RESUMEN
Biomimetic lipid membranes on solid supports have been used in a plethora of applications, including as biosensors, in research on membrane proteins or as interfaces in cell experiments. For many of these applications, structured lipid membranes, e.g., in the form of arrays with features of different functionality, are highly desired. The stability of these features on a given substrate during storage and in incubation steps is key, while at the same time the substrate ideally should also exhibit antifouling properties. Here, we describe the highly beneficial properties of a 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer for the stability of supported lipid membrane structures generated by dip-pen nanolithography with phospholipids (L-DPN). The MPC copolymer substrates allow for more stable and higher membrane stack structures in comparison to other hydrophilic substrates, like glass or silicon oxide surfaces. The structures remain highly stable under immersion in liquid and subsequent incubation and washing steps. This allows multiplexed functionalization of lipid arrays with antibodies via microchannel cantilever spotting (µCS), without the need of orthogonal binding tags for each antibody type. The combined properties of the MPC copolymer substrate demonstrate a great potential for lipid-based biomedical sensing and diagnostic platforms.
Asunto(s)
Lípidos de la Membrana/química , Membranas Artificiales , Metacrilatos/química , Fosforilcolina/análogos & derivados , Materiales Biomiméticos/química , Lípidos de la Membrana/síntesis química , Microscopía de Fuerza Atómica/métodos , Nanotecnología/métodos , Fosfolípidos/química , Fosforilcolina/química , Polímeros/química , Dióxido de Silicio/químicaRESUMEN
Multifunctional biomaterial surfaces can be created by controlling the competing adsorption of multiple proteins. To demonstrate this concept, bone morphogenetic protein 2 (BMP-2) and fibronectin were adsorbed to the hydrophobic surface of polychloro-para-xylylene. The resulting adsorption properties on the surface depended on the dimensional and steric characteristics of the selected protein molecule, the degree of denaturation of the adsorbed proteins, the associated adsorption of interphase water molecules within the protein layers, and the aggregation of proteins in a planar direction with respect to the adsorbent surface. Additionally, a defined surface composition was formed by the competing adsorption of multiple proteins, and this surface composition was directly linked to the composition of the protein mixture in the solution phase. Although the mechanism of this complex competing adsorption process is not fully understood, the adsorbed proteins were irreversibly adsorbed and were unaffected by the further adsorption of homologous or heterologous proteins. Moreover, synergistic biological activities, including cell osteogenesis and proliferation independently and specifically induced by BMP-2 or fibronectin, were observed on the modified surface, and these biological activities were positively correlated with the surface composition of the multiple adsorbed proteins. These results provide insights and important design parameters for prospective biomaterials and biointerfaces for (multi)functional modifications. The ability to control protein/interface properties will be beneficial for the processing of biomaterials for clinical applications and industrial products.
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Materiales Biocompatibles/farmacología , Proteína Morfogenética Ósea 2/farmacología , Fibronectinas/farmacología , Células Madre/efectos de los fármacos , Xilenos/farmacología , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Adsorción , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Materiales Biocompatibles/química , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Fibronectinas/metabolismo , Expresión Génica , Humanos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Osteonectina/genética , Osteonectina/metabolismo , Agregado de Proteínas , Células Madre/citología , Células Madre/metabolismo , Propiedades de Superficie , Andamios del Tejido , Xilenos/químicaRESUMEN
The ability to induce osteointegration was introduced to a parylene-C surface via the simple and intuitive process of protein adsorption mediated by hydrophobic interactions. In this way, bone morphogenetic protein (BMP)-2, fibronectin, and platelet-rich plasma (PRP) could be immobilized on parylene-C surfaces. This approach alleviates concerns related to the use of potentially harmful substances in parylene-C modification processes. The adsorbed protein molecules were quantitatively characterized with respect to adsorption efficacy and binding affinity, and the important biological activities of the proteins were also examined using both early and late markers of osteogenetic activity, including alkaline phosphatase expression, calcium mineralization and marker gene expression. Additionally, the adsorbed PRP exhibited potential as a substitute for expensive recombinant growth factors by effectively inducing comparable osteogenetic activity. In addition to the excellent biocompatibility of parylene-C and its ability to coat a wide variety of substrate materials, the modification of parylene-C via protein adsorption provides unlimited possibilities for installing specific biological functions, expanding the potential applications of this material to include various biointerface platforms.