , Galactose/farmacocinética , Masculino , Proteínas de Transporte de Monossacarídeos/sangue , Ratos , Ratos Wistar , Soroalbumina Bovina/análise , Soroalbumina Bovina/farmacocinética , Superóxido Dismutase/análise , Superóxido Dismutase/farmacocinéticaRESUMO
The roles of serum mannan binding protein (MBP) and the mannose receptor in the cellular uptake of mannosylated liposomes (Man-liposomes) by macrophages were studied. Man-liposomes were prepared by incorporating cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosylethyl)amino)butyl)formamide (Man-C4-Chol) into small unilamellar long circulating liposomes consisting of cholesterol (Chol) and distearoyl phosphatidylcholine (DSPC). In the in vitro cellular uptake study with cultured mouse peritoneal macrophages, [(3)H]Man-liposomes were taken up to a great extent, whereas no significant uptake was observed for [(3)H]cholesterol and DSPC liposomes without Man-C4-Chol (Bare-liposomes). The uptake of [(3)H]Man-liposomes was dose- and temperature-dependent and inhibited by an excess of mannosylated bovine serum albumin, suggesting their specific uptake via membrane mannose receptor-mediated endocytosis. Furthermore, it was demonstrated that (111)In-MBP binds strongly to Man-liposomes based on the recognition of Man-C4-Chol and markedly enhanced their uptake by macrophages. These results are supported by confocal laser microscopic images. In addition, in vivo hepatic uptake of (111)In-MBP was enhanced by Man-liposomes. On the other hand, the uptake of Man-liposomes was significantly reduced by preincubation with serum and further with MBP-depleted serum suggesting inhibitory effects of serum proteins such as albumin on mannose receptor-mediated endocytosis. The involvement of serum-type MBP and membrane mannose receptors in the uptake of Man-liposomes is thus suggested.
Assuntos
Proteínas de Transporte/metabolismo , Lectinas Tipo C , Lipossomos/metabolismo , Macrófagos/metabolismo , Lectinas de Ligação a Manose , Receptores de Superfície Celular/metabolismo , Animais , Células Cultivadas , Colectinas , Portadores de Fármacos , Radioisótopos de Índio , Fígado/metabolismo , Masculino , Manose , Receptor de Manose , Camundongos , Camundongos Endogâmicos ICR , Microscopia Confocal , Albumina SéricaRESUMO
The in vivo disposition behavior and pharmacokinetic characteristics of galactosylated (Gal), mannosylated (Man) and fucosylated (Fuc) liposomes were compared in this study. For the preparation of the glycosylated liposomes, cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiogalactosyle thyl)amino)a lkyl)formamide (Gal-C4-Chol) (Kawakami et al., Biochem. Biophys. Res. Commun. 252 (1998) 78-83) and its mannosylated and fucosylated derivatives (Man-C4-Chol and Fuc-C4-Chol, respectively) were synthesized. The glycosylated liposomes are composed of distearoylphosphatidylcholine (DSPC), cholesterol (Chol), and Gal-C4-Chol (or Man-C4-Chol or Fuc-C4-Chol) with the molar ratio of 60:35:5. After intravenous injection in mice, these three types of [(3)H]cholesteryl hexadecyl ether-labeled glycosylated liposomes were rapidly eliminated from the circulating blood and preferentially recovered in the liver. In contrast, DSPC/Chol (60:40) liposomes without glycosylation were retained for a long time in the circulating blood. The uptake ratios by parenchymal cells (PC) and nonparenchymal cells (NPC) (PC/NPC ratios) for 0.5% Gal, Man and Fuc liposomes were found to be 15.1, 0.6 and 0.2, respectively. The effect of predosing glycosylated proteins and liposomes on the hepatic uptake of 0.5% (3)H-labeled Gal, Man, and Fuc liposomes was investigated and the results support the conclusion that Gal, Man, and Fuc liposomes are taken up by the liver via asialoglycoprotein receptors in PC, mannose receptors in NPC, and fucose receptors in NPC, respectively. Interestingly, Gal liposomes were taken up by NPC rather than by PC at a high dose (5%). Together with the finding that 5% Gal liposomes inhibit the hepatic uptake of (3)H-labeled Fuc liposomes, this suggests that Gal-liposomes administered at a high dose will also be taken up by fucose receptors in NPC, that are considered to act as galactose particle receptors.
Assuntos
Fucose/química , Galactose/química , Lipossomos/farmacocinética , Manose/química , Animais , Terapia Genética , Glicolipídeos/farmacocinética , Glicosilação , Injeções Intravenosas , Células de Kupffer/metabolismo , Lipossomos/sangue , Lipossomos/química , Fígado/metabolismo , Camundongos , Tamanho da Partícula , Fatores de Tempo , Distribuição Tecidual , TrítioRESUMO
The effects of serum mannan binding proteins (MBP) in the transfection of plasmid DNA/Man-liposome complex via mannose receptor-mediated endocytosis was studied in vitro using cultured mouse peritoneal macrophages. Plasmid DNA encoding luciferase gene was complexed with cationic mannosylated liposomes (Man-liposomes), composed of cholesten-5-yloxy-N-(4-((1-imino-2-D-thiomannosylethyl)amino)alkyl)formamide (Man-C4-Chol) and dioleoyl phosphatidylethanolamine (DOPE). The transfection efficiency, as well as the binding and uptake of the plasmid DNA/Man-liposome complex, was investigated with or without serum MBP. The in vitro transfection efficiency of the complex was significantly reduced on increasing the amount of serum MBP. In addition, the cellular association of the complex was also reduced. These results indicate that serum MBP specifically binds to the mannose moieties on the complex and suppresses its cellular uptake, resulting in inhibition of the gene transfection in macrophages. Such an interaction is an obstacle to mannose receptor-mediated in vivo gene transfer to mannose receptor-positive cells using mannosylated gene carriers.
Assuntos
Proteínas de Transporte/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Lectinas Tipo C , Lipossomos/metabolismo , Macrófagos Peritoneais/metabolismo , Lectinas de Ligação a Manose , Manose/metabolismo , Transfecção/métodos , Animais , Células Cultivadas , Colectinas , DNA/metabolismo , Masculino , Receptor de Manose , Camundongos , Camundongos Endogâmicos ICR , Plasmídeos , Receptores de Superfície Celular/metabolismoRESUMO
Superoxide dismutase (SOD) was modified into cationized form (Cat-SOD) in order to enhance its pharmacological efficacy based on an electrostatic interaction. The inhibitory effect of Cat-SOD on superoxide anion release from inflammatory macrophages and its cellular interaction were studied in vitro. Cat-SOD exhibited an excellent inhibitory effect on superoxide anion release from the macrophages, and this effect surpassed those of native SOD and SOD modified with mannose (Man-SOD) which is taken up via mannose receptor-mediated endocytosis by macrophages. In the presence of colchicine, a microtubule-disruptive agent, the inhibitory effect of Cat-SOD was slightly impaired, whereas the effect of Man-SOD completely disappeared. The intracellular localization of fluorescein isothiocyanate-labeled SOD, Cat-SOD and Man-SOD observed by confocal laser microscopy supported the difference in their abilities to eliminate superoxide anions. The different sensitivities of Cat-SOD and Man-SOD to colchicine were also confirmed by the confocal laser microscopic images, suggesting their distinct intracellular trafficking pathways in the macrophages. In conclusion, Cat-SOD is desirable for its pharmacological activity, which is probably the result of its ability to be delivered to the vicinity of NADPH-oxidase which locates in the cell membrane and generates superoxide anions.
Assuntos
Macrófagos Peritoneais/metabolismo , Superóxido Dismutase/metabolismo , Superóxidos/metabolismo , Animais , Cátions/metabolismo , Membrana Celular/metabolismo , Diaminas/metabolismo , Fluoresceína-5-Isotiocianato , Radioisótopos de Índio , Macrófagos Peritoneais/enzimologia , CamundongosRESUMO
Our previous studies have demonstrated that serum would play an important role in the hepatic disposition of polystyrene microspheres (MS) and that complement C3 should be involved as the serum opsonin. In this study, we tried to identify the entity of other serum opsonins and dysopsonin for the hepatic uptake of MSs with particle sizes of 50 nm (MS-50) and 500 nm (MS-500) by isolated liver perfusion studies using a recirculation procedure in rats. Pretreatment of the liver by trypsin significantly suppressed the serum-dependent hepatic uptake of both MSs, suggesting that some protein components on the cell surface should be necessary for the serum-dependent phagocytosis of MSs. Pretreatment of the serum by the anti-fibronectin antibody resulted in a significant reduction in the hepatic disposition of MS-500 (49% of control), suggesting that fibronectin should also work as the opsonin for the hepatic uptake of MS-500. The hepatic disposition of both MSs in the presence of serum was inhibited by the addition of N-acetylgalactosamine into the perfusate, suggesting the possible involvement of lectin in the serum-dependent hepatic uptake of MSs. Furthermore, a more intensive hepatic disposition of MSs was observed in the presence of plasma compared with that in the presence of serum in the perfusate, suggesting the possible involvement of blood coagulation factors, such as fibrinogen, as the opsonin in the hepatic disposition of MSs.
Assuntos
Proteínas Sanguíneas/metabolismo , Fígado/metabolismo , Microesferas , Poliestirenos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Masculino , Ratos , Ratos WistarRESUMO
Hepatic uptake and biliary excretion of fluorescein isothiocyanate-labeled polystyrene microspheres with a particle size of 50 nm (MS-50) were studied in rats. Liver perfusion studies revealed that not only apo-E-mediated but also asialoglycoprotein receptor-mediated uptake is involved in the mechanism of the serum protein-dependent uptake of MS-50 in the liver. The uptake of MS-50 mediated by apo-E contributes more to the total uptake of MS-50 by the hepatocytes than that via asialoglycoprotein receptor in the presence of serum in the perfusate. Furthermore, it was found that MS-50 is substantially excreted into the bile by transcytosis. The extent of exocytosis of MS-50 taken up by the hepatocytes was much higher after MS-50 was endocytosed via asialoglycoprotein receptor than after taken up via the process mediated by apo-E. On the basis of these results, a possible regulation of the intracellular sorting of ligands, depending on the receptor-mediated uptake mechanism, was inferred.