RESUMEN
The vitreous humor is the first barrier encountered by intravitreally injected nanoparticles. Lipid-based nanoparticles in the vitreous are studied by evaluating their diffusion with single-particle tracking technology and by characterizing their protein coronae with surface plasmon resonance and high-resolution proteomics. Single-particle tracking results indicate that the vitreal mobility of the formulations is dependent on their charge. Anionic and neutral formulations are mobile, whereas larger (>200 nm) neutral particles have restricted diffusion, and cationic particles are immobilized in the vitreous. PEGylation increases the mobility of cationic and larger neutral formulations but does not affect anionic and smaller neutral particles. Convection has a significant role in the pharmacokinetics of nanoparticles, whereas diffusion drives the transport of antibodies. Surface plasmon resonance studies determine that the vitreal corona of anionic formulations is sparse. Proteomics data reveals 76 differentially abundant proteins, whose enrichment is specific to either the hard or the soft corona. PEGylation does not affect protein enrichment. This suggests that protein-specific rather than formulation-specific factors are drivers of protein adsorption on nanoparticles in the vitreous. In summary, our findings contribute to understanding the pharmacokinetics of nanoparticles in the vitreous and help advance the development of nanoparticle-based treatments for eye diseases.
Asunto(s)
Nanopartículas/química , Soluciones Oftálmicas/administración & dosificación , Enfermedades de la Retina/tratamiento farmacológico , Cuerpo Vítreo/metabolismo , Adsorción , Animales , Difusión , Composición de Medicamentos/métodos , Humanos , Inyecciones Intravítreas , Liposomas , Soluciones Oftálmicas/farmacocinética , Tamaño de la Partícula , Polietilenglicoles/química , Corona de Proteínas/análisis , Corona de Proteínas/metabolismo , Proteómica , Propiedades de Superficie , Sus scrofaRESUMEN
In this study, we present a novel in vitro approach that utilizes two surface-sensitive and label-free techniques, i.e. surface plasmon resonance (SPR) and quartz crystal microbalance (QCM), to study the interfacial events during liposome-target surface interactions. The flow channels of SPR and QCM devices were first synchronized via hydrodynamic modeling. Biotin-streptavidin was used as a model pair and self-assembled monolayers (SAMs) were utilized as model surfaces for targeted liposome-surface interaction studies. The interactions between biotin-liposomes and the streptavidin-biotin-SAM surfaces were investigated under controlled shear flows using the synchronized SPR and QCM devices. The response of the liposome interaction was monitored as a function of the flow rate. The affinity and the amount of bound liposome indicated that the increased flow rate improved the binding of the targeted liposomes to the model membrane surfaces. The combined use of the synchronized SPR and QCM devices for nanoparticle interaction studies clearly demonstrates the effect of the flow rate (or the shear stress) on the liposome binding. Our results suggest that the binding of liposomes to the model membranes is flow rate and shear stress regulated. Thus, the flow rate (or the shear stress), which is usually neglected, should be taken into account during the development and optimization of targeted liposome formulations. In addition, the water content within the liposome layer (including the water inside the liposomes and the water between the liposomes) had a significant influence on the visco-elasticity and the binding kinetics to the SAM surfaces.
Asunto(s)
Proteínas Bacterianas/química , Biotina/análogos & derivados , Liposomas/química , Nanopartículas/química , Biotina/química , Albúmina Sérica Bovina/química , Resonancia por Plasmón de Superficie , Agua/químicaRESUMEN
BACKGROUND: Epidermal growth factor receptor (EGFR) is overexpressed in many solid tumor types, such as ovarian carcinoma. Immunoliposome based drug targeting has shown promising results in drug delivery to the tumors. However, the ratio of tumor-to-normal tissue concentrations should be increased to minimize the adverse effects of cytostatic drugs. METHODOLOGY/PRINCIPAL FINDINGS: We studied the EGFR-targeted doxorubicin immunoliposomes using pre-targeting and local intraperitoneal (i.p.) administration of the liposomes. This approach was used to increase drug delivery to tumors as compared to direct intravenous (i.v.) administration of liposomes. EGFR antibodies were attached on the surface of PEG coated liposomes using biotin-neutravidin binding. Receptor mediated cellular uptake and cytotoxic efficacy of EGFR-targeted liposomes were investigated in human ovarian adenocarcinoma (SKOV-3 and SKOV3.ip1) cells. In vivo distribution of the liposomes in mice was explored using direct and pre-targeting approaches and SPECT/CT imaging. Targeted liposomes showed efficient and specific receptor-mediated binding to ovarian carcinoma cells in vitro, but the difference in cytotoxicity between targeted and non-targeted liposomes remained small. The relatively low cytotoxic efficacy is probably due to insufficient doxorubicin release from the liposomes rather than lack of target binding. Tumor uptake of targeted liposomes in vivo was comparable to that of non-targeted liposomes after both direct and pre-targeting administration. For both EGFR-targeted and non-targeted liposomes, the i.p. administration increased liposome accumulation to the tumors compared to i.v. injections. CONCLUSIONS/SIGNIFICANCE: Intraperitoneal administration of liposomes may be a beneficial approach to treat the tumors in the abdominal cavity. The i.p. pre-targeting method warrants further studies as a potential approach in cancer therapy.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Doxorrubicina/administración & dosificación , Terapia Molecular Dirigida/métodos , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados , Avidina/metabolismo , Biotina/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica , Cetuximab , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Estudios de Factibilidad , Femenino , Humanos , Liposomas , Ratones , Imagen Multimodal , Neoplasias Ováricas/diagnóstico por imagen , Neoplasias Ováricas/patología , Tomografía de Emisión de Positrones , Tomografía Computarizada por Rayos XRESUMEN
Drug nanocarriers are often derivatized with targeting moieties to achieve site specific delivery, however, the results from this approach have, as yet, not reached expectations. We have tested a new phage display based targeting moiety, the activated endothelium targeting peptide (AETP), for its vascular endothelium directed targeting efficiency, when anchored to a PEGylated liposome via maleimide chemistry. Our results have, however, not shown any evidence of improved targeting. We have hypothesized that the failure of the AETP moiety is due to its availability to target receptors being restricted, as a result of steric hindrance due to the PEG polymer, and possibly affinity for bloodstream proteins, particularly human serum albumin (HSA). In this context, molecular modeling was used to contrast the properties of the AETP moiety to those of the RGD targeting peptide, already found to be effective in previous trials. Our molecular dynamics simulation results indicate the AETP moiety is located within the PEG layer, and its hydrophobic nature causes it to be obscured by PEG to a greater extent than the more hydrophilic RGD targeting peptide. Protein-ligand docking results indicated similar affinities for HSA of both the AETP moiety and a PEG fragment, and a significantly lower affinity for the RGD peptide. We know of no means to investigate this experimentally with atomic level resolution, thus our use of computational methods to investigate this can be seen as a new tool for rational design in nanomedicine.
Asunto(s)
Liposomas/administración & dosificación , Liposomas/química , Terapia Molecular Dirigida/métodos , Péptidos/administración & dosificación , Péptidos/química , Polietilenglicoles/química , Secuencias de Aminoácidos , Animales , Células Cultivadas , Portadores de Fármacos/administración & dosificación , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Maleimidas/química , Ratones , Ratones Desnudos , Modelos Químicos , Simulación de Dinámica Molecular , Nanomedicina/métodos , Polietilenglicoles/administración & dosificación , Unión Proteica , Receptores de Superficie Celular/metabolismo , Distribución TisularRESUMEN
A series of amphiphilic star and linear block copolymers were synthesized using ATRP. The core consisted of either polystyrene (PS) or poly(n-butyl acrylate) (PBuA), having different glass-transition (T(g)) values. These polymers were used as macroinitiators in the polymerization of the cationic 2-(dimethylamino)ethyl methacrylate (DMAEMA). The polymers were used to study the effects of polymer architecture and flexibility on the self-assembling properties, DNA complexation, and transfection. All polymers formed core-shell micelles in aqueous solutions and condensed plasmid DNA. Linear PDMAEMA-PBuA-PDMAEMA has transfection efficiency comparable to PEI25K in ARPE19 cell line. Glassy state of the micellar core and star-shaped architecture decreased the DNA transfection compared with the rubbery and linear polymer structures. The polymers showed low cellular toxicity at low nitrogen/phosphate (n/p) ratios.
Asunto(s)
ADN/farmacología , Portadores de Fármacos/síntesis química , Células Epiteliales/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Técnicas de Transferencia de Gen , Metacrilatos/síntesis química , beta-Galactosidasa/farmacología , Acrilatos/química , Animales , Cationes/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Microscopía por Crioelectrón , ADN/genética , ADN/metabolismo , Portadores de Fármacos/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Haplorrinos , Humanos , Metacrilatos/metabolismo , Micelas , Plásmidos , Polietilenglicoles/química , Polímeros/química , Poliestirenos/química , Soluciones , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transfección , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Cationic polymers are efficient gene delivery vectors in in vitro conditions, but these carriers can fail in vivo due to interactions with extracellular polyanions, i.e. glycosaminoglycans (GAG). The aim of this study was to develop a stable gene delivery vector that is activated at the acidic endosomal pH. Cationic DNA/PEI complexes were coated by 1,2-dioleylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) (3:2 mol/mol) using two coating methods: detergent removal and mixing with liposomes prepared by ethanol injection. Only detergent removal produced lipid-coated DNA complexes that were stable against GAGs, but were membrane active at low pH towards endosome mimicking liposomes. In relation to the low cellular uptake of the coated complexes, their transfection efficacy was relatively high. PEGylation of the coated complexes increased their cellular uptake but reduced the pH-sensitivity. Detergent removal was thus a superior method for the production of stable, but acid activatable, lipid-coated DNA complexes.
Asunto(s)
ADN/administración & dosificación , Detergentes/química , Portadores de Fármacos/química , Glicosaminoglicanos/metabolismo , Lípidos/química , Transfección , Animales , Aniones , Bovinos , Línea Celular , Ésteres del Colesterol/química , ADN/genética , Estabilidad de Medicamentos , Técnicas de Transferencia de Gen , Haplorrinos , Concentración de Iones de Hidrógeno , Iminas/química , Liposomas , Tamaño de la Partícula , Fosfatidiletanolaminas/química , Plásmidos , Polietilenos/químicaRESUMEN
Nature has evolved proteins and enzymes to carry out a wide range of sophisticated tasks. Proteins modified with functional polymers possess many desirable physical and chemical properties and have applications in nanobiotechnology. Here we describe multivalent Newkome-type polyamine dendrons that function as synthetic DNA binding domains, which can be conjugated with proteins. These polyamine dendrons employ naturally occurring spermine surface groups to bind DNA with high affinity and are attached onto protein surfaces in a site-specific manner to yield well-defined one-to-one protein-polymer conjugates, where the number of dendrons and their attachment site on the protein surface are precisely known. This precise structure is achieved by using N-maleimido-core dendrons that selectively react via 1,4-conjugate addition with a single free thiol group on the protein surface--either Cys-34 of bovine serum albumin (BSA) or a genetically engineered cysteine mutant of Class II hydrophobin (HFBI). This reaction can be conducted in mild aqueous solutions (pH 7.2-7.4) and at ambient temperature, resulting in BSA- and HFBI-dendron conjugates. The protein-dendron conjugates constitute a specific biosynthetic diblock copolymer and bind DNA with high affinity, as shown by ethidium bromide displacement assay. Importantly, even the low-molecular-weight first-generation polyamine dendron (1 kDa) can bind a large BSA protein (66.4 kDa) to DNA with relatively good affinity. Preliminary gene transfection, cytotoxicity, and self-assembly studies establish the relevance of this methodology for in vitro applications, such as gene therapy and surface patterning. These results encourage further developments in protein-dendron block copolymer-like conjugates and will allow the advance of functional biomimetic nanoscale materials.