RESUMEN
Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.
Asunto(s)
Encéfalo/metabolismo , Sistemas de Liberación de Medicamentos , Liposomas/metabolismo , Nanopartículas/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Línea Celular , Portadores de Fármacos , Humanos , Liposomas/análisis , Liposomas/farmacocinética , Masculino , Nanopartículas/análisis , Péptidos/análisis , Péptidos/farmacocinética , Ratas Wistar , Distribución TisularRESUMEN
Poly(amidoamine)s with bioreducible disulfide linkages in the main chain (SS-PAAs) and pH-responsive, negatively charged citraconate groups in the sidechain have been designed for effective intracellular delivery and release of proteins with a net positive charge at neutral pH. Using lysozyme as a cationic model protein these water soluble polymers efficiently self-assemble into nanocomplexes by charge attraction. At pH5 (the endosomal pH) the amide linkages connecting the citraconate groups in the sidechains of the SS-PAAs are hydrolyzed by intramolecular catalysis, resulting in expulsion of the negative citraconate groups and formation of protonated amine groups, resulting in charge reversal of the polymeric carrier from negative to positive. The concomitant endosomal buffering effect and increased polymer-endosomal membrane interactions are considered to lead to increased protein delivery into the cytosol. Besides destabilization of the polymer-protein nanoparticles by the charge reversal effect, intracellular cleavage of disulfide linkages in the polymer ensure further unpacking of the protein in the cytosol. Cellinternalization and cytotoxicity experiments with primary human umbilical vein endothelial cells (HUVEC) showed that the SS-PAA-based nanocomplexes were essentially non-toxic, and that lysozyme is successfully internalized into HUVEC. The results indicate that these charge reversal SS-PAAs have excellent properties as non-toxic intracellular delivery systems for cationic proteins.
Asunto(s)
Células Endoteliales/química , Nanocápsulas/administración & dosificación , Nanocápsulas/química , Poliaminas/química , Proteínas/administración & dosificación , Proteínas/química , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Humanos , Ensayo de MaterialesRESUMEN
A poly(amidoamine) with disulfide linkages in the main chain and 4-hydroxybutyl and ω-carboxy-PEG groups (9:1 ratio) as side chains was prepared by Michael addition polymerization of cystamine bisacrylamide with 4-hydroxybutylamine and ω-carboxy-PEG-amine. To develop therapeutic protein formulations for improved delivery of antigen via the intranasal route, nanoparticles were prepared from this polymer by self-assembly with p24 or ovalbumin as the model proteins and CpG as the adjuvant. The nanoparticles incorporated the antigens and adjuvant from the feed solution with high efficiency (â¼90 %) and have sizes of 112 and 169â nm, respectively, with low positive surface charge (â¼+2â mV). Formulations of the nanoparticles were shown to be nontoxic and stable for at least 10 days at room temperature. Their capacity to pass through epithelial and endothelial cell layers was evaluated in vitro by using a respiratory mucosa-like barrier model in which monolayers of NCI H441 respiratory epithelial cells and ISO-HAS-1 endothelial cells were co-cultured on both sides of a transwell filter membrane. It was shown that p24 incorporated in the nanoparticles was transported with >140 % greater efficiency through the two contact-inhibited layers than p24 in its free form, whereas incorporation of ovalbumin in the nanoparticles leads to a 40 % decrease in transport efficiency relative to the free antigen.
Asunto(s)
Antígenos/química , Nanopartículas/química , Poliaminas/química , Poliaminas/farmacología , Mucosa Respiratoria/efectos de los fármacos , Administración Intranasal , Células Cultivadas , Humanos , Modelos Biológicos , Tamaño de la PartículaRESUMEN
In this study, we investigated nanoparticles formulated by self-assembly of a biodegradable poly(amidoamine) (PAA) and a fluorescently labeled peptide, in their capacity to internalize in endothelial cells and deliver the peptide, with possible applications for brain drug delivery. The nanoparticles were characterized in terms of size, surface charge, and loading efficiency, and were applied on human cerebral microvascular endothelial cells (hCMEC/D3) and human umbilical vein endothelial cells (Huvec) cells. Cell-internalization and cytotoxicity experiments showed that the PAA-based nanocomplexes were essentially nontoxic, and the peptide was successfully internalized into cells. The results indicate that these PAAs have an excellent property as nontoxic carriers for intracellular protein and peptide delivery, and provide opportunities for novel applications in the delivery of peptides to endothelial cells of the brain.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Nanopartículas/química , Oligopéptidos/química , Poliaminas/química , Encéfalo/metabolismo , Células Cultivadas , Portadores de Fármacos/química , Células Endoteliales/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Microvasos/citología , Nanopartículas/toxicidad , Oligopéptidos/administración & dosificación , Poliaminas/toxicidadRESUMEN
An effective intracellular protein delivery system was developed based on linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Two differently functionalized PAAs were synthesized by Michael-type polyaddition of 4-amino-1-butanol (ABOL) to cystamine bisacrylamide (CBA) and to bisacryloylpiperazine (BAP), yielding p(CBA-ABOL) and p(BAP-ABOL), respectively. These water-soluble PAAs efficiently condense human serum albumin (HSA) by self-assembly into stable nanoscaled and positively-charged complexes. The disulfide-containing p(CBA-ABOL)/HSA nanocomplexes exhibited high mucoadhesive properties and, while stable under neutral (extracellular) conditions, rapidly destabilized in a reductive (intracellular) environment due to the cleavage of the repetitive disulfide linkages in the CBA units of the polymer. Human-derived intestinal Caco-2/TC7 cells and HT29-MTX mucus secreting cells were exposed to these PAAs/HSA nanoparticles and the extent of their uptake and the localization within endosomal compartments were examined. The higher uptake of p(CBA-ABOL)/HSA than that of p(BAP-ABOL)/HSA suggests that the mucoadhesive properties of the p(CBA-ABOL) are beneficial to the uptake process. The transported HSA was located within early endosomes, lysosomes and the cytosol. The enhanced uptake of the p(CBA-ABOL)/HSA nanoparticles, observed in the presence of Cyclosporin A, a non-specific Multi Drug Resistance (MDR) blocker, indicates the possible efflux of these nanoparticles through MDR transporters. The results show that bioreducible PAAs have excellent properties for intracellular protein delivery, and should be applicative in oral protein delivery.
Asunto(s)
Proteínas Portadoras/metabolismo , Sistemas de Liberación de Medicamentos , Intestinos/citología , Poliaminas/metabolismo , Proteínas/metabolismo , Células CACO-2 , Endosomas/metabolismo , Citometría de Flujo , Células HT29 , Humanos , Nanopartículas/química , Transporte de Proteínas , Albúmina Sérica/metabolismoRESUMEN
An effective intracellular protein delivery system was developed based on functionalized linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Three differently functionalized PAAs were synthesized, two of these having repetitive disulfide bonds in the main chain, by Michael-type polyaddition of 4-amino-1-butanol (ABOL) to cystamine bisacrylamide (CBA), histamine (HIS) to CBA, and ABOL to bis(acryloyl)piperazine (BAP). These water-soluble PAAs efficiently condense ß-galactosidase by self-assembly into nanoscaled and positively-charged complexes. Stable under neutral extracellular conditions, the disulfide-containing nanocomplexes rapidly destabilized in a reductive intracellular environment. Cell-internalization and cytotoxicity experiments showed that the PAA-based nanocomplexes were essentially non-toxic. ß-Galactosidase was successfully internalized into cells, with up to 94% of the cells showing ß-galactosidase activity, whereas the enzyme alone was not taken up by the cells. The results indicate that these poly(amidoamine)s have excellent properties as highly potent and non-toxic intracellular protein carriers, which should create opportunities for novel applications in protein delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos , Poliaminas/química , Proteínas/administración & dosificación , Concentración de Iones de Hidrógeno , Nanopartículas , Poliaminas/administración & dosificación , beta-Galactosidasa/administración & dosificación , beta-Galactosidasa/química , beta-Galactosidasa/farmacocinética , beta-Galactosidasa/farmacologíaRESUMEN
To improve design processes in the field of nanomedicine, in vitro characterization of nanoparticles with systematically varied properties is of great importance. In this study, surface sensitive analytical techniques were used to evaluate the responsiveness of nano-sized drug-loaded polyelectrolyte complexes when adsorbed to model lipid membranes. Two bioreducible poly(amidoamine)s (PAAs) containing multiple disulfide linkages in the polymer backbone (SS-PAAs) were synthesized and used to form three types of nanocomplexes by self-assembly with human insulin, used as a negatively charged model protein at neutral pH. The resulting nanoparticles collapsed on top of negatively charged model membranes upon adsorption, without disrupting the membrane integrity. These structural rearrangements may occur at a cell surface which would prevent uptake of intact nanoparticles. By the addition of glutathione, the disulfide linkages in the polymer backbone of the SS-PAAs were reduced, resulting in fragmentation of the polymer and dissociation of the adsorbed nanoparticles from the membrane. A decrease in ambient pH also resulted in destabilization of the nanoparticles and desorption from the membrane. These mimics of intracellular environments suggest dissociation of the drug formulation, a process that releases the protein drug load, when the nanocomplexes reaches the interior of a cell.