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Immunotherapy has developed into an important modality of modern cancer treatment. Unfortunately, checkpoint inhibitor immunotherapies are currently delivered systemically and require frequent administration, which can result in toxicity and severe, sometimes fatal, adverse events. Localized delivery of immunomodulators for oral cancer and oral potentially malignant disorders offers the promise of maximum therapeutic potential and reduced systemic adverse effects. This review will discuss the limitations of current standard-of-care systemic therapies and highlight research advances in localized, intratumoral delivery platforms for immunotherapy for oral cancer and oral potentially malignant disorders.
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The most abundant natural collagens form heterotrimeric triple helices. Synthetic mimics of collagen heterotrimers have been found to fold slowly, even compared to the already slow rates of homotrimeric helices. These prolonged folding rates are not understood. Here we compare the stabilities, specificities and folding rates of three heterotrimeric collagen mimics designed through a computationally assisted approach. The crystal structure of one ABC-type heterotrimer verified a well-controlled composition and register and elucidated the geometry of pairwise cation-π and axial and lateral salt bridges in the assembly. This collagen heterotrimer folds much faster (hours versus days) than comparable, well-designed systems. Circular dichroism and NMR data suggest the folding is frustrated by unproductive, competing heterotrimer species and these species must unwind before refolding into the thermodynamically favoured assembly. The heterotrimeric collagen folding rate is inhibited by the introduction of preformed competing triple-helical assemblies, which suggests that slow heterotrimer folding kinetics are dominated by the frustration of the energy landscape caused by competing triple helices.
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Maintaining safe and potent pharmaceutical drug levels is often challenging. Multidomain peptides (MDPs) assemble into supramolecular hydrogels with a well-defined, highly porous nanostructure that makes them attractive for drug delivery, yet their ability to extend release is typically limited by rapid drug diffusion. To overcome this challenge, we developed self-assembling boronate ester release (SABER) MDPs capable of engaging in dynamic covalent bonding with payloads containing boronic acids (BAs). As examples, we demonstrate that SABER hydrogels can prolong the release of five BA-containing small-molecule drugs as well as BA-modified insulin and antibodies. Pharmacokinetic studies revealed that SABER hydrogels extended the therapeutic effect of ganfeborole from days to weeks, preventing Mycobacterium tuberculosis growth better than repeated oral administration in an infection model. Similarly, SABER hydrogels extended insulin activity, maintaining normoglycemia for six days in diabetic mice after a single injection. These results suggest that SABER hydrogels present broad potential for clinical translation.
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Progress in the design and synthesis of nanostructured self-assembling systems has facilitated the realization of numerous nanoscale geometries, including fibers, ribbons, and sheets. A key challenge has been achieving control across multiple length scales and creating macroscopic structures with nanoscale organization. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical macrostructures. Further, we demonstrate the ability to tune the nanostructure of macroscopic hydrogels through modulating phosphate buffer concentration during peptide self-assembly. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an extracellular matrix-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to decouple nanostructure from macrostructure and generate a gradient of anisotropic nanofibrous hydrogels. We anticipate that control of architecture at multiple length scales will be critical for a variety of applications, including the bottom-up tissue engineering explored here.
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Hidrogéis , Nanofibras , Peptídeos , Nanofibras/química , Peptídeos/química , Hidrogéis/química , Humanos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/síntese química , Anisotropia , AnimaisRESUMO
The de novo design of self-assembling peptides has garnered significant attention in scientific research. While alpha-helical assemblies have been extensively studied, exploration of polyproline type II (PPII) helices, such as those found in collagen, remains relatively limited. In this study, we focused on understanding the sequence-structure relationship in hierarchical assemblies of collagen-like peptides, using defense collagen SP-A as a model. By dissecting the sequence derived from SP-A and synthesizing short collagen-like peptides, we successfully constructed a discrete bundle of hollow triple helices. Mutation studies pinpointed amino acid sequences, including hydrophobic and charged residues that are critical for oligomer formation. These insights guided the de novo design of collagen-like peptides, resulting in the formation of diverse quaternary structures, including discrete and heterogenous bundled oligomers, 2D nanosheets, and pH-responsive nanoribbons. Our study represents a significant advancement in the understanding and harnessing of collagen higher-order assemblies beyond the triple helix.
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Oral squamous cell carcinoma (OSCC) presents significant treatment challenges due to its poor survival and intense pain at the primary cancer site. Cancer pain is debilitating, contributes to diminished quality of life, and causes opioid tolerance. The stimulator of interferon genes (STING) agonism has been investigated as an anti-cancer strategy. We have developed STINGel, an extended-release formulation that prolongs the availability of STING agonists, which has demonstrated an enhanced anti-tumor effect in OSCC compared to STING agonist injection. This study investigates the impact of intra-tumoral STINGel on OSCC-induced pain using two separate OSCC models and nociceptive behavioral assays. Intra-tumoral STINGel significantly reduced mechanical allodynia in the orofacial cancer model and alleviated thermal and mechanical hyperalgesia in the hind paw model. To determine the cellular signaling cascade contributing to the antinociceptive effect, we performed an in-depth analysis of immune cell populations via single-cell RNA-seq. We demonstrated an increase in M1-like macrophages and N1-like neutrophils after STINGel treatment. The identified regulatory pathways controlled immune response activation, myeloid cell differentiation, and cytoplasmic translation. Functional pathway analysis demonstrated the suppression of translation at neuron synapses and the negative regulation of neuron projection development in M2-like macrophages after STINGel treatment. Importantly, STINGel treatment upregulated TGF-ß pathway signaling between various cell populations and peripheral nervous system (PNS) macrophages and enhanced TGF-ß signaling within the PNS itself. Overall, this study sheds light on the mechanisms underlying STINGel-mediated antinociception and anti-tumorigenic impact.
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Toll-like receptors (TLRs) recognize pathogen- and damage-associated molecular patterns and, in turn, trigger the release of cytokines and other immunostimulatory molecules. As a result, TLR agonists are increasingly being investigated as vaccine adjuvants, though many of these agonists are small molecules that quickly diffuse away from the vaccination site, limiting their co-localization with antigens and, thus, their effect. Here, the small-molecule TLR7 agonist 1V209 is conjugated to a positively-charged multidomain peptide (MDP) hydrogel, K 2 , which was previously shown to act as an adjuvant promoting humoral immunity. Mixing the 1V209-conjugated K 2 50:50 with the unfunctionalized K 2 produces hydrogels that retain the shear-thinning and self-healing physical properties of the original MDP, while improving the solubility of 1V209 more than 200-fold compared to the unconjugated molecule. When co-delivered with ovalbumin as a model antigen, 1V209-functionalized K 2 produces antigen-specific IgG titers that were statistically similar to alum, the gold standard adjuvant, and a significantly lower ratio of Th2-associated IgG1 to Th1-associated IgG2a than alum, suggesting a more balanced Th1 and Th2 response. Together, these results suggest that K 2 MDP hydrogels functionalized with 1V209 are a promising adjuvant for vaccines against infectious diseases, especially those benefiting from a combined Th1 and Th2 immune response. Table of Contents: Activation of toll-like receptors (TLRs) stimulates a signaling cascade to induce an immune response. A TLR7 agonist was conjugated to an injectable peptide hydrogel, which was then used to deliver a model vaccine antigen. This platform produced antibody titers similar to the gold standard adjuvant alum and demonstrated an improved balance between Th1- and Th2-mediated immunity over alum.
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T cells have the ability to recognize and kill specific target cells, giving therapies based on their potential for treating infection, diabetes, cancer, and other diseases. However, the advancement of T cell-based treatments has been hindered by difficulties in their ex vivo activation and expansion, the number of cells required for sustained in vivo levels, and preferential localization following systemic delivery. Biomaterials may help to overcome many of these challenges by providing a combined means of proliferation, antigen presentation, and cell localization upon delivery. In this work, we studied self-assembling Multidomain Peptides (MDPs) as scaffolds for T cell culture, activation, and expansion. We evaluated the effect of different MDP chemistries on their biocompatibility with T cells and the maintenance of antigen specificity for T cells cultured in the hydrogels. We also examined the potential application of MDPs as scaffolds for T cell activation and expansion and the effect of MDP encapsulation on T cell phenotype. We found high cell viability when T cells were encapsulated in noncationic MDPs, O5 and D2, and superior retention of antigen specificity and tumor-reactivity were preserved in the anionic MDP, D2. Maintenance of antigen recognition by T cells in D2 hydrogels was confirmed by quantifying immune synapses of T Cells engaged with antigen-presenting cancer cells. When 3D cultured in anionic MDP D2 coloaded with anti-CD3, anti-CD28, IL2, IL7, and IL15, we observed successful T cell proliferation evidenced by upregulation of CD27 and CD107a. This study is the first to investigate the potential of self-assembling peptide-based hydrogels as 3D scaffolds for human T cell applications and demonstrates that MDP hydrogels are a viable platform for enabling T cell in vitro activation, expansion, and maintenance of antigen specificity and therefore a promising tool for future T cell-based therapies.
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Nanofibras , Neoplasias , Humanos , Hidrogéis/farmacologia , Hidrogéis/química , Linfócitos T , Peptídeos/química , Proliferação de CélulasRESUMO
Fibrous proteins that comprise the extracellular matrix (ECM) guide cellular growth and tissue organization. A lack of synthetic strategies able to generate aligned, ECM-mimetic biomaterials has hampered bottom-up tissue engineering of anisotropic tissues and led to a limited understanding of cell-matrix interactions. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical structures. We establish how modest changes in phosphate buffer concentration during peptide self-assembly can be used to tune their alignment and packing. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an ECM-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to generate a gradient of anisotropic nanofibrous hydrogels, which are used to better understand directed cell growth.
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Collagen mimetic peptides are composed of triple helices. Triple helical formation frequently utilizes charge pair interactions to direct protein assembly. The design of synthetic triple helices is challenging due to the large number of competing species and the overall fragile nature of collagen mimetics. A successfully designed triple helix incorporates both positive and negative criteria to achieve maximum specificity of the supramolecular assembly. Intrahelical charge pair interactions, particularly those involved in lysine-aspartate and lysine-glutamate pairs, have been especially successful both in driving helix specificity and for subsequent stabilization by covalent capture. Despite this progress, the important sequential and geometric relationships of charged residues in a triple helical context have not been fully explored for either supramolecular assembly or covalent capture stabilization. In this study, we compare the eight canonical axial and lateral charge pairs of lysine and arginine with glutamate and aspartate to their noncanonical, reversed charge pairs. These findings are put into the context of collagen triple helical design and synthesis.
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Ácido Aspártico , Lisina , Modelos Moleculares , Colágeno/química , Ácido GlutâmicoRESUMO
Self-assembled nanomaterials are promising candidates for drug delivery by providing a higher degree of spatiotemporal control compared to free drugs. However, challenges such as burst release, inadequate targeting, and drug-nanomaterial incompatibility leave room for improvement. The combination of orthogonal self-assembling systems can result in more useful materials that improve upon these weaknesses. In this work, we investigate an orthogonal self-assembling system of nanofibrous MultiDomain Peptide (MDP) hydrogels encapsulating liposomes. Both positively charged and negatively charged MDPs were prepared and mixed with positively charged, negatively charged, or zwitterionic liposomes for a total of six composites. We demonstrate that, despite both systems being amphiphilic, they are able to mix while retaining their independent identities. We show that changing the charge of either liposomes or MDPs does not hinder the self-assembly of either system or significantly affect their rheological properties. In all six cases, small molecules encapsulated in liposome-MDP composites resulted in slower release than was possible in MDP hydrogels alone. However, in one case, positively charged MDPs destabilized negatively charged liposomes and resulted in a unique release profile. Finally, we show that MDP hydrogels substantially decrease the release of chemotherapeutic doxorubicin from its liposomal formulation, Doxil, for 24 h. This work demonstrates the chemical compatibility of amphiphilic, orthogonally self-assembled systems and the range of their drug-delivering capabilities.
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Hidrogéis , Lipossomos , Lipossomos/química , Hidrogéis/química , Sistemas de Liberação de Medicamentos , Peptídeos/químicaRESUMO
Self-assembling peptides are a type of biomaterial rapidly emerging in the fields of biomedicine and material sciences due to their promise in biocompatibility and effectiveness at controlled release. These self-assembling peptides can form diverse nanostructures in response to molecular interactions, making them versatile materials. Once assembled, the peptides can mimic biological functions and provide a combinatorial delivery of therapeutics such as cytokines and drugs. These self-assembling peptides are showing success in biomedical settings yet face unique challenges that must be addressed to be widely applied in the clinic. Herein, we describe self-assembling peptides' characteristics and current applications in immunomodulatory therapeutics.
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The folding of collagen is a hierarchical process that starts with three peptides associating into the characteristic triple helical fold. Depending on the specific collagen in question, these triple helices then assemble into bundles reminiscent of α-helical coiled-coils. Unlike α-helices, however, the bundling of collagen triple helices is very poorly understood with almost no direct experimental data available. In order to shed light on this critical step of collagen hierarchical assembly, we have examined the collagenous region of complement component 1q. Thirteen synthetic peptides were prepared to dissect the critical regions allowing for its octadecameric self-assembly. We find that short peptides (under 40 amino acids) are able to self-assemble into specific (ABC)6 octadecamers. This requires the ABC heterotrimeric composition as the self-assembly subunit, but does not require disulfide bonds. Self-assembly into this octadecamer is aided by short noncollagenous sequences at the N-terminus, although they are not entirely required. The mechanism of self-assembly appears to begin with the very slow formation of the ABC heterotrimeric helix, followed by rapid bundling of triple helices into progressively larger oligomers, terminating in the formation of the (ABC)6 octadecamer. Cryo-electron microscopy reveals the (ABC)6 assembly as a remarkable, hollow, crown-like structure with an open channel approximately 18 Å at the narrow end and 30 Å at the wide end. This work helps to illuminate the structure and assembly mechanism of a critical protein in the innate immune system and lays the groundwork for the de novo design of higher order collagen mimetic peptide assemblies.
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Colágeno , Peptídeos , Sequência de Aminoácidos , Microscopia Crioeletrônica , Peptídeos/química , Colágeno/química , Conformação Proteica em alfa-HéliceRESUMO
3D printing has become one of the primary fabrication strategies used in biomedical research. Recent efforts have focused on the 3D printing of hydrogels to create structures that better replicate the mechanical properties of biological tissues. These pose a unique challenge, as soft materials are difficult to pattern in three dimensions with high fidelity. Currently, a small number of biologically derived polymers that form hydrogels are frequently reused for 3D printing applications. Thus, there exists a need for novel hydrogels with desirable biological properties that can be used as 3D printable inks. In this work, the printability of multidomain peptides (MDPs), a class of self-assembling peptides that form a nanofibrous hydrogel at low concentrations, is established. MDPs with different charge functionalities are optimized as distinct inks and are used to create complex 3D structures, including multi-MDP prints. Additionally, printed MDP constructs are used to demonstrate charge-dependent differences in cellular behavior in vitro. This work presents the first time that self-assembling peptides have been used to print layered structures with overhangs and internal porosity. Overall, MDPs are a promising new class of 3D printable inks that are uniquely peptide-based and rely solely on supramolecular mechanisms for assembly.
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Hidrogéis , Nanofibras , Hidrogéis/química , Nanofibras/química , Porosidade , Impressão Tridimensional , Peptídeos/química , Engenharia Tecidual/métodosRESUMO
Recently, there has been increased interest in using mannan as an immunomodulatory bioconjugate. Despite notable immunological and functional differences between the reduced (R-Man) and oxidized (O-Man) forms of mannan, little is known about the impact of mannan oxidation state on its in vivo persistence or its potential controlled release from biomaterials that may improve immunotherapeutic or prophylactic efficacy. Here, we investigate the impact of oxidation state on the in vitro and in vivo release of mannan from a biocompatible and immunostimulatory multidomain peptide hydrogel, K2(SL)6K2 (abbreviated as K2), that has been previously used for the controlled release of protein and small molecule payloads. We observed that O-Man released more slowly from K2 hydrogels in vitro than R-Man. In vivo, the clearance of O-Man from K2 hydrogels was slower than O-Man alone. We attributed the slower release rate to the formation of dynamic imine bonds between reactive aldehyde groups on O-Man and the lysine residues on K2. This imine interaction was also observed to improve K2 + O-Man hydrogel strength and shear recovery without significantly influencing secondary structure or peptide nanofiber formation. There were no observed differences in the in vivo release rates of O-Man loaded in K2, R-Man loaded in K2, and R-Man alone. These data suggest that, after subcutaneous injection, R-Man naturally persists longer in vivo than O-Man and minimally interacts with the peptide hydrogel. These results highlight a potentially critical, but previously unreported, difference in the in vivo behavior of O-Man and R-Man and demonstrate that K2 can be used to normalize the release of O-Man to that of R-Man. Further, since K2 itself is an adjuvant, a combination of O-Man and K2 could be used to enhance the immunostimulatory effects of O-Man for applications such as infectious disease vaccines and cancer immunotherapy.
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Nanofibras , Humanos , Nanofibras/química , Mananas , Preparações de Ação Retardada , Hidrogéis/química , Peptídeos/químicaRESUMO
Thermogelling hydrogels based on poly(N-isopropyl acrylamide) (p[NiPAAm]) and crosslinked with a peptide-bearing macromer poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) were fabricated to assess the role of hydrogel charge and lower critical solution temperature (LCST) over time in influencing cellular infiltration and tissue integration in an ex vivo cartilage explant model over 21 days. The p(NiPAAm)-based thermogelling polymer was synthesized to possess 0, 5, and 10 mol% dimethyl-γ-butyrolactone acrylate (DBA) to raise the LCST over time as the lactone rings hydrolyzed. Further, three peptides were designed to impart charge into the hydrogels via conjugation to the PdBT crosslinker. The positively, neutrally, and negatively charged peptides K4 (+), zwitterionic K2E2 (0), and E4 (-), respectively, were conjugated to the modular PdBT crosslinker and the hydrogels were evaluated for their thermogelation behavior in vitro before injection into the cartilage explant models. Samples were collected at days 0 and 21, and tissue integration and cellular infiltration were assessed via mechanical pushout testing and histology. Negatively charged hydrogels whose LCST changed over time (10 mol% DBA) were demonstrated to promote the greatest tissue integration when compared to the positive and neutral gels of the same thermogelling polymer formulation due to increased transport and diffusion across the hydrogel-tissue interface. Indeed, the negatively charged thermogelling polymer groups containing 5 and 10 mol% DBA demonstrated cellular infiltration and cartilage-like matrix deposition via histology. This study demonstrates the important role that material physicochemical properties play in dictating cell and tissue behavior and can inform future cartilage tissue engineering strategies.
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Cartilagem , Hidrogéis , Hidrogéis/farmacologia , Hidrogéis/química , Temperatura , Engenharia Tecidual , Polietilenoglicóis/química , Polímeros/química , Peptídeos/químicaRESUMO
Cation-π interactions play a significant role in the stabilization of globular proteins. However, their role in collagen triple helices is less well understood and they have rarely been used in de novo designed collagen mimetic systems. In this study, we analyze the stabilizing and destabilizing effects in pairwise amino acid interactions between cationic and aromatic residues in both axial and lateral sequential relationships. Thermal unfolding experiments demonstrated that only axial pairs are stabilizing, while the lateral pairs are uniformly destabilizing. Molecular dynamics simulations show that pairs with an axial relationship can achieve a near-ideal interaction distance, but pairs in a lateral relationship do not. Arginine-π systems were found to be more stabilizing than lysine-π and histidine-π. Arginine-π interactions were then studied in more chemically diverse ABC-type heterotrimeric helices, where arginine-tyrosine pairs were found to form the best helix. This work helps elucidate the role of cation-π interactions in triple helices and illustrates their utility in designing collagen mimetic peptides.
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Arginina , Colágeno , Estrutura Secundária de Proteína , Modelos Moleculares , Cátions/química , Colágeno/químicaRESUMO
Adjuvants play a critical role in enhancing vaccine efficacy; however, there is a need to develop new immunomodulatory compounds to address emerging pathogens and to expand the use of immunotherapies. Multidomain peptides (MDPs) are materials composed of canonical amino acids that form injectable supramolecular hydrogels under physiological salt and pH conditions. MDP hydrogels are rapidly infiltrated by immune cells in vivo and have previously been shown to influence cytokine production. Therefore, we hypothesized that these immunostimulatory characteristics would allow MDPs to function as vaccine adjuvants. Herein, we demonstrate that loading antigen into MDP hydrogels does not interfere with their rheological properties and that positively charged MDPs can act as antigen depots, as demonstrated by their ability to release ovalbumin (OVA) over a period of 7-9 days in vivo. Mice vaccinated with MDP-adjuvanted antigen generated significantly higher IgG titers than mice treated with the unadjuvanted control, suggesting that these hydrogels potentiate humoral immunity. Interestingly, MDP hydrogels did not elicit a robust cellular immune response, as indicated by the lower production of IgG2c and smaller populations of tetramer-positive CD8+ T splenocytes compared to mice vaccinated alum-adjuvanted OVA. Together, the data suggest that MDP hydrogel adjuvants strongly bias the immune response towards humoral immunity while evoking a very limited cellular immune response. As a result, MDPs may have the potential to serve as adjuvants for applications that benefit exclusively from humoral immunity.
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Hidrogéis , Imunidade Humoral , Camundongos , Animais , Ovalbumina , Adjuvantes Imunológicos/química , Antígenos , Peptídeos , Adjuvantes Farmacêuticos , Imunoglobulina G , Aminoácidos , CitocinasRESUMO
Transected peripheral nerve injury (PNI) affects the quality of life of patients, which leads to socioeconomic burden. Despite the existence of autografts and commercially available nerve guidance conduits (NGCs), the complexity of peripheral nerve regeneration requires further research in bioengineered NGCs to improve surgical outcomes. In this work, we introduce multidomain peptide (MDP) hydrogels, as intraluminal fillers, into electrospun poly(ε-caprolactone) (PCL) conduits to bridge 10 mm rat sciatic nerve defects. The efficacy of treatment groups was evaluated by electromyography and gait analysis to determine their electrical and motor recovery. We then studied the samples' histomorphometry with immunofluorescence staining and automatic axon counting/measurement software. Comparison with negative control group shows that PCL conduits filled with an anionic MDP may improve functional recovery 16 weeks postoperation, displaying higher amplitude of compound muscle action potential, greater gastrocnemius muscle weight retention, and earlier occurrence of flexion contracture. In contrast, PCL conduits filled with a cationic MDP showed the least degree of myelination and poor functional recovery. This phenomenon may be attributed to MDPs' difference in degradation time. Electrospun PCL conduits filled with an anionic MDP may become an attractive tissue engineering strategy for treating transected PNI when supplemented with other bioactive modifications.
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Collagen mimetic peptides (CMPs) are an excellent model to study the structural and biological properties of the extracellular matrix (ECM) due to ease of synthesis and variability in sequence. To ensure that synthetic materials accurately mimic the structure and function of natural collagen in the ECM, it is necessary to conserve the triple helix. However, CMP folding is subject to equilibrium, and frequently peptides exist in solution as both monomer and triple helix. Additionally, the stability of CMPs is highly dependent on peptide length and amino acid composition, leading to suboptimal performance. Here, we report the utility of covalent capture, a method to (a) direct the folding of a supramolecular triple helix and (b) form isopeptide bonds between the helix strands, in the design of an integrin-binding peptide with a GFOGER motif. Covalent capture effectively locked the triple helix and yielded a peptide with high thermal stability and a rapid folding rate. Compared to supramolecular triple helices bearing the same GFOGER-binding site, cell adhesion was substantially increased. In vitro assays using EDTA/Mg2+ and an anti-α2ß1 antibody demonstrated the preservation of the high specificity of the binding event. This covalently captured integrin-binding peptide provides a template for the future design of bioactive ECM mimics, which can overcome limitations of supramolecular approaches for potential drug and biomaterial designs.