RESUMEN
Biomolecules adsorbed on nanoparticles are known to confer a biological identity to nanoparticles, mediating the interactions with cells and biological barriers. However, how these molecules are presented on the particle surface in biological milieu remains unclear. The central aim of this study is to identify key protein recognition motifs and link them to specific cell-receptor interactions. Here, we employed an immuno-mapping technique to quantify epitope presentations of two major proteins in the serum corona, low-density lipoprotein and immunoglobulin G. Combining with a purpose-built receptor expression system, we show that both proteins present functional motifs to allow simultaneous recognition by low-density lipoprotein receptor and Fc-gamma receptor I of the corona. Our results suggest that the "labeling" of nanoparticles by biomolecular adsorption processes allows for multiple pathways in biological processes in which they may be "mistaken" for endogenous objects, such as lipoproteins, and exogenous ones, such as viral infections.
Asunto(s)
Nanopartículas/química , Corona de Proteínas/química , Receptores de IgG/química , Receptores de LDL/química , Adsorción , Sitios de Unión , Células Cultivadas , Mapeo Epitopo , Células HEK293 , Humanos , Inmunoglobulina G/química , Lipoproteínas LDL/química , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Characterizing the orientation of covalently conjugated proteins on nanoparticles, produced for in vitro and in vivo targeting, though an important feature of such a system, has proved challenging. Although extensive physicochemical characterization of targeting nanoparticles can be addressed in detail, relevant biological characterization of the nanointerface is crucial in order to select suitable nanomaterials for further in vitro or in vivo experiments. In this work, we adopt a methodology using antibody fragments (Fab) conjugated to gold nanoparticles (immunogold) to map the available epitopes on a transferrin grafted silica particle (SiO2-PEG8-Tf) as a proxy methodology to predict nanoparticle biological function, and therefore cellular receptor engagement. Data from the adopted method suggest that, on average, only â¼3.5% of proteins grafted on the SiO2-PEG8-Tf nanoparticle surface have a favorable orientation for recognition by the cellular receptor.
Asunto(s)
Anticuerpos/química , Oro/química , Nanopartículas del Metal/química , Dióxido de Silicio/química , Estructura Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Nanoparticles interacting with, or derived from, living organisms are almost invariably coated in a variety of biomolecules presented in complex biological milieu, which produce a bio-interface or 'biomolecular corona' conferring a biological identity to the particle. Biomolecules at the surface of the nanoparticle-biomolecule complex present molecular fragments that may be recognized by receptors of cells or biological barriers, potentially engaging with different biological pathways. Here we demonstrate that using intense fluorescent reporter binders, in this case antibodies bound to quantum dots, we can map out the availability of such recognition fragments, allowing for a rapid and meaningful biological characterization. The application in microfluidic flow, in small detection volumes, with appropriate thresholding of the detection allows the study of even complex nanoparticles in realistic biological milieu, with the emerging prospect of making direct connection to conditions of cell level and in vivo experiments.
Asunto(s)
Citometría de Flujo/métodos , Nanopartículas/química , Proteínas/química , Puntos Cuánticos , Algoritmos , Anticuerpos/química , Anticuerpos/metabolismo , Humanos , Nanopartículas/metabolismo , Unión Proteica , Proteínas/inmunología , Proteínas/metabolismo , Albúmina Sérica/química , Albúmina Sérica/inmunología , Albúmina Sérica/metabolismo , Espectrometría de Fluorescencia , Transferrina/química , Transferrina/inmunología , Transferrina/metabolismoRESUMEN
Nanoparticles (NPs) functionalized with two active targeting ligands have been proposed in drug delivery for their promising capability to stimulate different pathways with one object. Due to the multivalency, the construction and analysis of the effective surface of such bifunctional nanoparticles, however, is significantly more complex than for nanoparticles bearing only one ligand. Here, we optimize construction and analysis of bifunctional NPs containing recognizable combinations of human serum albumin (HSA), transferrin (Tf), and epidermal growth factor (EGF) on fluorescent silica NPs grafted via common polyethylene glycol (PEG) linkers as a model system. Combined with an overall protein quantification, a mapping of exposed recognizable sequences using monoclonal antibodies conjugated to gold nanoparticles (AuNPs) or quantum dots (QDs) for enhanced spectroscopic and microscopic detection revealed that active protein sequences can be one to two orders of magnitude lower than overall conjugated proteins while possessing specific cellular recognition. In addition, we found that common conjugation strategies lead to a large excess of non-specifically compared to covalently bound ligands and instabilities that may impact targeting. These can be avoided by certain synthetic conditions presented here for effective exploitation of multivalent surfaces in nanomedicine.
RESUMEN
A wide range of particles have been developed for different applications in drug-delivery, tissue engineering, or regenerative medicine. In contrast to traditional spherical particles, nonspherical (e.g., cylindrical) particles possess several structural and morphological advantages that make them attractive for specific applications. Here, we developed a top-down approach to process electrospun fibers into microsized cylinders (i.e., microcylinders) with high specific surface area and with or without surface porosity. To obtain these microcylinders, poly(l-lactic acid) (PLLA) solutions were subjected to electrospinning, followed by an aminolysis-based chemical scission procedure. The morphology, structure, and chemical composition of the microcylinders were then characterized. The specific surface area and surface porosity of the microcylinders were controlled by the volatility of the solvents, and their length was dependent on the duration of the aminolysis reaction. During aminolysis, the microcylinders became functionalized with amine groups, enabling potential further modifications by grafting with compounds containing desired chemical groups, for example, carboxyl, carbonyl, or hydroxyl functional groups. Additionally, the microcylinders showed in vitro biocompatible properties related to cell viability. These data demonstrate that PLLA microcylinders with high specific surface area, optional surface porosity, amine-based functional handles granting additional functionalization, and cytocompatible properties are candidate materials for future biomedical applications.
RESUMEN
The aim of this study is to verify if water-soluble porphyrins can be used as proteasome inhibitors. We have found that cationic porphyrins inhibit proteasome peptidase activities much more effectively than the corresponding anionic derivatives. The relevance of electrostatics in driving porphyin-proteasome interactions has been confirmed by the observation that the inhibitory efficiency of the cationic macrocycles decreases with the number of positive substituents. We have also investigated various metalloporphyrins, which differ due to the different propension of the central metal ion toward axial coordination. Our experimental results indicate that the naked cationic porphyrins are the most active in reversibly inhibiting the three main protease activities of the proteasome in the micromolar range. A spectroscopic characterization of porphyrin-proteasome interactions by UV-vis spectra parallels the results of inhibition assays: the higher the inhibitory effect the stronger the spectroscopic variations are. To interpret the action of porphyrins at a molecular level, we have performed calculations evidencing that cationic porphyrins may hinder the access to the canonical proteolytic site on the proteasome ß5 subunit. In particular, an inspection of the top-scoring docking modes shows that the tetracationic porphyrin blocks the catalytic pocket, close to the N termini of the ß5 proteasome subunit, more efficiently than its anionic counterpart. Proteasome inhibition activity of porphyrins unites their known anticancer properties making them suitable as a scaffold for the design of novel multitargeted molecules.