Therapeutic Peptides, Proteins and their Nanostructures for Drug Delivery and Precision Medicine.

Peptide and protein nanostructures with tunable structural features, multifunctionality, biocompatibility and biomolecular recognition capacity enable development of efficient targeted drug delivery tools for precision medicine applications. In this review article, we present various techniques employed for the synthesis and self-assembly of peptides and proteins into nanostructures. We discuss design strategies utilized to enhance their stability, drug-loading capacity, and controlled release properties, in addition to the mechanisms by which peptide nanostructures interact with target cells, including receptor-mediated endocytosis and cell-penetrating capabilities. We also explore the potential of peptide and protein nanostructures for precision medicine, focusing on applications in personalized therapies and disease-specific targeting for diagnostics and therapeutics in diseases such as cancer.

Peptide Hydrogels and Nanostructures Controlling Biological Machinery.

Peptides are versatile building blocks for the fabrication of various nanostructures that result in the formation of hydrogels and nanoparticles. Precise chemical functionalization promotes discrete structure formation, causing controlled bioactivity and physical properties for functional materials development. The conjugation of small molecules on amino acid side chains determines their intermolecular interactions in addition to their intrinsic peptide characteristics. Molecular information affects the peptide structure, formation, and activity. In this Perspective, peptide building blocks, nanostructure formation mechanisms, and the properties of these peptide materials are discussed with the results of recent publications. Bioinstructive and stimuli-responsive peptide materials have immense impacts on the nanomedicine field including drug delivery, cellular engineering, regenerative medicine, and biomedicine.

Supramolecular presentation of bioinstructive peptides on soft multilayered nanobiomaterials stimulates neurite outgrowth.

Peptide amphiphiles (PAs) have emerged as effective molecular building blocks for creating self-assembling nanobiomaterials for multiple biomedical applications. Herein, we report a straightforward approach to assemble soft bioinstructive platforms to recreate the native neural extracellular matrix (ECM) aiming for neuronal regeneration based on the electrostatic-driven supramolecular presentation of laminin-derived IKVAV-containing self-assembling PA (IKVAV-PA) on biocompatible multilayered nanoassemblies. Spectroscopic and microscopic techniques show that the co-assembly of positively charged low-molecular-weight IKVAV-PA with oppositely charged high-molecular-weight hyaluronic acid (HA) triggers the formation of ordered ß-sheet structures denoting a one-dimensional nanofibrous network. The successful functionalization of poly(L-lysine)/HA layer-by-layer nanofilms with an outer positively charged layer of self-assembling IKVAV-PA is demonstrated by the quartz crystal microbalance with dissipation monitoring and the nanofibrous morphological properties revealed by atomic force microscopy. The bioactive ECM-mimetic supramolecular nanofilms promote the enhancement of primary neuronal cells' adhesion, viability, and morphology when compared to the PA without the IKVAV sequence and PA-free biopolymeric multilayered nanofilms, and stimulate neurite outgrowth. The nanofilms hold great promise as bioinstructive platforms for enabling the assembly of customized and robust multicomponent supramolecular biomaterials for neural tissue regeneration.

Neuroactive Peptide Nanofibers for Regeneration of Spinal Cord after Injury.

The highly complex nature of spinal cord injuries (SCIs) requires design of novel biomaterials that can stimulate cellular regeneration and functional recovery. Promising SCI treatments use biomaterial scaffolds, which provide bioactive cues to the cells in order to trigger neural regeneration in the spinal cord. In this work, the use of peptide nanofibers is demonstrated, presenting protein binding and cellular adhesion epitopes in a rat model of SCI. The self-assembling peptide molecules are designed to form nanofibers, which display heparan sulfate mimetic and laminin mimetic epitopes to the cells in the spinal cord. These neuroactive nanofibers are found to support adhesion and viability of dorsal root ganglion neurons as well as neurite outgrowth in vitro and enhance tissue integrity after 6 weeks of injury in vivo. Treatment with the peptide nanofiber scaffolds also show significant behavioral improvement. These results demonstrate that it is possible to facilitate regeneration especially in the white matter of the spinal cord, which is usually damaged during the accidents using bioactive 3D nanostructures displaying high densities of laminin and heparan sulfate-mimetic epitopes on their surfaces.

Biotin Functionalized Self-Assembled Peptide Nanofiber as an Adjuvant for Immunomodulatory Response.

Biotinylated peptide amphiphile (Biotin-PA) nanofibers, are designed as a noncovalent binding location for antigens, which are adjuvants to enhance, accelerate, and prolong the immune response triggered by antigens. Presenting antigens on synthetic Biotin-PA nanofibers generated a higher immune response than the free antigens delivered with a cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN) (TLR9 agonist) adjuvant. Antigen attached Biotin-PA nanofibers trigger splenocytes to produce high levels of cytokines (IFN-γ, IL-12, TNF-α, and IL-6) and to exhibit a superior cross-presentation of the antigen. Both Biotin-PA nanofibers and CpG ODN induce a Th-1-biased IgG subclass response; however, delivering the antigen with Biotin-PA nanofibers induce significantly greater production of total IgG and subclasses of IgG compared to delivering the antigen with CpG ODN. Contrary to CpG ODN, Biotin-PA nanofibers also enhance antigen-specific splenocyte proliferation and increase the proportion of the antigen-specific CD8(+) T cells. Given their biodegradability and biocompatibility, Biotin-PA nanofibers have a significant potential in immunoengineering applications as a biomaterial for the delivery of a diverse set of antigens derived from intracellular pathogens, emerging viral diseases such as COVID-19, or cancer cells to induce humoral and cellular immune responses against the antigens.

Peptide gels for controlled release of proteins.

Treatment strategies in clinics have been shifting from small molecules to protein drugs due to the promising results of a highly specific mechanism of action and reduced toxicity. Despite their prominent roles in disease treatment, delivery of the protein therapeutics is challenging due to chemical instability, immunogenicity and biological barriers. Peptide hydrogels with spatiotemporally tunable properties have shown an outstanding potential to deliver complex protein therapeutics, maintain drug efficacy and stability over time, mimicking the extracellular matrix, and responding to external stimuli. In this review, we present recent advances in peptide hydrogel design strategies, protein release kinetics and mechanisms for protein drug delivery in cellular engineering, tissue engineering, immunotherapy and disease treatments.

N-Cadherin Mimetic Peptide Nanofiber System Induces Chondrogenic Differentiation of Mesenchymal Stem Cells.

Cadherins are vital for cell-to-cell interactions during tissue growth, migration, and differentiation processes. Both biophysical and biochemical inputs are generated upon cell-to-cell adhesions, which determine the fate of the mesenchymal stem cells (MSCs). The effect of cadherin interactions on the MSC differentiation still remains elusive. Here we combined the N-Cadherin mimetic peptide (HAV-PA) with the self-assembling E-PA and the resultant N-cadherin mimetic peptide nanofibers promoted chondrogenic differentiation of MSCs in conjunction with chondrogenic factors as a synthetic extracellular matrix system. Self-assembly of the precursor peptide amphiphile molecules HAV-PA and E-PA enable the organization of HAV peptide residues in close proximity to the cell interaction site, forming a supramolecular N-cadherin-like system. These bioactive peptide nanofibers not only promoted viability and enhanced adhesion of MSCs but also augmented the expression of cartilage specific matrix components compared to the nonbioactive control nanofibers. Overall, the N-cadherin mimetic peptide nanofiber system facilitated MSC commitment into the chondrogenic lineage presenting an alternative bioactive platform for stem-cell-based cartilage regeneration.

Dentin Phosphoprotein Mimetic Peptide Nanofibers Promote Biomineralization.

Dentin phosphoprotein (DPP) is a major component of the dentin matrix playing crucial role in hydroxyapatite deposition during bone mineralization, making it a prime candidate for the design of novel materials for bone and tooth regeneration. The bioactivity of DPP-derived proteins is controlled by the phosphorylation and dephosphorylation of the serine residues. Here an enzyme-responsive peptide nanofiber system inducing biomineralization is demonstrated. It closely emulates the structural and functional properties of DPP and facilitates apatite-like mineral deposition. The DPP-mimetic peptide molecules self-assemble through dephosphorylation by alkaline phosphatase (ALP), an enzyme participating in tooth and bone matrix mineralization. Nanofiber network formation is also induced through addition of calcium ions. The gelation process following nanofiber formation produces a mineralized extracellular matrix like material, where scaffold properties and phosphate groups promote mineralization. It is demonstrated that the DPP-mimetic peptide nanofiber networks can be used for apatite-like mineral deposition for bone regeneration.

The design and fabrication of supramolecular semiconductor nanowires formed by benzothienobenzothiophene (BTBT)-conjugated peptides.

π-Conjugated small molecules based on a [1]benzothieno[3,2-b]benzothiophene (BTBT) unit are of great research interest in the development of solution-processable semiconducting materials owing to their excellent charge-transport characteristics. However, the BTBT π-core has yet to be demonstrated in the form of electro-active one-dimensional (1D) nanowires that are self-assembled in aqueous media for potential use in bioelectronics and tissue engineering. Here we report the design, synthesis, and self-assembly of benzothienobenzothiophene (BTBT)-peptide conjugates, the BTBT-peptide (BTBT-C3-COHN-Ahx-VVAGKK-Am) and the C8-BTBT-peptide (C8-BTBT-C3-COHN-Ahx-VVAGKK-Am), as ß-sheet forming amphiphilic molecules, which self-assemble into highly uniform nanofibers in water with diameters of 11-13(±1) nm and micron-size lengths. Spectroscopic characterization studies demonstrate the J-type π-π interactions among the BTBT molecules within the hydrophobic core of the self-assembled nanofibers yielding an electrical conductivity as high as 6.0 × 10-6 S cm-1. The BTBT π-core is demonstrated, for the first time, in the formation of self-assembled peptide 1D nanostructures in aqueous media for potential use in tissue engineering, bioelectronics and (opto)electronics. The conductivity achieved here is one of the highest reported to date in a non-doped state.

Tenascin-C derived signaling induces neuronal differentiation in a three-dimensional peptide nanofiber gel.

The development of new biomaterials mimicking the neuronal extracellular matrix (ECM) requires signals for the induction of neuronal differentiation and regeneration. In addition to the biological and chemical cues, the physical properties of the ECM should also be considered while designing regenerative materials for nervous tissue. In this study, we investigated the influence of the microenvironment on tenascin-C signaling using 2D surfaces and 3D scaffolds generated by a peptide amphiphile nanofiber gel with a tenascin-C derived peptide epitope (VFDNFVLK). While tenascin-C mimetic PA nanofibers significantly increased the length and number of neurites produced by PC12 cells on 2D cell culture, more extensive neurite outgrowth was observed in the 3D gel environment. PC12 cells encapsulated within the 3D tenascin-C mimetic peptide nanofiber gel also exhibited significantly increased expression of neural markers compared to the cells on 2D surfaces. Our results emphasize the synergistic effects of the 3D conformation of peptide nanofibers along with the tenascin-C signaling and growth factors on the neuronal differentiation of PC12 cells, which may further provide more tissue-like morphology for therapeutic applications.

Promotion of neurite outgrowth by rationally designed NGF-ß binding peptide nanofibers.

Promotion of neurite outgrowth is an important limiting step for regeneration in nerve injury and depends strongly on the local expression of nerve growth factor (NGF). The rational design of bioactive materials is a promising approach for the development of novel therapeutic methods for nerve regeneration, and biomaterials capable of presenting NGF to nerve cells are especially suitable for this purpose. In this study, we show bioactive peptide amphiphile (PA) nanofibers capable of promoting neurite outgrowth by displaying high density binding epitopes for NGF. A high-affinity NGF-binding sequence was identified by phage display and combined with a beta-sheet forming motif to produce a self-assembling PA molecule. The bioactive nanofiber had higher affinity for NGF compared to control nanofibers and in vitro studies revealed that the NGF binding peptide amphiphile nanofibers (NGFB-PA nanofiber) significantly promote the neurite outgrowth of PC-12 cells. In addition, the nanofibers induced differentiation of PC-12 cells into neuron-like cells by enhancing NGF/high-activity NGF receptor (TrkA) interactions and activating MAPK pathway elements. The NGFB-PA nanofiber was further shown as a promising material to support axonal outgrowth from primary sensory neurons. These materials will pave the way for the development of new therapeutic agents for peripheral nervous system injuries.

Protective therapeutic effects of peptide nanofiber and hyaluronic acid hybrid membrane in in vivo osteoarthritis model.

Osteoarthritis (OA) is a condition where tissue function is lost through a combination of secondary inflammation and deterioration in articular cartilage. One of the most common causes of OA is age-related tissue impairment because of wear and tear due to mechanical erosion. Hyaluronic acid-based viscoelastic supplements have been widely used for the treatment of knee injuries. However, the current formulations of hyaluronic acid are unable to provide efficient healing and recovery. Here, a nanofiber-hyaluronic acid membrane system that was prepared by using a quarter of the concentration of commercially available hyaluronic acid supplement, Hyalgan®, was used for the treatment of an osteoarthritis model, and Synvisc®, which is another commercially available hyaluronic acid containing viscoelastic supplement, was used as a control. The results show that this system provides efficient protection of arthritic cartilage tissue through the preservation of cartilage morphology with reduced osteophyte formation, protection of the subchondral region from deterioration, and maintenance of cartilage specific matrix proteins in vivo. In addition, the hybrid nanofiber membrane enabled chondrocyte encapsulation and provided a suitable culturing environment for stem cell growth in vitro. Overall, our results suggest that this hybrid nanofibrous scaffold provides a potential platform the treatment of OA. STATEMENT OF SIGNIFICANCE: Osteoarthritis is a debilitating joint disease affecting millions of people worldwide. It occurs especially in knees due to aging, sport injuries or obesity. Although hyaluronic acid-based viscoelastic supplements are widely used, there is still no effective treatment method for osteoarthritis, which necessitates surgical operation as an only choice for severe cases. Therefore, there is an urgent need for efficient therapeutics. In this study, a nanofiber-HA membrane system was developed for the efficient protection of arthritic cartilage tissue from degeneration. This hybrid nanofiber system provided superior therapeutic activity at a relatively lower concentration of hyaluronic acid than Hyalgan® and Synvisc® gels, which are currently used in clinics. This work demonstrates for the first time that this hybrid nanofiber membrane scaffold can be utilized as a potential candidate for osteoarthritis treatment.

Biocompatible Electroactive Tetra(aniline)-Conjugated Peptide Nanofibers for Neural Differentiation.

Peripheral nerve injuries cause devastating problems for the quality of patients' lives, and regeneration following damage to the peripheral nervous system is limited depending on the degree of the damage. Use of nanobiomaterials can provide therapeutic approaches for the treatment of peripheral nerve injuries. Electroactive biomaterials, in particular, can provide a promising cure for the regeneration of nerve defects. Here, a supramolecular electroactive nanosystem with tetra(aniline) (TA)-containing peptide nanofibers was developed and utilized for nerve regeneration. Self-assembled TA-conjugated peptide nanofibers demonstrated electroactive behavior. The electroactive self-assembled peptide nanofibers formed a well-defined three-dimensional nanofiber network mimicking the extracellular matrix of the neuronal cells. Neurite outgrowth was improved on the electroactive TA nanofiber gels. The neural differentiation of PC-12 cells was more advanced on electroactive peptide nanofiber gels, and these biomaterials are promising for further use in therapeutic neural regeneration applications.

Peptide-Based Materials for Cartilage Tissue Regeneration.

Cartilaginous tissue requires structural and metabolic support after traumatic or chronic injuries because of its limited capacity for regeneration. However, current techniques for cartilage regeneration are either invasive or ineffective for long-term repair. Developing alternative approaches to regenerate cartilage tissue is needed. Therefore, versatile scaffolds formed by biomaterials are promising tools for cartilage regeneration. Bioactive scaffolds further enhance the utility in a broad range of applications including the treatment of major cartilage defects. This chapter provides an overview of cartilage tissue, tissue defects, and the methods used for regeneration, with emphasis on peptide scaffold materials that can be used to supplement or replace current medical treatment options.

Multivalent Presentation of Cationic Peptides on Supramolecular Nanofibers for Antimicrobial Activity.

Noncovalent and electrostatic interactions facilitate the formation of complex networks through molecular self-assembly in biomolecules such as proteins and glycosaminoglycans. Self-assembling peptide amphiphiles (PA) are a group of molecules that can form nanofibrous structures and may contain bioactive epitopes to interact specifically with target molecules. Here, we report the presentation of cationic peptide sequences on supramolecular nanofibers formed by self-assembling peptide amphiphiles for cooperative enhanced antibacterial activity. Antibacterial properties of self-assembled peptide nanofibers were significantly higher than soluble peptide molecules with identical amino acid sequences, suggesting that the tandem presentation of bioactive epitopes is important for designing new materials for bactericidal activity. In addition, bacteria were observed to accumulate more rapidly on peptide nanofibers compared to soluble peptides, which may further enhance antibacterial activity by increasing the number of peptide molecules interacting with the bacterial membrane. The cationic peptide amphiphile nanofibers were observed to attach to bacterial membranes and disrupt their integrity. These results demonstrate that short cationic peptides show a significant improvement in antibacterial activity when presented in the nanofiber form.

Three-Dimensional Laminin Mimetic Peptide Nanofiber Gels for In Vitro Neural Differentiation.

The extracellular matrix (ECM) provides biochemical signals and structural support for cells, and its functional imitation is a fundamental aspect of biomaterial design for regenerative medicine applications. The stimulation of neural differentiation by a laminin protein-derived epitope in two-dimensional (2D) and three-dimensional (3D) environments is investigated. The 3D gel system is found to be superior to its 2D counterpart for the induction of neural differentiation, even in the absence of a crucial biological inducer in nerve growth factor (NGF). In addition, cells cultured in 3D gels exhibits a spherical morphology that is consistent with their form under in vivo conditions. Overall, the present study underlines the impact of bioactivity, dimension, and NGF addition, as well as the cooperative effects thereof, on the neural differentiation of PC-12 cells. These results underline the significance of 3D culture systems in the development of scaffolds that closely replicate in vivo environments for the formation of cellular organoid models in vitro.

Supramolecular Peptide Nanofiber Morphology Affects Mechanotransduction of Stem Cells.

Chirality and morphology are essential factors for protein function and interactions with other biomacromolecules. Extracellular matrix (ECM) proteins are also similar to other proteins in this sense; however, the complexity of the natural ECM makes it difficult to study these factors at the cellular level. The synthetic peptide nanomaterials harbor great promise in mimicking specific ECM molecules as model systems. In this work, we demonstrate that mechanosensory responses of stem cells are directly regulated by the chirality and morphology of ECM-mimetic peptide nanofibers with strictly controlled characteristics. Structural signals presented on l-amino acid containing cylindrical nanofibers (l-VV) favored the formation of integrin ß1-based focal adhesion complexes, which increased the osteogenic potential of stem cells through the activation of nuclear YAP. On the other hand, twisted ribbon-like nanofibers (l-FF and d-FF) guided the cells into round shapes and decreased the formation of focal adhesion complexes, which resulted in the confinement of YAP proteins in the cytosol and a corresponding decrease in osteogenic potential. Interestingly, the d-form of twisted-ribbon like nanofibers (d-FF) increased the chondrogenic potential of stem cells more than their l-form (l-FF). Our results provide new insights into the importance and relevance of morphology and chirality of nanomaterials in their interactions with cells and reveal that precise control over the chemical and physical properties of nanostructures can affect stem cell fate even without the incorporation of specific epitopes.

Hierarchical Self-Assembly of Histidine-Functionalized Peptide Amphiphiles into Supramolecular Chiral Nanostructures.

Controlling the hierarchical organization of self-assembling peptide amphiphiles into supramolecular nanostructures opens up the possibility of developing biocompatible functional supramolecular materials for various applications. In this study, we show that the hierarchical self-assembly of histidine- (His-) functionalized PAs containing d- or l-amino acids can be controlled by both solution pH and molecular chirality of the building blocks. An increase in solution pH resulted in the structural transition of the His-functionalized chiral PA assemblies from nanosheets to completely closed nanotubes through an enhanced hydrogen-bonding capacity and π-π stacking of imidazole ring. The effects of the stereochemistry and amino acid sequence of the PA backbone on the supramolecular organization were also analyzed by CD, TEM, SAXS, and molecular dynamics simulations. In addition, an investigation of chiral mixtures revealed the differences between the hydrogen-bonding capacities and noncovalent interactions of PAs with d- and l-amino acids.

Diabetic wound regeneration using heparin-mimetic peptide amphiphile gel in db/db mice.

There is an urgent need for more efficient treatment of chronic wounds in diabetic patients especially with a high risk of leg amputation. Biomaterials capable of presenting extracellular matrix-mimetic signals may assist in the recovery of diabetic wounds by creating a more conducive environment for blood vessel formation and modulating the immune system. In a previous study, we showed that glycosaminoglycan-mimetic peptide nanofibers are able to increase the rate of closure in STZ-induced diabetic rats by induction of angiogenesis. The present study investigates the effect of a heparin-mimetic peptide amphiphile (PA) nanofiber gel on full-thickness excisional wounds in a db/db diabetic mouse model, with emphasis on the ability of the PA nanofiber network to regulate angiogenesis and the expression of pro-inflammatory cytokines. Here, we showed that the heparin-mimetic PA gel can support tissue neovascularization, enhance the deposition of collagen and expression of alpha-smooth muscle actin (α-SMA), and eliminate the sustained presence of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the diabetic wound site. As the absence of neovascularization and overexpression of pro-inflammatory markers are a hallmark of diabetes and interfere with wound recovery by preventing the healing process, the heparin-mimetic PA treatment is a promising candidate for acceleration of diabetic wound healing by modulating angiogenesis and local immune response.

Angiogenic peptide nanofibers repair cardiac tissue defect after myocardial infarction.

Myocardial infarction remains one of the top leading causes of death in the world and the damage sustained in the heart eventually develops into heart failure. Limited conventional treatment options due to the inability of the myocardium to regenerate after injury and shortage of organ donors require the development of alternative therapies to repair the damaged myocardium. Current efforts in repairing damage after myocardial infarction concentrates on using biologically derived molecules such as growth factors or stem cells, which carry risks of serious side effects including the formation of teratomas. Here, we demonstrate that synthetic glycosaminoglycan (GAG) mimetic peptide nanofiber scaffolds induce neovascularization in cardiovascular tissue after myocardial infarction, without the addition of any biologically derived factors or stem cells. When the GAG mimetic nanofiber gels were injected in the infarct site of rodent myocardial infarct model, increased VEGF-A expression and recruitment of vascular cells was observed. This was accompanied with significant degree of neovascularization and better cardiac performance when compared to the control saline group. The results demonstrate the potential of future clinical applications of these bioactive peptide nanofibers as a promising strategy for cardiovascular repair. STATEMENT OF SIGNIFICANCE: We present a synthetic bioactive peptide nanofiber system can enhance cardiac function and enhance cardiovascular regeneration after myocardial infarction (MI) without the addition of growth factors, stem cells or other biologically derived molecules. Current state of the art in cardiac repair after MI utilize at least one of the above mentioned biologically derived molecules, thus our approach is ground-breaking for cardiovascular therapy after MI. In this work, we showed that synthetic glycosaminoglycan (GAG) mimetic peptide nanofiber scaffolds induce neovascularization and cardiomyocyte differentiation for the regeneration of cardiovascular tissue after myocardial infarction in a rat infarct model. When the peptide nanofiber gels were injected in infarct site at rodent myocardial infarct model, recruitment of vascular cells was observed, neovascularization was significantly induced and cardiac performance was improved. These results demonstrate the potential of future clinical applications of these bioactive peptide nanofibers as a promising strategy for cardiovascular repair.