Peptide-based self-assembled monolayers (SAMs): what peptides can do for SAMs and vice versa.

Self-assembled monolayers (SAMs) represent highly ordered molecular materials with versatile biochemical features and multidisciplinary applications. Research on SAMs has made much progress since the early begginings of Au substrates and alkanethiols, and numerous examples of peptide-displaying SAMs can be found in the literature. Peptides, presenting increasing structural complexity, stimuli-responsiveness, and biological relevance, represent versatile functional components in SAMs-based platforms. This review examines the major findings and progress made on the use of peptide building blocks displayed as part of SAMs with specific functions, such as selective cell adhesion, migration and differentiation, biomolecular binding, advanced biosensing, molecular electronics, antimicrobial, osteointegrative and antifouling surfaces, among others. Peptide selection and design, functionalisation strategies, as well as structural and functional characteristics from selected examples are discussed. Additionally, advanced fabrication methods for dynamic peptide spatiotemporal presentation are presented, as well as a number of characterisation techniques. All together, these features and approaches enable the preparation and use of increasingly complex peptide-based SAMs to mimic and study biological processes, and provide convergent platforms for high throughput screening discovery and validation of promising therapeutics and technologies.

Peptide Amphiphile Hydrogels Based on Homoternary Cucurbit[8]uril Host-Guest Complexes.

Supramolecular hydrogels based on peptide amphiphiles (PAs) are promising materials for tissue engineering and model extracellular matrixes for biological studies. While PA hydrogels are conventionally formed via electrostatic screening, new hydrogelation mechanisms might help to improve the design and functionality of these materials. Here, we present a host-guest-mediated PA hydrogelation method that relies on the formation of a host-guest homoternary complex with cucurbit[8]uril (CB[8]) and aromatic amino-acid-bearing PA nanofibers. As a result of the host-guest cross-linking between PA nanofibers, hierarchical morphologies and increased stiffness were found when host-guest-mediated PA hydrogels were compared to their ion-based equivalents. Additionally, both families of hydrogels exhibited similar biocompatibilities. These results demonstrate that CB[8]-mediated hydrogelation can be used as an alternative cross-linking method to upgrade the design of PA materials and extend their biomedical applications.

Unravelling the Enzymatic Degradation Mechanism of Supramolecular Peptide Nanofibers and Its Correlation with Their Internal Viscosity.

Enzyme-responsive supramolecular peptide biomaterials have attracted growing interest for disease diagnostics and treatments. However, it remains unclear whether enzymes target the peptide assemblies or dissociated peptide monomers. To gain further insight into the degradation mechanism of supramolecular peptide amphiphile (PA) nanofibers, cathepsin B with both exopeptidase and endopeptidase activities was exploited here for degradation studies. Hydrolysis was found to occur directly on the PA nanofibers as only surface amino acid residues were cleaved. The number of cleaved residues and the degradation efficiency was observed to be negatively correlated with the internal viscosity of the PA nanofibers, quantified to be between 200-800 cP (liquid phase) using fluorescence lifetime imaging microscopy combined with an environmentally sensitive molecular rotor, BODIPY-C10. These findings enhance our understanding on the enzymatic degradation of supramolecular PA nanofibers and have important implications for the development of PA probes for the real-time monitoring of disease-related enzymes.

Supramolecular Hydrogels for Protein Delivery in Tissue Engineering.

Therapeutic proteins, such as growth factors (GFs), have been used in tissue engineering (TE) approaches for their ability to provide signals to cells and orchestrate the formation of functional tissue. However, to be effective and minimize off-target effects, GFs should be delivered at the target site with temporal control. In addition, protein drugs are typically sensitive water soluble macromolecules with delicate structure. As such, hydrogels, containing large amounts of water, provide a compatible environment for the direct incorporation of proteins within the hydrogel network, while their release rate can be tuned by engineering the network chemistry and density. Being formed by transient crosslinks, afforded by non-covalent interactions, supramolecular hydrogels offer important advantages for protein delivery applications. This review describes various types of supramolecular hydrogels using a repertoire of diverse building blocks, their use for protein delivery and their further application in TE contexts. By reviewing the recent literature on this topic, the merits of supramolecular hydrogels are highlighted as well as their limitations, with high expectations for new advances they will provide for TE in the near future.

Photoconfigurable, Cell-Remodelable Disulfide Cross-linked Hyaluronic Acid Hydrogels.

Dynamic photoresponsive synthetic hydrogels offer important advantages for biomaterials design, from the ability to cure hydrogels and encapsulate cells in situ to the light-mediated control of cell-spreading and tissue formation. We report the facile and effective photocuring and photoremodeling of disulfide-cross-linked hyaluronic acid hydrogels, based on photo-oxidation of corresponding thiol residues and their radical-mediated photodegradation. We find that the mechanical properties of disulfide hydrogels and the extent of their photoremodeling can be tuned by controlling the photo-oxidation and photodegradation reactions, respectively. This enables not only the photopatterning of the mechanical properties of hydrogels but also their self-healing and photomediated healing. Finally, we demonstrate the ability to encapsulate mesenchymal stromal cells within these materials and to regulate their protrusion and spreading in 3D matrices by controlling the mechanical properties of the disulfide networks. Therefore, synthetically accessible photoconfigurable disulfide hydrogels offer interesting opportunities for the design of soft biomaterials and the regulation of cell encapsulation and matrix remodeling for tissue engineering.

Interaction of Antimicrobial Lipopeptides with Bacterial Lipid Bilayers.

The resistance of pathogens to traditional antibiotics is currently a global issue of enormous concern. As the discovery and development of new antibiotics become increasingly challenging, synthetic antimicrobial lipopeptides (AMLPs) are now receiving renewed attention as a new class of antimicrobial agents. In contrast to traditional antibiotics, AMLPs act by physically disrupting the cell membrane (rather than targeting specific proteins), thus reducing the risk of inducing bacterial resistance. In this study, we use microsecond-timescale atomistic molecular dynamics simulations to quantify the interaction of a short AMLP (C16-KKK) with model bacterial lipid bilayers. In particular, we investigate how fundamental transmembrane properties change in relation to a range of lipopeptide concentrations. A number of structural, mechanical, and dynamical features are found to be significantly altered in a non-linear fashion. At 10 mol% concentration, lipopeptides have a condensing effect on bacterial bilayers, characterized by a decrease in the area per lipid and an increase in the bilayer order. Higher AMLP concentrations of 25 and 40 mol% destabilize the membrane by disrupting the bilayer core structure, inducing membrane thinning and water leakage. Important transmembrane properties such as the lateral pressure and dipole potential profiles are also affected. Potential implications on membrane function and associated proteins are discussed.

Enhancing the Potency of Antimicrobial Peptides through Molecular Engineering and Self-Assembly.

Healthcare-associated infections resulting from bacterial attachment and biofilm formation on medical implants are posing significant challenges in particular with the emergence of bacterial resistance to antibiotics. Here, we report the design, synthesis and characterization of self-assembled nanostructures, which integrate on their surface antibacterial peptides. The antibacterial WMR peptide, which is a modification of the native sequence of the myxinidin, a marine peptide isolated from the epidermal mucus of hagfish, was used considering its enhanced activity against Gram-negative bacteria. WMR was linked to a peptide segment of aliphatic residues (AAAAAAA) containing a lipidic tail (C19H38O2) attached to the ε-amino of a terminal lysine to generate a peptide amphiphile (WMR PA). The self-assembly of the WMR PA alone, or combined with coassembling shorter PAs, was studied using spectroscopy and microscopy techniques. The designed PAs were shown to self-assemble into stable nanofiber structures and these nanoassemblies significantly inhibit biofilm formation and eradicate the already formed biofilms of Pseudomonas aeruginosa (Gram-negative bacteria) and Candida albicans (pathogenic fungus) when compared to the native WMR peptide. Our results provide insights into the design of peptide based supramolecular assemblies with antibacterial activity, and establish an innovative strategy to develop self-assembled antimicrobial materials for biomedical applications.

Mimicking the endothelial glycocalyx through the supramolecular presentation of hyaluronan on patterned surfaces.

The glycocalyx is the immediate pericellular matrix that surrounds many cell types, including endothelial cells (ECs), and is typically composed of glycans (glycosaminoglycans, proteoglycans, and glycoproteins). The endothelial glycocalyx is rich in hyaluronic acid (HA), which plays an important role in the maintenance of vascular integrity, although fundamental questions about the precise molecular regulation mechanisms remain unanswered. Here, we investigate the contribution of HA to the regulation of endothelial function using model surfaces. The peptide sequence GAHWQFNALTVR, previously identified by phage display with strong binding affinity for HA and named Pep-1, was thiolated at the N-terminal to form self-assembled monolayers (SAMs) on gold (Au) substrates, and microcontact printing (µCP) was used to develop patterned surfaces for the controlled spatial presentation of HA. Acetylated Pep-1 and a scrambled sequence of Pep-1 were used as controls. The SAMs and HA-coated surfaces were characterized by X-ray photoelectron spectroscopy (XPS), contact angle measurements, and quartz crystal microbalance with dissipation (QCM-D) monitoring, which confirmed the binding and presence of thiolated peptides on the Au surfaces and the deposition of HA. Fluorescence microscopy showed the localization of fluorescently labelled HA only on areas printed with Pep-1 SAMs. Cell culture studies demonstrated that low molecular weight HA improved the adhesion of human umbilical vein endothelial cells (HUVECs) to the substrate and also stimulated their migration. This research provides insight into the use of SAMs for the controlled presentation of HA with defined size in cultures of HUVECs to study their functions.

Empowering the Potential of Cell-Penetrating Peptides for Targeted Intracellular Delivery via Molecular Self-Assembly.

Cell-penetrating peptides (CPPs) have been widely explored as an effective tool to deliver a variety of molecules and nanoparticles into cells due to their intrinsic property to translocate across cell membranes. CPPs are easier to synthesize and functionalize, and their incorporation into delivery vehicles could be achieved by both non-covalent and covalent methods. Recent advances in molecular self-assembly have demonstrated the possibility to fabricate various nanostructures with precise control over the shape, size and presentation of diverse functionalities. Through rational design, CPPs could be used as a building block for the nanostructure formation via self-assembly, while providing the functionality for intracellular delivery. In this book chapter, we will describe strategies to design self-assembling CPP conjugates and illustrate how their self-assembled nanostructures are manipulated for effective intracellular delivery. Fundamental knowledge on CPPs and molecular self-assembly will also be described.

Dual targeted immunotherapy via in vivo delivery of biohybrid RNAi-peptide nanoparticles to tumour-associated macrophages and cancer cells.

Lung cancer is associated with very poor prognosis and considered one of the leading causes of death worldwide. Here, we present highly potent and selective bio-hybrid RNAi-peptide nanoparticles that can induce specific and long-lasting gene therapy in inflammatory tumour associated macrophages (TAMs), via an immune modulation of the tumour milieu combined with tumour suppressor effects. Our data prove that passive gene silencing can be achieved in cancer cells using regular RNAi NPs. When combined with M2 peptide-based targeted immunotherapy that immuno-modulates TAMs cell-population, a synergistic effect and long-lived tumour eradication can be observed along with increased mice survival. Treatment with low doses of siRNA (ED50 0.0025-0.01 mg/kg) in a multi and long-term dosing system substantially reduced the recruitment of inflammatory TAMs in lung tumour tissue, reduced tumour size (∼95%) and increased animal survival (∼75%) in mice. Our results suggest that it is likely that the combination of silencing important genes in tumour cells and in their supporting immune cells in the tumour microenvironment, such as TAMs, will greatly improve cancer clinical outcomes.

Structuring supramolecular hyaluronan hydrogels via peptide self-assembly for modulating the cell microenvironment.

The use of synthetic extracellular matrices (ECMs) in fundamental in vitro cell culture studies has been instrumental for investigating the interplay between cells and matrix components. To provide cells with a more native environment in vitro, it is desirable to design matrices that are biomimetic and emulate compositional and structural features of natural ECMs. Here, the supramolecular fabrication of peptide-hyaluronan (HA) hydrogels is presented as potential ECM surrogates, combining native HA and rationally designed cationic amphipatic peptides [(KI)nK, lysine (K), isoleucine (I), n â€‹= â€‹2-6] whose mechanical properties and microstructure are tunable by the peptide sequence. (KI)nK peptides adopt ß-sheet configuration and self-assemble into filamentous nanostructures triggered by pH or ionic strength. The self-assembly propensity of (KI)nK peptides increases with the sequence length, forming single phase hydrogels (shorter peptides) or with phase separation (longer peptides) in presence of the anionic polyelectrolyte HA through electrostatic complexations. The gel phase formed in (KI)nK-HA complexes exhibits viscoelastic behavior and triggers the formation of human mesenchymal stem cell (MSC) spheroids which disassemble over the time. It is anticipated that these (KI)nK-HA hydrogels with tunable physical and biochemical properties offer a promising platform for in vitro applications and in stem cell therapy.

Improving cancer immunotherapy via co-delivering checkpoint blockade and thrombospondin-1 downregulator.

The use of checkpoint-blockade antibodies is still restricted in several malignancies due to the modest efficacy, despite considerable success in anti-tumor immunotherapy. The poor response of cancer cells to immune destruction is an essential contributor to the failure of checkpoint therapy. We hypothesized that combining checkpoint therapy with natural-product chemosensitizer could enhance immune response. Herein, a targeted diterpenoid derivative was integrated with the checkpoint blockade (anti-CTLA-4) to improve immunotherapy using thermosensitive liposomes as carriers. In vivo, the liposomes enabled the co-delivery of the two drug payloads into the tumor. Consequently, the regulatory T cell proliferation was restrained, the cytotoxic T cell infiltration was enhanced, and the profound immunotherapeutic effect was achieved. In addition, the immunotherapeutic effect of another clinically used checkpoint antibody, anti-PD-1, also benefited from the diterpenoid derivative. Of note, our mechanism study revealed that the targeted diterpenoid derivative increased the sensitivity of cancer cells to immune attack via THBS1 downregulation and the resultant destruction of THBS1-CD47 interaction. Collectively, co-delivering THBS1 inhibitor and checkpoint blockade is promising to boost cancer immunotherapy. We first time discovered that THBS1 suppression could strengthen checkpoint therapy.

Microfluidic fabrication of self-assembled peptide-polysaccharide microcapsules as 3D environments for cell culture.

We report a mild cell encapsulation method based on self-assembly and microfluidics technology. Xanthan gum, an anionic polysaccharide, was used to trigger the self-assembly of a positively charged multidomain peptide. The self-assembly resulted in the formation of a nanofibrous matrix and using a microfluidic device, microcapsules with homogeneous size were fabricated. The properties and performance of xanthan-peptide microcapsules were optimized by changing peptide/polysaccharide ratio and their effects on the microcapsule permeability and mechanical stability were analyzed. The effect of microcapsule formulation on viability and proliferation of encapsulated chondrocytic (ATDC5) cells was also investigated. The encapsulated cells were metabolically active, showing an increased viability and proliferation over 21 days of in vitro culture, demonstrating the long-term stability of the self-assembled microcapsules and their ability to support and enhance the survival of encapsulated cells over a prolonged time. Self-assembling materials combined with microfluidics demonstrated to be an innovative approach in the fabrication of cytocompatible matrix for cell microencapsulation and delivery.

Embracing complexity in biomaterials design.

Animate materials, man-made materials behaving like living systems, are attracting enormous interest across a range of sectors, from construction and transport industry to medicine. In this leading opinion article, we propose that embracing complexity in biomaterials design offers untapped opportunities to create biomaterials with innovative life-like properties that extend their capabilities and unleash new paradigms in medical treatment.

Targeted delivery of baicalein-p53 complex to smooth muscle cells reverses pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is an uncommon and deadly cardiopulmonary disease. PAH stems essentially from pulmonary artery (PA) remodeling induced predominantly by over-proliferation of PA smooth muscle cells (PASMCs) and inflammation. However, effective treatments are still missing in the clinic because the available drugs consisting of vasodilators are aimed to attenuate PAH symptoms rather than inhibit the remodeling process. Here, we aimed to specifically co-deliver apoptotic executor gene p53 and anti-inflammatory baicalein to PASMCs to alleviate PAH. The targeted co-delivery system was prepared through a carrier-free approach, which was prepared by loading the conjugate, NLS (nuclear localization signal) peptide-p53 gene, onto the baicalein pure crystals, followed by coating with glucuronic acid (GA) for targeting the glucose transport-1 (GLUT-1). The co-delivery system developed has a 200-nm diameter with a rod shape and a drug-loading capacity of 62% (w/w). The prepared system was shown to target PASMCs in vitro and enabled effective gene transfection, efficient apoptosis, and inflammation suppression. In vivo, via targeting the axis lung-PAs-PASMCs, the co-delivery reversed monocrotaline-induced PAH by reducing pulmonary artery pressure, downregulating the proinflammatory cytokine TNF-α, and inhibiting remodeling of both PAs and right ventricular. The potent efficacy may closely correlate with the activation of the signaling axis Bax/Bcl-2/Cas-3. Overall, our results indicate that the co-delivery system holds a significant potential to target the axis of lung-PAs-PASMCs and treat PAH.

Modeling the Tumor Microenvironment of Ovarian Cancer: The Application of Self-Assembling Biomaterials.

Ovarian cancer (OvCa) is one of the leading causes of gynecologic malignancies. Despite treatment with surgery and chemotherapy, OvCa disseminates and recurs frequently, reducing the survival rate for patients. There is an urgent need to develop more effective treatment options for women diagnosed with OvCa. The tumor microenvironment (TME) is a key driver of disease progression, metastasis and resistance to treatment. For this reason, 3D models have been designed to represent this specific niche and allow more realistic cell behaviors compared to conventional 2D approaches. In particular, self-assembling peptides represent a promising biomaterial platform to study tumor biology. They form nanofiber networks that resemble the architecture of the extracellular matrix and can be designed to display mechanical properties and biochemical motifs representative of the TME. In this review, we highlight the properties and benefits of emerging 3D platforms used to model the ovarian TME. We also outline the challenges associated with using these 3D systems and provide suggestions for future studies and developments. We conclude that our understanding of OvCa and advances in materials science will progress the engineering of novel 3D approaches, which will enable the development of more effective therapies.

Polymyxin B-Triggered Assembly of Peptide Hydrogels for Localized and Sustained Release of Combined Antimicrobial Therapy.

Repurposing old antibiotics into more effective and safer formulations is an emergent approach to tackle the growing threat of antimicrobial resistance. Herein, a peptide hydrogel is reported for the localized and sustained release of polymyxin B (PMB), a decade-old antibiotic with increasing clinical utility for treating multidrug-resistant Gram-negative bacterial infections. The hydrogel is assembled by additing PMB solution into a rationally designed peptide amphiphile (PA) solution and its mechanical properties can be adjusted through the addition of counterions, envisioning its application in diverse infection scenarios. Sustained release of PMB from the hydrogel over a 5-day period and prolonged antimicrobial activities against Gram-negative bacteria are observed. The localized release of active PMB from the hydrogel is shown to be effective in vivo for treating Pseudomonas aeruginosa infection in the Galleria mellonella burn wound infection model, dramatically reducing the mortality from 93% to 13%. Complementary antimicrobial activity against Gram-positive Staphylococcus aureus and enhanced antimicrobial effect against the Gram-negative Acinetobacter baumannii are observed when an additional antibiotic fusidic acid is incorporated into the hydrogen network. These results demonstrate the potential of the PMB-triggered PA hydrogel as a versatile platform for the localized and sustained delivery of combined antimicrobial therapies.