RESUMEN
BACKGROUND: Decaarginine-polyethylene glycol-conjugated 3,5-bis(dodecyloxy)benzamide/plasmid DNA [Arg10-polyethylene glycol (PEG)-lipid/plasmid DNA (pDNA)] complexes (designated R10B/DNA complexes) are efficient nonviral carriers for pDNA delivery into human cervical carcinoma HeLa cells. Previous reports indicated that these complexes formed at a relatively low R10B/DNA ratio and showed high transgene expression efficiency. However, the intracellular behaviour of the two different nanostructures, which leads to differences in gene delivery, remains to be elucidated. METHODS: R10B/DNA complexes prepared at a N/P ratio of 8.5/1 or 42.5/1, corresponding to 5 µm or 25 µm R10B, respectively, were added to HeLa cells, and their uptake and subsequent intracellular fate were examined by cell imaging using electron microscopy (EM) and correlative light-electron microscopy (CLEM). RESULTS: EM and CLEM analyses revealed that R10B/DNA complexes formed at the lower N/P ratio were mainly taken up by the cells through macropinocytosis, whereas R10B/DNA complexes formed at the higher N/P ratio bound to protruding membrane structures or permeated into the cells by a different pathway. In cells expressing the transgene, R10B/DNA complexes were observed both in macropinosomes and in the cytoplasm. In addition, these cells had macropinosomes with disrupted membranes. These results suggest that cellular uptake through macropinocytosis and subsequent disruption of the macropinosome membrane may be a critical step for R10B-mediated gene delivery. CONCLUSIONS: We have shown that the existence of R10B/DNA complexes in macropinosomes at the early stages of gene delivery correlates with high efficiency R10B-mediated gene delivery. This finding will provide valuable insights for the engineering of more efficient gene delivery systems based on oligoarginine-mediated carriers.
Asunto(s)
Técnicas de Transferencia de Gen , Péptidos/química , Polietilenglicoles/química , Polietileno/química , ADN/administración & dosificación , ADN/química , Células HeLa , Humanos , Lípidos/química , Liposomas , TransfecciónRESUMEN
Oligoarginine conjugates are highly efficient vectors for the delivery of plasmid DNA into cells. Decaarginine-conjugated lipid (Arg10-PEG-lipid) was synthesized and the effects of Arg10-PEG-lipid concentration at a fixed DNA concentration on transfection efficiency and the structure of the complexes were studied below and above critical micelle concentration (CMC), and at the lipid nitrogen/DNA phosphate (N/P) ratio corresponding to transfection, respectively. Arg10-PEG-lipid at the concentration below CMC showed stronger interaction with DNA by fluorescence intensity distribution analysis, and significantly higher luciferase and green fluorescent protein expression than that above CMC. A phase-contrast cryo-transmission electron microscope (cryo-TEM) experiment showed that the morphology of the complexes depended on the N/P ratio. At a low N/P ratio corresponding to that in transfection at a lipid concentration below CMC, a net-like structure developed in which plasmid DNA was involved. A further increase in the N/P ratio, a large fibrous nanostructure of complexes, was also observed. Without DNA, these structures were not obtained. The cellular uptake mechanism of complexes using flow cytometry with inhibitors suggested that complexes with two different morphologies showed similar cellular uptake and uptake mechanism, macropinocytosis. Differences in transfection efficiency of the complexes may be explained by a large fibrous nanostructure inhibiting the cellular internalization of complexes or the release of DNA from macropinosomes into cytoplasm. Arg10-PEG-lipid/DNA complexes formed a favorable nanostructure for gene delivery, depending on the N/P ratio in water.
Asunto(s)
Arginina/química , ADN/química , Lípidos/química , Nanoestructuras , Polietilenglicoles/química , Transfección , Fluorescencia , Células HeLa , Humanos , Microscopía ElectrónicaRESUMEN
Arthrobacter endo-beta-N-acetylglucosaminidase (Endo-A), a member of glycoside hydrolase (GH) family 85, catalyses the hydrolysis and transglycosylation of asparagine-linked oligosaccharides of glycoproteins with retention of anomeric configuration. Glu-173 of Endo-A is a catalytically essential amino acid residue, and the corresponding residue is conserved in all GH family 85 members. The catalytic activity of Endo-A E173A mutant was rescued by the addition of sodium azide or sodium formate. Furthermore, the produced beta-glycosyl azide (Man(5)GlcNAc-beta-N(3)) retained the anomeric configuration, indicating that Glu-173 is the catalytic acid-base residue of Endo-A. This is the first identification of the catalytic residue for GH family 85 endo-beta-N-acetylglucosaminidases.
Asunto(s)
Arthrobacter/enzimología , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Mutación , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia de Carbohidratos , Catálisis , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/química , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/genética , Datos de Secuencia MolecularRESUMEN
Previous studies have shown that different calcitonins interact with planar lipid membranes to form ion channels. In this study, glycosylation of eel calcitonin (eCt) at different positions (Ct3-GlcNAc, Ct14-GlcNAc, Ct20-GlcNAc, Ct26-GlcNAc) is shown to preserve molecular structure and slightly change the energy of incorporation and channel formation in planar lipid bilayers made up of palmitoyl-oleoyl-phosphatidylcholine:dioleoyl phosphatidyl-glycerol (85:15, w:w). The voltage needed to form channels decreased as the attached carbohydrate moved toward the C-terminal (eCt = Ct3-GlcNAc > Ct14-GlcNAc = Ct20-GlcNAc > Ct26-GlcNAc). Interestingly, all the Cts tested maintain the characteristic voltage-conductance dependence found for other Cts, the only channel properties modified concern ion selectivity, that shift toward anion selectivity (eCt = 0.97, Ct3-GlcNAc = 0.49, Ct14-GlcNAc = 0.41, Ct20-GlcNAc = 0.36, Ct26-GlcNAc = 0.47). These aspects would be useful in managing peptide properties for biotechnological and therapeutic applications considering the physiological nature of this peptide.
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Calcitonina/química , Calcitonina/metabolismo , Membrana Celular/metabolismo , Canales Iónicos/biosíntesis , Canales Iónicos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fosfolípidos/metabolismo , Secuencia de Aminoácidos , Animales , Membrana Celular/química , Permeabilidad de la Membrana Celular , Anguilas , Glicosilación , Membrana Dobles de Lípidos/química , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
PURPOSE: To establish a novel formulation tumor-targeted drug carrier of lipophilic antitumor antibiotics, aclacinomycin A (ACM), folate-linked microemulsions were prepared and investigated both in vitro and in vivo. EXPERIMENTAL DESIGN: Three kinds of folate-linked microemulsions with different polyethylene glycol (PEG) chain lengths loading ACM were formulated with 0.24 mol% folate-PEG(2000)-distearoylphosphatidylethanolamine (DSPE), folate-PEG(5000)-DSPE, and folate-lipid (without PEG linker) in microemulsions. In vitro studies were done in a human nasopharyngeal cell line, KB, which overexpresses the folate receptor (FR), and a human hepatoblastoma cell line, [FR(-)] HepG2. In vivo experiments were done in a KB xenograft by systemic administration of folate-linked microemulsions loading ACM. RESULTS: The association of folate-linked microemulsions to KB cells could be blocked by 2 mmol/L free folic acid. Selective FR-mediated cytotoxicity of folate-linked microemulsions loading ACM was obtained in KB but not in HepG2 cells. The association of the folate-PEG(5000)-linked microemulsion and folate-PEG(2000)-linked microemulsion with the cells was 200- and 4-fold higher, whereas their cytotoxicity was 90- and 3.5-fold higher than those of nonfolate microemulsion, respectively. The folate-PEG(5000)-linked microemulsions showed 2.6-fold higher accumulation in solid tumors 24 hours after i.v. injection and greater tumor growth inhibition than free ACM. CONCLUSION: These findings suggest that a folate-linked microemulsion is feasible for tumor-targeted ACM delivery. This study shows that folate modification with a sufficiently long PEG chain on emulsions is an effective way of targeting emulsion to tumor cells.
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Aclarubicina/administración & dosificación , Aclarubicina/farmacocinética , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/farmacocinética , Carcinoma/patología , Ácido Fólico/química , Hepatoblastoma/patología , Neoplasias Hepáticas/patología , Neoplasias Nasofaríngeas/patología , Polietilenglicoles/química , Química Farmacéutica , Emulsiones , Humanos , Lípidos/química , Nanoestructuras , Fosfatidiletanolaminas/química , Células Tumorales CultivadasRESUMEN
Naturally occurring glycopeptides and glycoproteins usually contain more than one glycosylation site, and the structure of the carbohydrate attached is often different from site to site. Therefore, synthetic methods for preparing peptides and proteins that are glycosylated at multiple sites, possibly with different carbohydrate structures, are needed. Here, we report a chemo-enzymatic approach for accomplishing this. Complex-type oligosaccharides were introduced to the calcitonin derivatives that contained two N-acetyl-D-glucosamine (GlcNAc) residues at different sites by treatment with Mucor hiemalis endo-beta-N-acetylglucosaminidase. Using this enzymatic transglycosylation reaction, three glycopeptides were produced, a calcitonin derivative with the same complex-type carbohydrate at two sites, and two calcitonin derivatives each with one complex-type carbohydrate and one GlcNAc. Starting from the derivatives with one complex-type carbohydrate and one GlcNAc, a high-mannose-type oligosaccharide was successfully transferred to the remaining GlcNAc using another endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae. Thus, we were able to obtain glycopeptides containing not only two complex-type carbohydrates, but also both complex and high-mannose-type oligosaccharides in a single molecule. Using the resultant glycosylated calcitonin derivatives, the effects of di-N-glycosylation on the structure and the activity of calcitonin were studied. The effect appeared to be predictable from the results of mono-N-glycosylated calcitonin derivatives.
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Calcitonina/síntesis química , Anguilas , Secuencia de Aminoácidos , Animales , Calcitonina/química , Calcitonina/metabolismo , Secuencia de Carbohidratos , Células Cultivadas , Glicosilación , Glicosiltransferasas/química , Masculino , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/química , Ratones , Datos de Secuencia Molecular , Oligosacáridos/química , RatasRESUMEN
The design, synthesis, and evaluation of in vitro gene delivery efficacy of a novel series of oligo-Arg-lipid conjugates are described. 3,5-Bis(dodecyloxy)benzamide (BDB) was employed as the lipid component, and a poly(ethylene glycol) (PEG) spacer was introduced between the C-terminal of oligo-Arg and the amide group of BDB. Four derivatives with various oligo-Arg lengths (ArgN-PEG-BDB; N = 4, 6, 8, 10: the number of arginine residues) were prepared, and the effect of oligo-Arg length on the gene transfection was investigated in HeLa cells. Transfection efficiency increased as the number of arginine residues increased. Arg10-PEG-BDB showed the highest transfection efficiency, without severe toxicity to cells. These findings well corresponded to the cellular association of the Arg-PEG-BDB/DNA complex determined by flow cytometry. Even in the presence of serum, Arg10-PEG-BDB achieved appreciable cellular association and attained high gene expression. Thus, Arg10-PEG-BDB is potentially a simple and useful gene delivery tool, because one need only to mix it with plasmid DNA and apply the complexes to the cells even in a serum-containing medium.
Asunto(s)
Diseño de Fármacos , Técnicas de Transferencia de Gen , Lípidos , Liposomas , Péptidos , Polietilenglicoles , Citometría de Flujo , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Lípidos/administración & dosificación , Lípidos/síntesis química , Lípidos/química , Liposomas/administración & dosificación , Liposomas/síntesis química , Liposomas/química , Luciferasas/genética , Estructura Molecular , Péptidos/administración & dosificación , Péptidos/síntesis química , Péptidos/química , Plásmidos/química , Polietilenglicoles/administración & dosificación , Polietilenglicoles/síntesis química , Polietilenglicoles/química , TransfecciónRESUMEN
A novel plasmal conjugate of galactosylsphingosine (psychosine), Gro1(3)-O-plasmal-O-6Galbeta-sphingosine (glyceroplasmalopsychosine), was analyzed by electrospray ionization and liquid secondary ion mass spectrometry with low- or high-energy collision-induced dissociation (CID). In the product ion spectra of the [M + H](+) ions, [M + H - glycerol](+) ions arising from the loss of a glycerol were predominant. Unexpectedly, CID of the [M + H - glycerol](+) ion produced an outstanding ion, [(M + H - glycerol) - Hex](+), which required the loss of the galactose from inside the molecule. This ion was greatly reduced in the spectra of N,N-dimethyl derivatives, indicating that the [(M + H - glycerol) - Hex](+) ion is formed from an intramolecular rearrangement with migration of the plasmal residue to the free amino group of sphingosine. It would be expected that the rearrangement occurs simultaneously with the elimination of glycerol or a rearranged [M + H](+) ion leads to the elimination of glycerol, to form a Schiff base-type [M + H - glycerol](+) ion, from which the terminal galactose could be removed by the normal mechanism of glycosidic cleavage. On the other hand, the [M + Na - glycerol](+) ion derived from the sodiated molecule did not produce an ion corresponding to the rearrangement reaction, possibly owing to a higher stability of the sodiated ions against conformational changes.
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Plasmalógenos/metabolismo , Psicosina/análogos & derivados , Psicosina/metabolismo , Animales , Encéfalo/metabolismo , Química Encefálica , Cationes/análisis , Cationes/metabolismo , Bovinos , Plasmalógenos/análisis , Psicosina/análisis , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Coating of 3,4,5-tris(dodecyloxy)benzyl-beta-D-galactopyranoside (TDOB-Gal) on a dish for suspension culture increased the specific ammonia consumption rate of primary rat hepatocytes to 2.4 times that in the case of rat hepatocytes cultured in a dish without coating while there was no increase in the specific urea production rate. TDOB-beta-D-glucopyranoside (TDOB-Glc), -alpha-D-mannnoside, -beta-D-mannoside, 2-acetamido-2-deoxy-beta-D-glucopyranoside, and 2-acetamido-2-deoxy-beta-D-galactopyranoside had almost no influence on the above-mentioned specific rates. In the ammonia loading assay, cells on the dish with TDOB-Gal and -Glc coatings produced glucose, suggesting gluconeogenesis.
RESUMEN
A new class of injectable nanocomposite thermogels having excellent cell-compatibility were developed through cooperative self-assembly of biodegradable poly(lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(lactide-co-glycolide) copolymer micelles and clay nanosheets for effective cell delivery. This study will be valuable for the establishment of injectable cell delivery technology.
RESUMEN
Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm-oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for ß-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from ß-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm-oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle.
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Cell adhesive peptides derived from extracellular matrix components are potential candidates to afford bio-adhesiveness to cell culture scaffolds for tissue engineering. Previously, we covalently conjugated bioactive laminin peptides to polysaccharides, such as chitosan and alginate, and demonstrated their advantages as biomaterials. Here, we prepared functional polysaccharide matrices by mixing laminin active peptides and agarose gel. Several laminin peptide/agarose matrices showed cell attachment activity. In particular, peptide AG73 (RKRLQVQLSIRT)/agarose matrices promoted strong cell attachment and the cell behavior depended on the stiffness of agarose matrices. Fibroblasts formed spheroid structures on the soft AG73/agarose matrices while the cells formed a monolayer with elongated morphologies on the stiff matrices. On the stiff AG73/agarose matrices, neuronal cells extended neuritic processes and endothelial cells formed capillary-like networks. In addition, salivary gland cells formed acini-like structures on the soft matrices. These results suggest that the peptide/agarose matrices are useful for both two- and three-dimensional cell culture systems as a multifunctional biomaterial for tissue engineering.
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Materiales Biocompatibles , Laminina/metabolismo , Péptidos/metabolismo , Sefarosa/metabolismo , Ingeniería de Tejidos , Secuencia de Aminoácidos , Animales , Adhesión Celular , Células Cultivadas , Ácido Edético , Heparina , Humanos , Laminina/química , Ratones , Datos de Secuencia Molecular , Neuritas , Células PC12 , Péptidos/química , RatasRESUMEN
Previously, we have reported that decaarginine-conjugated PEG-lipids (R10B) efficiently delivered plasmid DNA (pDNA) into human cervical carcinoma HeLa cells via macropinocytosis; however, the mechanism of cellular uptake by R10B was not evaluated in other cell lines. In this study, we investigated the internalization mechanism by R10B/pDNA complex (R10B-lipoplex) in human prostate tumor PC-3 and human nasopharyngeal tumor KB cells, and compared with that in HeLa cells. Although it was necessary for R10B-lipoplex to associate with heparan sulfate (HS) on the cell surface in all cell lines, the R10B-lipoplex was internalized primarily through clathrin-mediated endocytosis in PC-3 and KB cells, and macropinosytosis in HeLa cells. In HeLa cells, treatment with the R10B-lipoplex induced the formation of lamellipodia for macropinocytosis, but did not in KB and PC-3 cells. Furthermore, the highest transfection efficiency by R10B-lipoplex was observed in HeLa cells. These findings indicated that the R10B-lipoplex induced the formation of lamellipodia in HeLa cells after binding to HS on the cells and was then internalized by macropinocytosis, which could induce high gene expression because of escaping degradation in lysosomes. Cell physiology might be a critical factor in cellular internalization and efficient transfection by cell penetration peptide.
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Arginina/química , Membrana Celular/metabolismo , ADN/metabolismo , Lípidos/química , Péptidos/química , Pinocitosis , Polietilenglicoles/química , Actinas/metabolismo , Arginina/análogos & derivados , Línea Celular Tumoral , Clatrina/metabolismo , ADN/química , Femenino , Genes Reporteros , Células HeLa , Heparitina Sulfato/metabolismo , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Masculino , Seudópodos/metabolismo , TransfecciónRESUMEN
Oligoarginine-conjugated lipids ((Arg)n-PEG-lipid) (n=4, 6, 8, and 10: number of arginine residues) are novel gene delivery vectors. We prepared two oligoarginine-modified liposomes using (Arg)n-lipid without and with poly(ethylene glycol) (PEG) spacer ((Arg)n-L and (Arg)n-PEG-L), and investigated the effect of PEG spacer and oligoarginine length of liposomes on cellular uptake, gene transfection, and its mechanism in HeLa cells, using complexes with plasmid DNA (DNA) or oligodeoxynucleotide. Transfection efficiency increased as the number of arginine residues increased and Arg10-PEG-L/DNA complexes (lipoplexes) showed the highest gene transfection efficiency among (Arg)n- and (Arg)n-PEG-lipoplexes. Arg4- and Arg4-PEG-lipoplexes were taken up greatly into cells, but showed lower transfection efficiency than Arg10- and Arg10-PEG-lipoplexes, respectively. The different gene expression by Arg4-L to Arg10-L with or without PEG spacer may be explained by the different intracellular uptake mechanism. The main cellular uptake mechanism of Arg10-L and Arg10-PEG-L was the macropinocytosis pathway, whereas that of Arg4-L and Arg4-PEG-L was not. PEG spacer was more effective for intracellular trafficking than Arg length and surface charge of lipoplex which depends on Arg length at the almost same size of lipoplexes. The findings suggested that Arg10-PEG-L was a superior vector since Arg10 induced the macropinocytosis uptake pathway.
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Péptidos/química , Polietilenglicoles/química , Transfección/métodos , ADN/administración & dosificación , ADN/genética , Fluoresceína-5-Isotiocianato , Expresión Génica , Células HeLa , Humanos , Liposomas , Luciferasas/genética , Microscopía Confocal , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/genética , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
T-antigen (Galbeta1-3GalNAc-Thr/Ser) also known as Thomsen-Friedenreich (TF) antigen is the core 1 structure of O-linked mucin type glycans. In normal epithelium, the disaccharide structure is masked by terminal carbohydrate moieties, but is uncovered in most primary and metastatic epithelial malignant tumors. For the purpose of establishing cancer diagnosis and therapeutics, anti-T-antigen antibodies were isolated from a phage library displaying human single chain antibodies. A strategy similar to the previously published method (Sakai et al. Biochemistry. 2007; 46:253-262) was used to screen T-antigen specific antibodies, except that a different type of glycolipid was used for panning and screening. Eleven phage clones were isolated and characterized by DNA sequencing and ELISA, which revealed 4 groups of clones with T-antigen binding activity. One single chain antibody (scFv) protein, derived from phage clone 1G11, was expressed in Escherichia coli and purified to near homogeneity by two column chromatographies. ELISA and surface plasmon resonance analyses revealed that the purified 1G11 scFv bound to the T-antigen moiety of the neoglycolipid used. This study not only demonstrated the validity of our previously introduced strategy employing the phage display technology in constructing human scFvs against various carbohydrate antigens, but also provided us with various scFv genes that can lead to future development of antibody-based therapeutics.
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Anticuerpos/aislamiento & purificación , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Secuencia de Bases , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Biblioteca de Péptidos , Análisis de Secuencia de ADN , Resonancia por Plasmón de SuperficieRESUMEN
Camptothecin (CPT) is a strong antitumor agent, but its use limited by its low solubility and the instability of the active lactone form. To overcome these difficulties, liposomes incorporating CPT (CPT liposomes) were designed and tested. CPT liposomes were formulated by the addition of 3,5-bis(dodecyloxy)benzoic acid (DB) to polyethylene glycol-containing liposomes, and by coating the surface of the liposomes with human serum albumin (HSA, HSA-DB-L). HSA-DB-L successfully entrapped CPT with about 80% efficiency and with a particle size of about 150 nm. HSA-DB-L showed attenuated drug release and storage stability. Pharmacokinetics studies in mice showed that i.v. injection of HSA-DB-L (2.5 mg/kg) led to prolonged circulation in the plasma; the area under the curve was 22-fold higher than that of CPT solution. The tumor growth in mice with subcutaneous transplantation of colon 26 tumor cells was significantly inhibited after a single i.v. injection of HSA-DB-L at a dose of 15 mg/kg without any significant body weight loss. HSA-DB-L increased the accumulation of CPT in tumor tissue significantly (9.6-fold) more efficiently than CPT solution 24 h after i.v. injection. These findings suggest that HSA-DB-L could increase the stability and the antitumor effect of CPT. CPT delivery by novel liposome formulations is a potential approach for effective treatment of cancer.
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Antineoplásicos Fitogénicos/uso terapéutico , Benzoatos/uso terapéutico , Camptotecina/uso terapéutico , Liposomas/uso terapéutico , Neoplasias/tratamiento farmacológico , Albúmina Sérica/uso terapéutico , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacocinética , Benzoatos/química , Benzoatos/farmacocinética , Camptotecina/química , Camptotecina/farmacocinética , Estabilidad de Medicamentos , Femenino , Humanos , Liposomas/química , Liposomas/farmacocinética , Masculino , Ratones , Ratones Endogámicos , Neoplasias/metabolismo , Neoplasias/patología , Albúmina Sérica/química , Albúmina Sérica/farmacocinética , Distribución Tisular , Carga Tumoral/efectos de los fármacosRESUMEN
Camptothecin (CPT) has anticancer activity. While only the lactone form of CPT is biologically active, this form exhibits poor aqueous solubility. Pharmaceutical formulation of CPT incorporated in liposomes is of significant importance to develop the therapeutic utilization of CPT. The aim of this study was to increase incorporation efficiency and stability of CPT in liposomes composed of hydrogenated soybean phosphatidylcholine, cholesterol, and oleic acid (7 : 3 : 1, molar ratio), by incorporating three kinds of artificial lipids (DBs) (DB-liposome); 4-n-(M12B), 3,5-bis(B12B) and 3,4,5-tris(dodecyloxy)benzoic acid (T12B). The interaction of CPT with DB in the state of liposomes, was examined. In DB-liposomes presenting mean diameters of 150 nm, incorporation efficiency of CPT up to 55% and final drug to lipid molar ratio up to 0.07 were obtained when the liposomes were prepared at a feeding ratio of 1/30 (w/w) CPT/total lipid. However, in the optimal formulations, incorporated DB mol% was different; T12B and D12B were incorporated about one third and half mol% of M12B, respectively. Moreover, we demonstrated that T12B stabilized CPT in liposomes significantly compared with other DBs as measured by CPT release, and by steady state fluorescence polarization degree of CPT using intrinsic fluorescence of CPT. These findings suggested that in addition of contribution of phenyl group of DB, dodecyloxy group may interact strongly with lactone ring of CPT. The capacity to contain CPT interacted with DBs may be limited in liposomes. T12B may be incorporated in the interior of the bilayers, resulting in increase of incorporation stability of CPT. This finding demonstrates a potential application of the novel liposome formulation of CPT in drug delivery.
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Antineoplásicos Fitogénicos/química , Camptotecina/química , Lípidos/química , Liposomas/química , Algoritmos , Anisotropía , Portadores de Fármacos , Excipientes , Inmunoensayo de Polarización FluorescenteRESUMEN
One obstacle to effective gene therapy lies in low transfection efficiency by non-viral vectors. To meet this challenge, we developed cell-penetrating peptide-based gene delivery vectors. A novel oligoarginine lipid ((Arg)n-B, n=4, 10) conjugated to 3,5-bis(dodecyloxy)benzamide (BDB) lipid with a poly(ethylene glycol) (PEG) spacer was synthesized. Oligoarginine lipid-coated vector was prepared by the addition of (Arg)n-B to DNA/protamine complex (PD) ((Arg)n-B-PD). Transfection efficiency of (Arg)n-B-PD was compared with that of (Arg)n-B/DNA complex ((Arg)n-B/D) for in vitro and in xenograft tumor of human cervical carcinoma HeLa by intratumoral injection. Transfection efficiency in tumors and in vitro greatly depended on the charge ratios of (Arg)n-B to DNA and the length of Arg residues. In vitro, positively charged Arg10-B-PD showed the highest transfection efficiency. In contrast, in tumor transfection, negatively charged Arg4-B-PD showed the highest transfection efficiency, about 2-, 16- and 23-fold higher than PD alone, Arg10-B-PD and a commercial gene transfection reagent, respectively. This result suggests that negatively charged tetraarginine-conjugated-PEG lipid-coated PD is a promising gene delivery vector for intratumoral injection.
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Arginina/química , ADN/metabolismo , Terapia Genética/métodos , Lípidos/química , Neoplasias/terapia , Oligopéptidos/química , Polietilenglicoles/química , Protaminas/química , Transfección/métodos , Animales , ADN/administración & dosificación , ADN/química , Femenino , Células HeLa , Humanos , Inyecciones Intralesiones , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/genética , Neoplasias/metabolismo , Tamaño de la Partícula , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Fullerene derivative C60TT, which is substituted with the low-molecular-weight organogelator tris(dodecyloxy)benzamide, formed nanowire structures on application of the Langmuir-Blodgett (LB) method. The surface morphology of the C60TT LB film was dependent on the holding time before deposition at a surface pressure of 5 mN m(-1); it changed from a homogeneous monolayer to a bilayer fibrous structure via a fibrous monolayer structure, which was estimated to have dimensions of 1.2 nm in height, 8 nm in width, and 5-10 microm in length. From the structural and spectroscopic data, it is inferred that close packing of the fullerene moiety occurs along with intermolecular hydrogen bonding within the monolayer fibrous structure. The morphological changes in the LB film are explained kinetically by the Avrami theory, based on the decrease in the surface area of the monolayer at the air/water interface. The growth of the quasi-one-dimensional fibrous monolayer structures at holding times from 0 to 0.2 h is considered to be an interface-controlled process, whereas the growth of the quasi-one-dimensional bilayer fibrous structures from 0.2 to 18 h is thought to be a diffusion-controlled process.
Asunto(s)
Benzamidas/química , Fulerenos/química , Membranas Artificiales , Nanocables/química , Silicatos de Aluminio/química , Geles/química , Enlace de Hidrógeno , Microscopía de Fuerza Atómica , Modelos Moleculares , Estructura Molecular , Peso Molecular , Espectrofotometría Ultravioleta/métodos , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Propiedades de Superficie , Factores de TiempoRESUMEN
Phage-display technology is probably the best available strategy to produce antibodies directed against various carbohydrate moieties since conventional hybridoma technologies have yielded mostly low-affinity antibodies against a limited number of carbohydrate antigens. Because of difficulties in immobilization of carbohydrate antigens onto plastic plates, however, the same procedures used for protein antigens cannot be readily applied. We adapted phage-display technology to generate human single chain antibodies (scFvs) using neoglycolipids as antigens. This study describes the isolation and characterization of phage-displayed antibodies (phage Abs) that recognized nonreducing terminal mannose residues. We first constructed a phage Ab library with a large repertoire using CDR shuffling and VL/VH shuffling methods with unique vector constructs. The library was subjected to four rounds of panning against neoglycolipids synthesized from mannotriose (Man3) and dipalmitoylphosphatidylethanolamine (DPPE) by reductive amination. Of 672 clones screened by enzyme-linked immunosorbent assay (ELISA) using Man3-DPPE as an antigen, 25 positive clones encoding scFvs with unique amino acid sequences were isolated as candidates for phage Abs against Man3 residues. TLC-overlay assays and surface plasmon resonance analyses revealed that selected phage Abs bound to neoglycolipids bearing mannose residues at nonreducing termini. In addition, binding of the phage Ab to RNase B carrying high mannose type oligosaccharides but not to fetuin carrying complex type and O-linked oligosaccharides was confirmed. Furthermore, first round characterization of scFvs expressed from respective phages indicated good affinity and specificity for nonreducing terminal mannose residues. These results demonstrated the usefulness of this strategy in constructing human scFv against various carbohydrate antigens. Further studies on the purification and characterization of these scFvs are presented in an accompanying paper in this issue.