RESUMO
Nerve regeneration scaffolds often consist of soft hydrogels modified with extracellular matrix (ECM) proteins or fragments, as well as linear and cyclic peptides. One of the commonly used integrin-mediated cell adhesive peptide sequences is Arg-Gly-Asp (RGD). Despite its straightforward coupling mechanisms to artificial extracellular matrix (aECM) constructs, linear RGD peptides suffer from low stability towards degradation and lack integrin selectivity. Cyclization of RGD improves the affinity towards integrin subtypes but lacks selectivity. In this study, a new class of short bicyclic peptides with RGD in a cyclic loop and 'random screened' tri-amino acid peptide sequences in the second loop is investigated as a biochemical cue for cell growth inside three-dimensional (3D) synthetic poly(ethylene glycol) (PEG)-based Anisogels. These peptides impart high integrin affinity and selectivity towards either αvß3 or α5ß1 integrin subunits. Enzymatic conjugation of such bicyclic peptides to the PEG backbone enables the formulation of an aECM hydrogel that supports nerve growth. Furthermore, different proteolytic cleavable moieties are incorporated and compared to promote cell migration and proliferation, resulting in enhanced cell growth with different degradable peptide crosslinkers. Mouse fibroblasts and primary nerve cells from embryonic chick dorsal root ganglions (DRGs) show superior growth in bicyclic RGD peptide conjugated gels selective towards αvß3 or α5ß1, compared to monocyclic or linear RGD peptides, with a slight preference to αvß3 selective bicyclic peptides in the case of nerve growth. Synthetic Anisogels, modified with bicyclic RGD peptides and containing short aligned, magneto-responsive fibers, show oriented DRG outgrowth parallel to the fibers. This report shows the potential of PEG hydrogels coupled with bicyclic RGD peptides as an aECM model and paves the way for a new class of integrin selective biomolecules for cell growth and nerve regeneration.
Assuntos
Oligopeptídeos , Peptídeos , Animais , Hidrogéis , Camundongos , PolietilenoglicóisRESUMO
We report the identification of high-affinity and selectivity integrin α5ß1-binding bicyclic peptides via "designed random libraries", that is, the screening of libraries comprising the universal integrin-binding sequence Arg-Gly-Asp (RGD) in the first loop in combination with a randomized sequence (XXX) in the second loop. Screening of first-generation libraries for α5ß1-binding peptides yielded a triple-digit nanomolar bicyclic α5ß1-binder (CT3RGDcT3AYGCT3, IC50 = 406 nM). Next-generation libraries were designed by partially varying the structure of the strongest first-generation lead inhibitor and screened for improved affinities and selectivities for this receptor. In this way, we identified three high-affinity α5ß1-binders (CT3RGDcT3AYJCT3, J = d-Leu, IC50 = 90 nM; CT3RGDcT3AYaCT3, IC50 = 156 nM; CT3RGDcT3AWGCT3, IC50 = 173 nM), of which one even showed a higher α5ß1-affinity than the 32 amino acid benchmark peptide knottin-RGD (IC50 = 114 nM). Affinity for α5ß1-integrin was confirmed by SPFS analysis showing a Kd of 4.1 nM for Cy5-labeled RGD-bicycle CT3RGDcT3AYJCT3 (J = d-Leu) and a somewhat higher Kd (9.0 nM) for Cy5-labeled knottin-RGD. The α5ß1-bicycles, for example, CT3RGDcT3AYJCT3 (J = d-Leu), showed excellent selectivities over αvß5 (IC50 ratio α5ß1/αvß5 between <0.009 and 0.039) and acceptable selectivities over αvß3 (IC50 ratios α5ß1/αvß3 between 0.090 and 0.157). In vitro staining of adipose-derived stem cells with Cy5-labeled peptides using confocal microscopy revealed strong binding of the α5ß1-selective bicycle CT3RGDcT3AWGCT3 to integrins in their natural environment, illustrating the high potential of these RGD bicycles as markers for α5ß1-integrin expression.
Assuntos
Oligopeptídeos/análise , Biblioteca de Peptídeos , Receptores de Vitronectina/química , Técnicas de Química Combinatória , Oligopeptídeos/síntese químicaRESUMO
We describe the identification of bicyclic RGD peptides with high affinity and selectivity for integrin αvß3 via high-throughput screening of partially randomized libraries. Peptide libraries (672 different compounds) comprising the universal integrin-binding sequence Arg-Gly-Asp (RGD) in the first loop and a randomized sequence XXX (X being one of 18 canonical l-amino acids) in the second loop, both enclosed by either an l- or d-Cys residue, were converted to bicyclic peptides via reaction with 1,3,5-tris(bromomethyl)benzene (T3). Screening of first-generation libraries yielded lead bicyclic inhibitors displaying submicromolar affinities for integrin αvß3 (e.g., CT3HEQcT3RGDcT3, IC50 = 195 nM). Next generation (second and third) libraries were obtained by partially varying the structure of the strongest lead inhibitors and screening for improved affinities and selectivities. In this way, we identified the highly selective bicyclic αvß3-binders CT3HPQcT3RGDcT3 (IC50 = 30 nM), CT3HPQCT3RGDcT3 (IC50 = 31 nM), and CT3HSQCT3RGDcT3 (IC50 = 42 nM) with affinities comparable to that of a knottin-RGD-type peptide (32 amino acids, IC50 = 38 nM) and outstanding selectivities over integrins αvß5 (IC50 > 10000 nM) and α5ß1 (IC50 > 10000 nM). Affinity measurements using surface plasmon-enhanced fluorescence spectroscopy (SPFS) yielded Kd values of 0.4 and 0.6 nM for the Cy5-labeled bicycle CT3HPQcT3RGDcT3 and RGD "knottin" peptide, respectively. In vitro staining of HT29 cells with Cy5-labeled bicycles using confocal microscopy revealed strong binding to integrins in their natural environment, which highlights the high potential of these peptides as markers of integrin expression.
Assuntos
Integrina alfaVbeta3/química , Oligopeptídeos/química , Sequência de Aminoácidos , Aminoácidos/química , Regulação da Expressão Gênica , Células HT29 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Integrina alfaVbeta3/genética , Imagem Óptica/métodos , Biblioteca de Peptídeos , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
Biomaterial design in tissue engineering aims to identify appropriate cellular microenvironments in which cells can grow and guide new tissue formation. Despite the large diversity of synthetic polymers available for regenerative medicine, most of them fail to fully match the functional properties of their native counterparts. In contrast, the few biological alternatives employed as biomaterials lack the versatility that chemical synthesis can offer. Herein, we studied the HUVEC adhesion and proliferation properties of elastin-like recombinamers (ELRs) that were covalently functionalized with each three high-affinity and selectivity α v ß 3- and α 5 ß 1-binding bicyclic RGD peptides. Next to the bicycles, ELRs were also functionalized with various integrin-binding benchmark peptides, i.e. knottin-RGD, cyclo-[KRGDf] and GRGDS, allowing for better classification of the obtained results. Covalent functionalization with the RGD peptides, as validated by MALDI-TOF analysis, guarantees flexibility and minimal steric hindrance for interactions with cellular integrins. In addition to the covalently modified RGD-ELRs, we also synthesized another benchmark ELR comprising RGD as part of the backbone. HUVEC adhesion and proliferation analysis using the PicoGreen® assay revealed a higher short-term adhesion and proliferative capacity of cells on ELR surfaces functionalized with high affinity, integrin-binding bicyclic RGD-peptides compared with the ELRs containing RGD in the backbone.
Assuntos
Materiais Biocompatíveis/química , Adesão Celular/efeitos dos fármacos , Elastina/química , Engenharia Genética/métodos , Integrina alfaVbeta3/química , Oligopeptídeos/química , Receptores de Vitronectina/química , Proliferação de Células , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos/química , Polímeros/química , Ligação Proteica , Medicina Regenerativa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Engenharia TecidualRESUMO
We describe a highly sensitive competition ELISA to measure integrin-binding of RGD-peptides in high-throughput without using cells, ECM-proteins, or antibodies. The assay measures (nonlabeled) RGD-peptides' ability to inhibit binding of a biotinylated "knottin"-RGD peptide to surface-immobilized integrins and, thus, enables quantification of the binding strength of high-, medium-, and low-affinity RGD-binders. We introduced the biotinylated knottin-RGD peptide instead of biotinylated cyclo[RGDfK] (as reported by Piras et al.), as integrin-binding was much stronger and clearly detectable for all three integrins. In order to maximize sensitivity and cost-efficiency, we first optimized several parameters, such as integrin-immobilization levels, knottin-RGD concentration, buffer compositions, type of detection tag (biotin, His- or cMyc-tag), and spacer length. We thereby identified two key factors, that is, (i) the critical spacer length (longer than Gly) and (ii) the presence of Ca2+ and Mg2+ in all incubation and washing buffers. Binding of knottin-RGD peptide was strongest for αvß3 but also detectable for both αvß5 and α5ß1, while binding of biotinylated cyclo[RGDfK] was very weak and only detectable for αvß3. For assay validation, we finally determined IC50 values for three unlabeled peptides, that is: (i) linear GRGDS, (ii) cyclo[RGDfK], and (iii) the knottin-RGD itself for binding to three different integrin receptors (αvß3, αvß5, α5ß1). Major benefits of the novel assay are (i) the extremely low consumption of integrin (50 ng/peptide), (ii) the fact that neither antibodies/ECM-proteins nor integrin-expressing cells are required for detection, and (iii) its suitability for high-throughput screening of (RGD-)peptide libraries.