RESUMEN
The sensitivity of microorganisms to low pH can be utilized in food protection by preparing coatings based on macromolecular acids. Due to limited diffusivity of macromolecules low pH occurs primarily at the surface, while the interior parts of the food remain unaffected. This principle is demonstrated using food approved alginic acid in various types of coatings (aqueous, emulsions, dispersions, dry coating) on a wide range of foods including meat, fish, chicken, shrimp and boiled rice. Significant delay or inhibition of the natural flora is generally demonstrated, particularly when exposed to 'temperature abuse'. Specifically, we show that the coatings reduce or inhibit regrowth of pathogens (Bacillus cereus, B. weihenstephanensis, Listeria monocytogenes serotype 1 and Staphylococcus aureus). In special cases like boiled rice, alginic acid may largely replace acetic acid for acidification and preservation, as demonstrated studying regrowth of added spores of B. cereus. Most formulations allow easy removal prior to further processing (cooking, frying). Temporary side effects such as 'acid cooking' obtained for high acid concentrations on sensitive surfaces (e.g. salmon) disappear during processing, recovering the normal taste and texture. The coating is hence suitable for a large variety of foods.
Asunto(s)
Bacterias/efectos de los fármacos , Microbiología de Alimentos/métodos , Conservación de Alimentos/métodos , Carne/microbiología , Animales , Bacillus cereus/efectos de los fármacos , Bacillus cereus/crecimiento & desarrollo , Bacterias/crecimiento & desarrollo , Recuento de Colonia Microbiana , Manipulación de Alimentos/normas , Listeria monocytogenes/efectos de los fármacos , Oryza/microbiología , Salmón/microbiología , Staphylococcus aureus/fisiología , TemperaturaRESUMEN
Chitosan is widely explored as a gene delivery vehicle due to its ability to condense DNA, facilitate transport, and subsequent release allowing gene expression, as well as protecting the DNA. Here, we investigate the enhancement of chitosan-DNA dispersion stability while maintaining transfection efficacy by PEGylation of chitosan. Molecular properties of fully deacetylated chitosans and degree of PEGylation were investigated with respect to compaction of DNA, stability and transfection efficacy. Each of the three chitosan samples with varying chain lengths was PEGylated at three different degrees. The chitosans with degree of PEGylation from 0.6 to 1.9% made polyplexes with DNA. PBS induced colloidal aggregation of polyplexes with initial radius of about 100 nm observed for nonPEGylated chitosans was suppressed for 1.9% PEGylated chitosans. The observed increase in transfection efficacy coinciding with increased polyplex colloidal stability suggests that aggregation of gene-delivery packages may reduce the transfection efficacy.
Asunto(s)
Quitosano/análogos & derivados , Quitosano/química , Polietilenglicoles/química , Transfección , Quitosano/síntesis química , Coloides , ADN/química , ADN/genética , Etidio/química , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Sustancias Intercalantes/química , Luciferasas/biosíntesis , Luciferasas/genética , Plásmidos/química , Plásmidos/genética , Polietilenglicoles/síntesis química , PolimerizacionRESUMEN
The blood-brain barrier (BBB), composed of tightly organized endothelial cells, limits the availability of drugs to therapeutic targets in the central nervous system. The barrier is maintained by membrane bound efflux pumps efficiently transporting specific xenobiotics back into the blood. The efflux pump P-glycoprotein (P-gp), expressed at high levels in brain endothelial cells, has several drug substrates. Consequently, siRNA mediated silencing of the P-gp gene is one possible strategy how to improve the delivery of drugs to the brain. Herein, we investigated the potential of siRNA-chitosan nanoparticles in silencing P-gp in a BBB model. We show that the transfection of rat brain endothelial cells mediated effective knockdown of P-gp with subsequent decrease in P-gp substrate efflux. This resulted in increased cellular delivery and efficacy of the model drug doxorubicin.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Quitosano/química , Portadores de Fármacos/química , Células Endoteliales/efectos de los fármacos , Nanopartículas/química , ARN Interferente Pequeño/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Transporte Biológico , Barrera Hematoencefálica/citología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/química , Doxorrubicina/farmacología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Cinética , Ratas , TransfecciónRESUMEN
The mechanism involved in the ultrasoundenhanced intracellular delivery of fluorescein-isothiocyanate (FITC)-dextran (molecular weight 4 to 2000 kDa) and liposomes containing doxorubicin (Dox) was studied using HeLa cells and an ultrasound transducer at 300 kHz, varying the acoustic power. The cellular uptake and cell viability were measured using flow cytometry and confocal microscopy. The role of endocytosis was investigated by inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis. Microbubbles were found to be required during ultrasound treatment to obtain enhanced cellular uptake. The percentage of cells internalizing Dox and dextran increased with increasing mechanical index. Confocal images and flow cytometric analysis indicated that the liposomes were disrupted extracellularly and that released Dox was taken up by the cells. The percentage of cells internalizing dextran was independent of the molecular weight of dextrans, but the amount of the small 4-kDa dextran molecules internalized per cell was higher than for the other dextrans. The inhibition of endocytosis during ultrasound exposure resulted in a significant decrease in cellular uptake of dextrans. Therefore, the improved uptake of Dox and dextrans may be a result of both sonoporation and endocytosis.
Asunto(s)
Dextranos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Fluoresceína-5-Isotiocianato/análogos & derivados , Liposomas/administración & dosificación , Sonicación/métodos , Análisis de Varianza , Supervivencia Celular/efectos de los fármacos , Dextranos/química , Dextranos/farmacocinética , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Doxorrubicina/farmacocinética , Endocitosis/efectos de los fármacos , Citometría de Flujo , Fluoresceína-5-Isotiocianato/administración & dosificación , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/farmacocinética , Células HeLa , Humanos , Liposomas/química , Liposomas/farmacocinética , Microburbujas , Microscopía Confocal , UltrasonidoRESUMEN
The success of gene therapy depends on efficient delivery of DNA and requires a vector. A promising non-viral vector is chitosan. We tailored chitosan to optimize it for transfection by synthesizing self-branched and trisaccharide-substituted chitosan oligomers (SBTCO), which show superior transfection efficacy compared with linear chitosan (LCO). The aim of the work was to compare the cellular uptake and endocytic pathways of polyplexes formed by LCO and SBTCO. Both polyplexes were taken up by the majority of the cells, but the uptake of LCO was lower than SBTCO polyplexes. LCO polyplexes were internalized through both clathrin-dependent and clathrin-independent pathways, whereas SBTCO polyplexes were primarily taken up by clathrin-independent endocytosis. The different level of cellular uptake and the distinct endocytic pathways, may explain the difference in transfection efficacy. This was supported by the observation that photochemical internalization increased the transfection by LCO polyplexes considerably, whereas no effect on transfection was found for SBTCO polyplexes.
Asunto(s)
Caveolas/metabolismo , Quitosano/química , Quitosano/metabolismo , Clatrina/metabolismo , ADN/metabolismo , Endocitosis , Nanopartículas , Caveolas/efectos de los fármacos , Clorpromazina/farmacología , ADN/genética , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Endocitosis/efectos de los fármacos , Genisteína/farmacología , Células HeLa , Humanos , Hidrazonas/farmacología , Propiedades de Superficie , Temperatura , TransfecciónRESUMEN
Multichannel microscopy is frequently used to study intermolecular interactions and spatial relationships between biomolecules and organelles or vesicles in cells. Based on multichannel images, quantitative colocalization analysis can provide valuable information about cellular internalization, vesicular transport, and the intracellular kinetics and location of biomolecules. However, such analyses should be performed carefully, because quantitative colocalization parameters have different interpretations and can be highly affected by image quality. We use quantitative three-dimensional colocalization analysis of deconvolved and chromatic-registered confocal images to study the dissociation of double-labeled pDNA-chitosan polyplexes in HeLa cells and their colocalization with early endosomes. Two chitosans that form polyplexes with highly different transfection efficacies are compared. Pearson's correlation coefficient, Manders' colocalization coefficients, and the intensity correlation quotient are estimated to determine the intracellular localization of polyplexes, free pDNA, and free chitosans. Differences are observed in the amount of uptake, and in the intracellular pathways and rates of dissociation for the two chitosans. The results support previous findings that polyplexes formed by self-branched, glycosylated chitosan oligomers are more favorable for cellular uptake and intracellular trafficking to the nucleus compared with polyplexes formed by linear chitosans.
Asunto(s)
Algoritmos , Quitosano/metabolismo , Interpretación de Imagen Asistida por Computador/métodos , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Plásmidos/metabolismo , Fracciones Subcelulares/metabolismo , Células HeLa , Humanos , Aumento de la Imagen/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Chitosan has gained increasing interest for siRNA delivery. Although chitosan covers a family of structurally different polysaccharides, most siRNA delivery studies have been performed with conventional partially N-acetylated chitosans. Herein, the purpose was to identify fundamental chitosan molecular properties favoring siRNA delivery and efficient gene silencing in mammalian cells. Nanoparticles were prepared from well-defined chitosans of various chemical compositions, degrees of polymerization (DP(n)) and chain architectures. Structure-activity relationships were determined by the cellular uptake of siRNA and the knockdown efficiency at mRNA and protein levels. Additionally, the nanoparticle cytotoxicity was evaluated on the basis of cellular metabolic activity and membrane integrity. Our results show that the most efficient gene silencing was achieved using fully de-N-acetylated chitosans with intermediate chain lengths (DP(n) 100-300). These chitosans mediated efficient siRNA delivery at low siRNA concentrations and, in several cell lines, potent long-term silencing of both exogenous and endogenous target genes, with minimal cytotoxicity.
Asunto(s)
Quitosano/administración & dosificación , Portadores de Fármacos/administración & dosificación , Nanopartículas/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Silenciador del Gen , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Transfección/métodosRESUMEN
Diffusion through the extracellular matrix (ECM) is a critical step for the delivery of nanoparticles and genes. Gene delivery requires a carrier that protects the nucleic acid from degradation and facilitates transport. Chitosan is a promising carrier. To increase the circulation time, PEGylation of the carrier is performed. However, the effect of PEGylation on the transport and stability of gene delivery systems in the ECM has only been studied in solutions containing ECM components. We used polymerized collagen and collagen-hyaluronic acid (HA) gels to study the effects of PEGylation on the diffusion and stability of chitosan-DNA polyplexes. We found that PEGylation of the polyplexes was required for diffusion to occur, and PEGylation increased the dissociation between DNA and chitosan to some extent. The presence of HA had a contradictory role: it decreased the penetration depth of PEGylated polyplexes into the gels and increased the diffusion of the polyplexes being mixed into the gels.
Asunto(s)
Materiales Biocompatibles/química , Quitosano , Colágeno/química , ADN/metabolismo , Matriz Extracelular/metabolismo , Geles/química , Polietilenglicoles/química , Materiales Biocompatibles/metabolismo , Quitosano/química , Quitosano/metabolismo , Colágeno/metabolismo , Difusión/efectos de los fármacos , Estabilidad de Medicamentos , Matriz Extracelular/química , Fluoresceína-5-Isotiocianato/análisis , Geles/metabolismo , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Microscopía Confocal , Nanopartículas , Tamaño de la Partícula , Plásmidos/metabolismo , Polietilenglicoles/farmacología , Espectrometría de Fluorescencia , Coloración y EtiquetadoRESUMEN
Chitosan possesses many characteristics of an ideal gene delivery system. However, the transfection efficiency of conventional chitosans is generally found to be low. In this study, we investigated the self-branching of chitosans as a strategy to improve its gene transfer properties without compromising its safety profile. Self-branched (SB) and self-branched trisaccharide-substituted (SBTCO) chitosans with molecular weights of 11-71 kDa were synthesized, characterized, and compared with their linear counterparts with respect to transfection efficiency, cellular uptake, formulation stability, and cytotoxicity. Our studies show that in contrast with unmodified linear chitosans that were unable to transfect HeLa cells, self-branched chitosans mediated high transfection efficiencies. The most efficient chitosan, SBTCO30, yielded gene expression levels two and five times higher than those of Lipofectamine and Exgen, respectively, and was nontoxic to cells. Nanoparticles formed with SBTCO chitosans exhibited a higher colloidal stability of formulation, efficient internalization without excessive cell surface binding, and low cytotoxicity.
Asunto(s)
Quitosano/química , Transfección/métodos , Quitosano/farmacocinética , Coloides/química , Coloides/farmacocinética , Expresión Génica , Técnicas de Transferencia de Gen , Células HeLa , Humanos , Peso Molecular , Nanopartículas/química , Transfección/normasRESUMEN
Chitosan is an attractive gene delivery vehicle, but the criteria and strategies for the design of efficient chitosan gene delivery systems remain unclear. The purpose of this work was to investigate how the strength of the charge-based interaction between chitosan and DNA determines the gene expression levels and to design chitosan vectors with an optimized balance between polyplex stability and polyplex unpacking. Using 21 formulations based on low molecular weight chitosans with constant charge density and a number-average degree of polymerization (DPn) in the range of 21-88 (M(w) 4.7-33kDa), we studied the relationship between the chain length and the formulation properties, cellular uptake of polyplexes and gene transfer efficacy. We were able to identify a narrow interval of DPn31-42 that mediated the maximum level of transgene expression. An increase in chain length and/or the amino-phosphate (A/P) ratio reduced and delayed transgene expression. Compared to DPn31, transfection with the same amount of DPn72 or DPn88 resulted in 10-fold-lower expression levels. The gene transfer pattern correlated with the ability of heparin to release DNA from the polyplexes. As a tool to facilitate the unpacking of the polyplexes, we substituted the chitosans with uncharged oligosaccharides that reduced the interaction with DNA. The substitution of chitosans that originally yielded too stable polyplexes, such as DPn72 and DPn88 resulted in a 5-10-fold enhancement of the expression levels. However, the substitution of chitosans shorter than DP28 completely abolished transfection. Tailoring of the chain length and the substitution of chitosan were shown to be feasible tools to modulate the electrostatic interactions between the chitosan and DNA and to design chitosans with an optimized balance between polyplex stability and polyplex unpacking.
Asunto(s)
Quitosano/química , ADN/administración & dosificación , ADN/química , Portadores de Fármacos/síntesis química , Riñón/fisiología , Transfección/métodos , Línea Celular , Humanos , Ensayo de MaterialesRESUMEN
Development of efficient local delivery systems for the auditory organ has an important role in clinical practice for the management of inner ear disorders using pharmacological means. Chitosan, a biodegradable polymer, is a good drug carrier with bioadhesive properties. The aim of this study was to investigate the feasibility of using chitosan to deliver drugs to the inner ear across the round window membrane (RWM). Three structurally different chitosans loaded with a tracer drug, neomycin, were injected into the middle ear cavity of albino guinea pigs (n=35). After 7 days the effect of chitosans and neomycin was compared among the treatment groups. The hearing organ was analysed for hair cell loss and the RWM evaluated in term of thickness. All tested chitosan formulations successfully released the loaded neomycin which then diffused across the RWM, and exerted ototoxic effect on the cochlear hair cells in a degree depending on the concentrations used. Chitosans per se had no noxious effect on the cochlear hair cells. It is concluded that the chitosans, and especially glycosylated derivative, are safe and effective carriers for inner ear therapy.
Asunto(s)
Materiales Biocompatibles/administración & dosificación , Quitosano/administración & dosificación , Portadores de Fármacos/administración & dosificación , Oído Interno/efectos de los fármacos , Vehículos Farmacéuticos/administración & dosificación , Animales , Materiales Biocompatibles/química , Quitosano/análogos & derivados , Quitosano/química , Relación Dosis-Respuesta a Droga , Portadores de Fármacos/síntesis química , Cobayas , Células Ciliadas Auditivas Internas/efectos de los fármacos , Células Ciliadas Auditivas Internas/patología , Instilación de Medicamentos , Neomicina/administración & dosificación , Neomicina/efectos adversos , Ventana Redonda/efectos de los fármacos , Ventana Redonda/patologíaRESUMEN
Chitosan can be used as a nonviral gene delivery vector for which DNA condensation and transfection efficacy strongly depend on structural parameters. In this study, we characterized the condensation of DNA by three molecularly tailored chitosans, including linear, trisaccharide substituted-, and self-branched trisaccharide substituted chitosan oligomers. No significant differences could be detected in the hydrodynamic diameters formed by the various chitosans as analyzed by dynamic light scattering. However, atomic force microscopy revealed that self-branched chitosan formed complexes with a higher ratio of globules to rods, and the heights of both globules and rods were larger than for complexes formed by the other chitosans. Using an amino/phosphate ratio of 10, fluorescence correlation spectroscopy measurements showed that self-branched chitosan exhibited a lower fraction (30%) of bound chitosan than the other chitosans. YOYO-1 was a superior fluorescent DNA-label compared to Cy5 and PicoGreen, since labeling with YOYO-1 had least effect on the size and structure of the complexes.
Asunto(s)
Quitosano , Técnicas de Transferencia de Gen , Vectores Genéticos , Colorantes Fluorescentes , Microscopía de Fuerza Atómica , Dispersión de Radiación , Espectrometría de Fluorescencia/métodosRESUMEN
Chitosan is a promising biomaterial with an attractive safety profile; however, its application potential for gene delivery is hampered by poor compatibility at physiological pH values. Here we have tailored the molecular architecture of chitosan to improve the functional properties and gene transfer efficacy of chitosan oligomers and have developed self-branched glycosylated chitosan oligomer (SB-TCO) substituted with a trisaccharide containing N-acetylglucosamine, AAM. SB-TCO was prepared by controlled depolymerization of chitosan, followed by simultaneous branching and AAM substitution. The product was fully soluble at physiological pH and complexed plasmid DNA into polyplexes of high colloidal and physical stability. SB-TCO displayed high transfection efficacy in HEK293 cells, reaching transfection efficiencies of up to 70%, and large amounts of transgene were produced. Gene transfer efficacy was confirmed in HepG2 cells, where gene expression levels mediated by SB-TCO were up to 10 and 4 times higher than those obtained with unsubstituted and substituted linear oligomers, respectively. The rapid onset of transgene expression in both cell lines indicates efficient DNA release and transcription from SB-TCO polyplexes. In comparison with 22 kDa linear PEI-based transfection reagent used as the control, SB-TCO possessed higher gene transfer efficacy, significantly lower cytotoxicity, and improved serum compatibility.
Asunto(s)
Quitosano/química , Técnicas de Transferencia de Gen , Materiales Biocompatibles/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Glicosilación , Humanos , Plásmidos , SolubilidadRESUMEN
Electrostatic interactions between synthetic polyelectrolytes and proteins can lead to the formation of dense, macroion-rich liquid phases, with equilibrium microheterogeneities on length scales up to hundreds of nanometers. The effects of pH and ionic strength on the rheological and optical properties of these coacervates indicate microstructures sensitive to protein-polyelectrolyte interactions. We report here on the properties of coacervates obtained for bovine serum albumin (BSA) with the biopolyelectrolyte chitosan and find remarkable differences relative to coacervates obtained for BSA with poly(diallyldimethylammonium chloride) (PDADMAC). Coacervation with chitosan occurs more readily than with PDADMAC. Viscosities of coacervates obtained with chitosan are more than an order of magnitude larger and, unlike those with PDADMAC, show temperature and shear rate dependence. For the coacervates with chitosan, a fast relaxation time in dynamic light scattering, attributable to relatively unrestricted protein diffusion in both systems, is diminished in intensity by a factor of 3-4, and the consequent dominance by slow modes is accompanied by a more heterogeneous array of slow apparent diffusivities. In place of a small-angle neutron scattering Guinier region in the vicinity of 0.004 A-1, a 10-fold increase in scattering intensity is observed at lower q. Taken together, these results confirm the presence of dense domains on length scales of hundreds of nanometers to micrometers, which in coacervates prepared with chitosan are less solidlike, more interconnected, and occupy a larger volume fraction. The differences in properties are thus correlated with differences in mesophase structure.
Asunto(s)
Quitosano/química , Polietilenos/química , Compuestos de Amonio Cuaternario/química , Albúmina Sérica Bovina/química , Animales , Bovinos , Microscopía por Crioelectrón , Difusión , Electrólitos , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Transmisión , Nefelometría y Turbidimetría , Neutrones , Reología , Análisis Espectral , Volumetría , ViscosidadRESUMEN
The aim of this study was to improve the gene delivery efficacy of chitosan oligomer polyplexes by introducing a trisaccharide branch that targets cell-surface lectins. For this purpose, chitosan oligomers were substituted by a trisaccharide with the N-acetylglucosamine residue at the free end, and the ability of the trisaccharide-substituted chitosan oligomers (TCO) polyplexes to transfect various cell lines in vitro and lung tissue after in vivo administration to mice was investigated. Live-cell confocal microscopy showed improved cellular uptake in HEK 293 cells (11-fold, p<0.001) for the TCO polyplexes compared with the linear chitosan oligomers. Colloidal stability was also enhanced with the substituted form, which suggests that the trisaccharide branch stabilised the polyplexes by means of a steric stabilisation mechanism. Interestingly, gene expression levels in the human liver hepatocyte (HepG2) cells were 10-fold higher with the TCO polyplexes than those mediated by polyethyleneimine. A similar improvement was obtained in a human bronchial epithelial cell line (16HBE14o-). Transfection with the TCO was significantly inhibited (by 30-80%), for all the cell lines tested, in the presence of the free trisaccharide branch, confirming lectin-mediated uptake. Finally, in vivo studies showed that, 24 h after lung administration to mice, luciferase gene expression was 4-fold higher with the TCO than with the corresponding linear chitosan oligomers.
Asunto(s)
Quitosano/química , Técnicas de Transferencia de Gen , Pulmón/metabolismo , Trisacáridos/química , Animales , Tampones (Química) , Secuencia de Carbohidratos , Línea Celular , Química Farmacéutica , Coloides , ADN/administración & dosificación , ADN/química , Sistemas de Liberación de Medicamentos , Ensayo de Cambio de Movilidad Electroforética , Humanos , Intubación Intratraqueal , Lectinas , Luciferasas/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Tamaño de la Partícula , Relación Estructura-Actividad , TransfecciónRESUMEN
The interactions between DNA and chitosans varying in fractional content of acetylated units (FA), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on FA, and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.
Asunto(s)
Materiales Biocompatibles/química , Quitosano/química , ADN/química , Electrólitos/química , Vectores Genéticos , Animales , Cationes , Bovinos , Electroforesis , Etidio/química , Etidio/farmacología , Técnicas de Transferencia de Gen , Concentración de Iones de Hidrógeno , Sustancias Intercalantes , Iones , Luz , Sustancias Macromoleculares/química , Microscopía de Fuerza Atómica , Peso Molecular , Conformación de Ácido Nucleico , Polietileneimina , Polilisina , Polímeros/química , Unión Proteica , Conformación Proteica , Dispersión de Radiación , Electricidad Estática , Timo/metabolismo , Factores de TiempoRESUMEN
Chitosan-based gene delivery systems are promising candidates for non-viral gene therapy. A wide range of chitosans has been studied to optimize the properties of the DNA-chitosan complexes to yield high transfection efficiencies. An important parameter to control is the polyplex stability to allow transport towards the cells, subsequent internalization and release of DNA intracellularly. The stability of the DNA-chitosan complexes was here studied after exposure to heparin and hyaluronic acid (HA) using atomic force microscopy (AFM) and ethidium bromide (EtBr) fluorescence assay. To study the effect of polycation chain length on the polyplex stability, chitosans with a degree of polymerization (DP) varying from approximately 10 to approximately 1000 were employed for DNA compaction. Whereas HA was unable to dissociate the complexes, the degree of dissociation caused by heparin depended on both the chitosan chain length and the amount of chitosan used for complexation. When increasing the chitosan concentration, larger heparin concentrations were required for polyplex dissociation. Furthermore, increasing the chitosan chain length yielded more stable complexes. Varying the chitosan chain length thus provides a tool for controlling the ability of the polyplex to deliver therapeutic gene vectors to cells.
Asunto(s)
Quitosano/química , ADN/química , Terapia Genética , Glicosaminoglicanos/farmacología , Sistemas de Liberación de Medicamentos , Microscopía de Fuerza AtómicaRESUMEN
Three types of well-characterized chitosans of different composition were applied to flocculate 8 different bacterial species. The aim of this study was to relate chitosan structure and flocculation characteristic to general bacterial characteristics such as the cell surface charge and hydrophobicity. Large differences in the flocculation efficiency of chitosan were found between different bacterial suspensions, both regarding the effective chitosan concentrations and the optimal type of chitosan. However, no correlation was observed between general surface characteristics of bacteria and flocculation by chitosan of different composition. It may be concluded that purely electrostatic interactions did not play a dominant role in flocculation of Gram-negative bacteria in this study. The presence of GlcNAc residues had clearly beneficial effects on flocculation in such cases.