Self-assembling Peptide-based Hydrogels Research Articles

A Review on the Rheological Properties of Single Amino Acids and Short Dipeptide Gels.

Self-assembled peptide-based hydrogels have attracted considerable interest from the research community. Particularly, low molecular weight gelators (LMWGs) consisting of amino acids and short peptides are highly suitable for biological applications owing to their facile synthesis and scalability, as well as their biocompatibility, biodegradability, and stability in physiological conditions. However, challenges in understanding the structure-property relationship and lack of design rules hinder the development of new gelators with the required properties for several applications. Hereby, in the plethora of peptide-based gelators, this review discusses the mechanical properties of single amino acid and dipeptide-based hydrogels. A mutual analysis of these systems allows us to highlight the relationship between the gel mechanical properties and amino acid sequence, preparation methods, or N capping groups. Additionally, recent advancements in the tuning of the gels' rheological properties are reviewed. In this way, the present review aims to help bridge the knowledge gap between structure and mechanical properties, easing the selection or design of peptides with the required properties for biological applications.

Effects of amyloid-β-mimicking peptide hydrogel matrix on neuronal progenitor cell phenotype

Self-assembling peptide-based hydrogels have become a highly attractive scaffold for three-dimensional (3D) in vitro disease modeling as they provide a way to create tunable matrices that can resemble the extracellular matrix (ECM) of various microenvironments. Alzheimer's disease (AD) is an exceptionally complex neurodegenerative condition; however, our understanding has advanced due to the transition from two-dimensional (2D) to 3D in vitro modeling. Nonetheless, there is a current gap in knowledge regarding the role of amyloid structures, and previously developed models found long-term difficulty in creating an appropriate model involving the ECM and amyloid aggregates. In this report, we propose a multi-component self-assembling peptide-based hydrogel scaffold to mimic the amyloid-beta (β) containing microenvironment. Characterization of the amyloid-β-mimicking hydrogel (Col-HAMA-FF) reveals the formation of β-sheet structures as a result of the self-assembling properties of phenylalanine (Phe, F) through π−π stacking of the residues, thus mimicking the amyloid-β protein nanostructures. We investigated the effect of the amyloid-β-mimicking microenvironment on healthy neuronal progenitor cells (NPCs) compared to a natural-mimicking matrix (Col-HAMA). Our results demonstrated higher levels of neuroinflammation and apoptosis markers when NPCs were cultured in the amyloid-like matrix compared to a natural brain matrix. Here, we provided insights into the impact of amyloid-like structures on NPC phenotypes and behaviors. This foundational work, before progressing to more complex plaque models, provides a promising scaffold for future investigations on AD mechanisms and drug testing. Statement of significanceIn this study, we engineered two multi-component hydrogels: one to mimic the natural extracellular matrix (ECM) of the brain and one to resemble an amyloid-like microenvironment using a self-assembling peptide hydrogel. The self-assembling peptide mimics β-amyloid fibrils seen in amyloid-β protein aggregates. We report on the culture of neuronal progenitor cells within the amyloid-mimicking ECM scaffold to study the impact through marker expressions related to inflammation and DNA damage. This foundational work, before progressing to more complex plaque models, offers a promising scaffold for future investigations on AD mechanisms and drug testing.

Nanofibrous MultiDomain Peptide Hydrogels Provide T Cells a 3D, Cytocompatible Environment for Cell Expansion and Antigen-Specific Killing.

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.

Unveiling the Role of Capping Groups in Naphthalene N-Capped Dehydrodipeptide Hydrogels.

Self-assembled peptide-based hydrogels are archetypical nanostructured materials with a plethora of foreseeable applications in nanomedicine and as biomaterials. N-protected di- and tri-peptides are effective minimalist (molecular) hydrogelators. Independent variation of the capping group, peptide sequence and side chain modifications allows a wide chemical space to be explored and hydrogel properties to be tuned. In this work, we report the synthesis of a focused library of dehydrodipeptides N-protected with 1-naphthoyl and 2-naphthylacetyl groups. The 2-naphthylacetyl group was extensively reported for preparation of peptide-based self-assembled hydrogels, whereas the 1-naphthaloyl group was largely overlooked, owing presumably to the lack of a methylene linker between the naphthalene aromatic ring and the peptide backbone. Interestingly, dehydrodipeptides N-capped with the 1-naphthyl moiety afford stronger gels, at lower concentrations, than the 2-naphthylacetyl-capped dehydrodipeptides. Fluorescence and circular dichroism spectroscopy showed that the self-assembly of the dehydrodipeptides is driven by intermolecular aromatic π-π stacking interactions. Molecular dynamics simulations revealed that the 1-naphthoyl group allows higher order aromatic π-π stacking of the peptide molecules than the 2-naphthylacetyl group, together with hydrogen bonding of the peptide scaffold. The nanostructure of the gel networks was studied by TEM and STEM microscopy and was found to correlate well with the elasticity of the gels. This study contributes to understanding the interplay between peptide and capping group structure on the formation of self-assembled low-molecular-weight peptide hydrogels. Moreover, the results presented here add the 1-naphthoyl group to the palette of capping groups available for the preparation of efficacious low-molecular-weight peptide-based hydrogels.

Mechanisms and influencing factors of peptide hydrogel formation and biomedicine applications of hydrogels.

Self-assembled peptide-based hydrogels have shown great potential in bio-related applications due to their porous structure, strong mechanical stability, high biocompatibility, and easy functionalization. Herein, the structure and characteristics of hydrogels and the mechanism of action of several regular secondary structures during gelation are investigated. The factors influencing the formation of peptide hydrogels, especially the pH responsiveness and salt ion induction are analyzed and summarized. Finally, the biomedical applications of peptide hydrogels, such as bone tissue engineering, cell culture, antigen presentation, antibacterial materials, and drug delivery are reviewed.

Sensing Cells-Peptide Hydrogel Interaction In Situ via Scanning Ion Conductance Microscopy.

Peptide-based hydrogels were shown to serve as good matrices for 3D cell culture and to be applied in the field of regenerative medicine. The study of the cell-matrix interaction is important for the understanding of cell attachment, proliferation, and migration, as well as for the improvement of the matrix. Here, we used scanning ion conductance microscopy (SICM) to study the growth of cells on self-assembled peptide-based hydrogels. The hydrogel surface topography, which changes during its formation in an aqueous solution, were studied at nanoscale resolution and compared with fluorescence lifetime imaging microscopy (FLIM). Moreover, SICM demonstrated the ability to map living cells inside the hydrogel. A zwitterionic label-free pH nanoprobe with a sensitivity > 0.01 units was applied for the investigation of pH mapping in the hydrogel to estimate the hydrogel applicability for cell growth. The SICM technique that was applied here to evaluate the cell growth on the peptide-based hydrogel can be used as a tool to study functional living cells.

Tunable Self-Assembled Peptide Hydrogel Sensor for Pharma Cold Supply Chain.

Defrost sensors are a crucial element for proper functioning of the pharmaceutical cold chain. In this paper, the self-assembled peptide-based hydrogels were used to construct a sensitive defrost sensor for the transportation and storage of medications and biomaterials. The turbidity of the peptide hydrogel was employed as a marker of the temperature regime. The gelation kinetics under different conditions was studied to detect various stages of hydrogel structural transitions aimed at tuning the system properties. The developed sensor can be stored at room temperature for a long period, irreversibly indicates whether the product has been thawed, and can be adjusted to a specific temperature range and detection time.

Co-assembled C13-dipeptide hydrogels by Gallic Acid (CA) and epigallocatechin gallate (EGCG) with antibacterial activity

The self-assembled peptide-based hydrogels have been widely utilized in foods. However, the self-assembled hydrogels still have some shortages such as low mechanical properties and hydrogel stabilities. Herein, we designed two co-assembled C13-WS hydrogels conjugated by Gallic Acid (CA) and Epigallocatechin Gallate (EGCG). The introduction of CA and EGCG into C13-WS hydrogels can significantly improve the mechanical properties and hydrogel stabilities. The storage modulus (G’) values of co-assembled C13-WS/CA and C13-WS/EGCG were 13.33 KPa and 16 KPa, respectively. From transmission electron microscope (TEM) results, CA and EGCG contributed to the rearrangement of C13-WS molecules during co-assembly. Moreover, the self-/co-assembled C13-WS hydrogels showed antimicrobial activity against E. coli, P. aeruginosa, and S. epidermidis. The introduction of CA and EGCG significantly enhanced the antibacterial activity of C13-WS hydrogels. Our findings provide a novel strategy for the development of stable supramolecular antibacterial short peptide hydrogels through co-assembly.

Controlling Doxorubicin Release from a Peptide Hydrogel through Fine-Tuning of Drug-Peptide Fiber Interactions.

Hydrogels are versatile materials that have emerged in the last few decades as promising candidates for a range of applications in the biomedical field, from tissue engineering and regenerative medicine to controlled drug delivery. In the drug delivery field, in particular, they have been the subject of significant interest for the spatially and temporally controlled delivery of anticancer drugs and therapeutics. Self-assembling peptide-based hydrogels, in particular, have recently come to the fore as potential candidate vehicles for the delivery of a range of drugs. In order to explore how drug–peptide interactions influence doxorubicin (Dox) release, five β-sheet-forming self-assembling peptides with different physicochemical properties were used for the purpose of this study, namely: FEFKFEFK (F8), FKFEFKFK (FK), FEFEFKFE (FE), FEFKFEFKK (F8K), and KFEFKFEFKK (KF8K) (F: phenylalanine; E: glutamic acid; K: lysine). First, Dox-loaded hydrogels were characterized to ensure that the incorporation of the drug did not significantly affect the hydrogel properties. Subsequently, Dox diffusion out of the hydrogels was investigated using UV absorbance. The amount of drug retained in F8/FE composite hydrogels was found to be directly proportional to the amount of charge carried by the peptide fibers. When cation−π interactions were used, the position and number of end-lysine were found to play a key role in the retention of Dox. In this case, the amount of Dox retained in F8/KF8K composite hydrogels was linked to the amount of end-lysine introduced, and an end-lysine/Dox interaction stoichiometry of 3/1 was obtained. For pure FE and KF8K hydrogels, the maximum amount of Dox retained was also found to be related to the overall concentration of the hydrogels and, therefore, to the overall fiber surface area available for interaction with the drug. For 14 mM hydrogel, ∼170–200 μM Dox could be retained after 24 h. This set of peptides also showed a broad range of susceptibilities to enzymatic degradation opening the prospect of being able to control also the rate of degradation of these hydrogels. Finally, the Dox released from the hydrogel was shown to be active and affect 3T3 mouse fibroblasts viability in vitro. Our study clearly shows the potential of this peptide design as a platform for the formulation of injectable or sprayable hydrogels for controlled drug delivery.

Self-Assembling Peptide-Based Hydrogels for Wound Tissue Repair.

Wound healing is a long‐term, multistage biological process that includes hemostasis, inflammation, proliferation, and tissue remodeling and requires intelligent designs to provide comprehensive and convenient treatment. The complexity of wounds has led to a lack of adequate wound treatment materials, which must systematically regulate unique wound microenvironments. Hydrogels have significant advantages in wound treatment due to their ability to provide spatiotemporal control over the wound healing process. Self‐assembling peptide‐based hydrogels are particularly attractive due to their innate biocompatibility and biodegradability along with additional advantages including ligand‐receptor recognition, stimulus‐responsive self‐assembly, and the ability to mimic the extracellular matrix. The ability of peptide‐based materials to self‐assemble in response to the physiological environment, resulting in functionalized microscopic structures, makes them conducive to wound treatment. This review introduces several self‐assembling peptide‐based systems with various advantages and emphasizes recent advances in self‐assembling peptide‐based hydrogels that allow for precise control during different stages of wound healing. Moreover, the development of multifunctional self‐assembling peptide‐based hydrogels that can regulate and remodel the wound immune microenvironment in wound therapy with spatiotemporal control has also been summarized. Overall, this review sheds light on the future clinical and practical applications of self‐assembling peptide‐based hydrogels.

The Effect of RADA16-I and CDNF on Neurogenesis and Neuroprotection in Brain Ischemia-Reperfusion Injury.

Scaffold materials, neurotrophic factors, and seed cells are three elements of neural tissue engineering. As well-known self-assembling peptide-based hydrogels, RADA16-I and modified peptides are attractive matrices for neural tissue engineering. In addition to its neuroprotective effects, cerebral dopamine neurotrophic factor (CDNF) has been reported to promote the proliferation, migration, and differentiation of neural stem cells (NSCs). However, the role of RADA16-I combined with CDNF on NSCs remains unknown. First, the effect of RADA16-I hydrogel and CDNF on the proliferation and differentiation of cultured NSCs was investigated. Next, RADA16-I hydrogel and CDNF were microinjected into the lateral ventricle (LV) of middle cerebral artery occlusion (MCAO) rats to activate endogenous NSCs. CDNF promoted the proliferation of NSCs, while RADA16-I induced the neural differentiation of NSCs in vitro. Importantly, both RADA16-I and CDNF promoted the proliferation, migration, and differentiation of endogenous NSCs by activating the ERK1/2 and STAT3 pathways, and CDNF exerted an obvious neuroprotective effect on brain ischemia-reperfusion injury. These findings provide new information regarding the application of the scaffold material RADA16-I hydrogel and the neurotrophic factor CDNF in neural tissue engineering and suggest that RADA16-I hydrogel and CDNF microinjection may represent a novel therapeutic strategy for the treatment of stroke.

Cationic self-assembled peptide-based molecular hydrogels for extended ocular drug delivery

The physiological barriers and clearance mechanism of the eye challenge the therapeutic delivery for treating various ocular disorders effectively. Here, we show the use of a cationic peptide (i.e., Nap-FFKK) as the molecular hydrogelator for generating supramolecular hydrogels spontaneously in a pH value of 5-7 which allows it to function as a promising ocular drug vehicle. The cationic peptide-based hydrogel hardly exhibited the cytotoxicity against human corneal epithelial cell (i.e., HCEC) from the in vitro cytotoxicity assay. Moreover, the single topical instillation of the hydrogel resulted in high ocular tolerance and biocompatibility. In vivo corneal distribution of the cationic peptide-based hydrogel showed that it dramatically increased the retention and the adhesion on the surface of cornea, compared to the anionic peptide-based analogue, owing to the ionic interactions with mucin on the ocular surface. In addition, we also synthesized environment-sensitive fluorophore-conjugated analogues (i.e., NBD-FFKK and NBD-FFD) to visualize the uptake of hydrogels in HCEC cells, revealing that the cationic peptide-based hydrogel displayed the better in vitro cellular uptake than the anionic peptide-based hydrogel. More importantly, the resulting cationic Nap-FFKK supramolecular hydrogel displayed a superior ocular bioavailability over that of anionic Nap-FFD supramolecular hydrogel, as indicated by in vivo pharmacokinetics study. This work, as a systematic investigation of ionic peptide-based molecular hydrogels in the ocular application, illustrates a new and powerful supramolecular approach for antagonizing clinically difficult ocular drug delivery. Statement of significanceHere we show the use of a cationic peptide as the molecular hydrogelator for generating supramolecular hydrogels, which allows it to function as a promising ocular drug vehicle for antagonizing the therapeutic delivery difficulties associated with the physiological barriers and clearance mechanism of the eye. The in vitro and in vivo studies of the hydrogel both show high ocular tolerance and biocompatibility. Moreover, the in vivo corneal distribution of the hydrogel exhibits the increased retention and adhesion on the surface of cornea. This work, as an investigation of cationic peptide-based molecular hydrogels in the ocular application, illustrates a powerful supramolecular approach for overcoming clinically difficult ocular drug delivery.

Supramolecular ultra-short carboxybenzyl-protected dehydropeptide-based hydrogels for drug delivery

Self-assembled peptide-based hydrogels are promising materials for biomedical research owing to biocompatibility and similarity to the extracellular matrix, amenable synthesis and functionalization and structural tailoring of the rheological properties. Wider developments of self-assembled peptide-based hydrogels in biomedical research and clinical translation are hampered by limited commercial availability allied to prohibitive costs.In this work a focused library of Cbz-protected dehydrodipeptides Cbz-L-Xaa-Z-ΔPhe-OH (Xaa= Met, Phe, Tyr, Ala, Gly) was synthesised and evaluated as minimalist hydrogels. The Cbz-L-Met-Z-ΔPhe-OH and Cbz-L-Phe-Z-ΔPhe-OH hydrogelators were comprehensively evaluated regarding molecular aggregation and self-assembly, gelation, biocompatibility and as drug carriers for delivery of the natural compound curcumin and the clinically important antitumor drug doxorubicin. Drug release profiles and FRET studies of drug transport into small unilamellar vesicles (as biomembrane models) demonstrated that the Cbz-protected dehydropeptide hydrogels are effective nanocarriers for drug delivery. The expedite and scalable synthesis (in 3 steps), using commercially available reagents and amenable reaction conditions, makes Cbz-protected dehydrodipeptide hydrogels, widely available at affordable cost to the research community.

Cationic Self-Assembled Peptide-Based Molecular Hydrogels for Extended Ocular Drug Delivery

Cationic Self-Assembled Peptide-Based Molecular Hydrogels for Extended Ocular Drug Delivery

Self-Assembling Peptide-Based Hydrogels in Angiogenesis.

Ischemic diseases, especially in the heart and the brain, have become a serious threat to human health. Growth factor and cell therapy are emerging as promising therapeutic strategies; however, their retention and sustainable functions in the injured tissue are limited. Self-assembling peptide (SAP)-based hydrogels, mimicking the extracellular matrix, are therefore introduced to encapsulate and controllably release cells, cell-derived exosomes or growth factors, thus promoting angiogenesis and tissue recovery after ischemia. We will summarize the classification, composition and structure of SAPs, and the influencing factors for SAP gelation. Moreover, we will describe the functionalized SAPs, and the combinatorial therapy of cells, exosomes or growth factors with functionalized SAPs for angiogenic process as well as its advantage in immunogenicity and injectability. Finally, an outlook on future directions and challenges is provided.

On the Mechanism of Self-Assembly by a Hydrogel-Forming Peptide

Self-assembling peptide-based hydrogels are a class of tunable soft materials that have been shown to be highly useful for a number of biomedical applications. The dynamic formation of the supramolecular fibrils that compose these materials has heretofore remained poorly characterized. A better understanding of this process would provide important insights into the behavior of these systems and could aid in the rational design of new peptide hydrogels. Here, we report the determination of the microscopic steps that underpin the self-assembly of a hydrogel-forming peptide, SgI37-49. Using theoretical models of linear polymerization to analyze the kinetic self-assembly data, we show that SgI37-49 fibril formation is driven by fibril-catalyzed secondary nucleation and that all the microscopic processes involved in SgI37-49 self-assembly display an enzyme-like saturation behavior. Moreover, this analysis allows us to quantify the rates of the underlying processes at different peptide concentrations and to calculate the time evolution of these reaction rates over the time course of self-assembly. We demonstrate here a new mechanistic approach for the study of self-assembling hydrogel-forming peptides, which is complementary to commonly used materials science characterization techniques.

Supramolecular Nanofibrous Peptide/Polymer Hydrogels for the Multiplexing of Bioactive Signals.

The ability to provide multiple functions within a single scaffold biomaterial is a major goal in tissue engineering. Self-assembling peptide-based hydrogels are gaining significant attention as three-dimensional biomaterials because they provide a network of nanofibers similar to the native extracellular matrix while allowing the presentation of multiple biochemical cues for cell signaling. Herein, we combine a positively charged peptide amphiphile (PA) and the negatively charged synthetic polymer poly(sodium 4-styrenesulfonate) (PSS) to fabricate hybrid hydrogels through supramolecular self-assembly. PSS/PA hydrogels show rather high mechanical stiffness while being stable in buffered environment. The sulfonate functionality in PSS promotes hydrogel mineralization which can be controlled if undertaken in standard osteogenic medium. Loading proteins with different charges in the hydrogels reveals their ability to retain and sustain their release and indicates their potential for the controlled delivery of growth factors. Human mesenchymal stem cells encapsulated in PSS/PA hydrogels remain viable. The biomimetic nanofibrous structure of the hydrogels, together with multiplexing of bioactive signals, can provide a suitable environment for stem cell differentiation.

Enhancement of the Stability and Anti-DPPIV Activity of Hempseed Hydrolysates Through Self-Assembling Peptide-Based Hydrogels.

Although there is an increasing interest for bioactive food protein hydrolysates as valuable ingredients for functional food and dietary supplement formulations, their potential applications are hampered by their insufficient stability in physiological conditions. In this study, an innovative strategy based on nanomaterials was developed in order to increase the hempseed hydrolysate stability and the anti-diabetic properties, through their encapsulation into ionic self-complementary RADA16 peptide based-hydrogels. Atomic force microscope (AFM) morphological analysis indicated that the new nanomaterials were composed of a nanofibril network, whose increased diameter in respect to native RADA16 suggests the presence of transient non-covalent interactions among the RADA16 supramolecular assemblies and the embedded hempseed peptides. Structural analysis by FT-IR spectroscopy indicated typical β-sheet signatures. The RADA16-hempseed protein hydrolysate hydrogel was shown to act as a novel dipeptidyl peptidase IV (DPPIV) inhibitor in different biological assays. Finally, this nanoformulation was used as a drug delivery system of the anti-diabetic drug sitagliptin, helping to reduce its dosage and eventually associated side-effects.

Recent advances of self-assembling peptide-based hydrogels for biomedical applications.

Peptide-based hydrogels have been proven to be preeminent biomedical materials due to their high water content, tunable mechanical stability, great biocompatibility and excellent injectability. The ability of peptide-based hydrogels to provide extracellular matrix-mimicking environments opens up opportunities for their biomedical applications in fields such as drug delivery, tissue engineering, and wound healing. In this review, we first describe several methods commonly used for the fabrication of robust peptide-based hydrogels, including spontaneous hydrogelation, enzyme-controlled hydrogelation and cross-linking-enhanced hydrogelation. We then introduce some representative studies on their applications in drug delivery and antitumor therapy, antimicrobial and wound healing materials, and 3D bioprinting and tissue engineering. We hope that this review facilitates the advances of hydrogels in biomedical applications.

Synthesis of Self-Assembling Peptide-Based Hydrogels for Regenerative Medicine Using Solid-Phase Peptide Synthesis.

Peptide self-assembly is an important field in biomaterials in which short peptides are designed to aggregate into nanostructures that often form hydrogels. These peptides are typically made using solid-phase peptide synthesis (SPPS), a technique in which amino acids are added sequentially to a growing chain. This technique has been used to synthesize peptides with more than 100 amino acids. However, self-assembling peptides are designed to aggregate in solution, which often reduces coupling efficiency during synthesis and makes purification more difficult. Here, an outline of solid-phase peptide synthesis is provided, along with steps that can be used to improve the synthetic yield and purification of self-assembling peptides for regenerative medicine applications.