RESUMEN
Compared to soluble cytokines, surface-tethered ligands can deliver biological signalling with precise control of spatial positioning and concentration. A strategy that immobilises ligand molecules on a surface in a uniform orientation using non-cleavable linkages under physiological conditions would enhance the specific and systemic delivery of signalling in the local environment. We used mixed self-assembled monolayers (SAMs) of oxyamine- and oligo(ethylene glycol)-terminated thiols on gold to covalently install aldehyde- or ketone-functionalised ligands via oxime conjugation. Characterisation by electrochemistry and X-ray photoelectron spectroscopy showed quantitative immobilisation of the ligands on SAM surfaces. The thrombopoietin mimetic peptide, RILL, was immobilised on SAMs and the bioactivity of the substrate was demonstrated by culturing factor-dependent cells. We also optimised the immobilisation and wash conditions so that the peptide was not released into the culture medium and the immobilised RILL could be re-used for consecutive cell cultures. The surface also supported the growth of haematopoietic CD34+ cells comparable to the standard thrombopoietin-supplemented culture. Furthermore, the RILL-immobilised SAM surface was as effective in expanding uncommitted CD34+ cells as standard culture. The stimulatory effect of surface-tethered ligands in haematopoietic stem cell expansion supports the use of ligand immobilisation strategies to replicate the haematopoietic stem cell niche.
Asunto(s)
Antígenos CD34/metabolismo , Proteínas Inmovilizadas/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas Electroquímicas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Inmunofenotipificación , Ligandos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/farmacología , Espectroscopía de Fotoelectrones , Compuestos de Sulfhidrilo/química , Propiedades de SuperficieRESUMEN
Brain tissue engineering has the potential to harness existing elements of neurogenesis within the adult brain to overcome a microenvironment that is otherwise inhibitory to regeneration, especially following severe tissue damage. This study investigates the ability of electrospun poly ε-caprolactone (PCL) to re-direct the migratory pathway of endogenous neuroblasts from the disrupted subventricular zone (SVZ). A small molecule non-peptide ligand (BDNF-mimetic) that mimicked the trophic properties of brain-derived neurotrophic factor (BDNF) was incorporated into electrospun PCL scaffolds to improve neuroblast survival and promote neuroblast migration towards the implant. PCL scaffolds were able to support neuroblast infiltration and migration along the implant tract. In the presence of the BDNF-mimetic, neuroblasts were able to migrate towards the implant via the parenchyma, and their persistence within the implants was prolonged. In addition, the BDNF-mimetic improved implant integration and increased local neuronal plasticity by increasing neurite sprouting at the tissue-implant interface. SMI32+ neurites were observed inside scaffolds at 21 days but not 8 days post implantation, indicating that at least some of the infiltrated neuroblasts had differentiated into neurons.
Asunto(s)
Materiales Biomiméticos/farmacología , Factor Neurotrófico Derivado del Encéfalo/química , Encéfalo/patología , Movimiento Celular/efectos de los fármacos , Nanofibras/química , Neuronas/patología , Andamios del Tejido/química , Animales , Astrocitos/patología , Diferenciación Celular/efectos de los fármacos , Proteínas de Dominio Doblecortina , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/patología , Implantes Experimentales , Inflamación/patología , Masculino , Microglía/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Nanofibras/ultraestructura , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuropéptidos/metabolismo , Poliésteres/química , Ratas , Ratas WistarRESUMEN
Total synthesis of proteins can be challenging despite assembling techniques, such as native chemical ligation (NCL) and expressed protein ligation (EPL). Especially, the combination of recombinant protein expression and chemically addressable solid-phase peptide synthesis (SPPS) is well suited for the redesign of native protein structures. Incorporation of analytical probes and artificial amino acids into full-length natural protein domains, such as the sequence-specific DNA binding zinc-finger motifs, are of interest combining selective DNA recognition and artificial function. The semi-synthesis of the natural 90 amino acid long sequence of the zinc-finger domain of Zif268 is described including various chemically modified constructs. Our approach offers the possibility to exchange any amino acid within the third zinc finger. The realized modifications of the natural sequence include point mutations, attachment of a fluorophore, and the exchange of amino acids at different positions in the zinc finger by artificial amino acids to create additional metal binding sites. The individual constructs were analyzed by circular dichroism (CD) spectroscopy with respect to the integrity of the zinc-finger fold and DNA binding.