Stimulus-Responsive Peptide Assembly: Design and Biomedical Applications

Introduction to SRPNs

Peptides play a crucial role in various physiological processes and can form self-assembled peptide (SAP) nanomaterials with diverse structures. Self-assembly of stimulus-responsive peptide nanomaterials (SRPNs) is driven by external stimuli such as temperature, pH, metal ions, and other factors. By modifying the amino acid side chains, peptides can respond to specific substances, enabling SRPNs to function in various biomedical applications. The flexibility and designability of peptides allow for the creation of SRPNs that can undergo behavioral changes under conditions like pH, temperature, enzymes, reactive oxygen species (ROS), and light, making them useful for drug delivery, bioimaging, tumor diagnostics, and biosensors.

SRPNs can be tailored for highly targeted applications, such as drug release or photothermal therapy (PTT) in tumor microenvironments (TME) with acidic conditions. ROS-based SRPNs are also beneficial for inflammatory microenvironments. Additionally, DNA or RNA modifications of SRPNs facilitate gene delivery and programmed apoptosis. The size of SRPNs can adapt based on stimuli, improving their therapeutic efficacy.

Design of Stimuli-Responsive Peptides (SRPs)

By incorporating specific functional components, peptides can be easily chemically modified to form SRPs that respond to both external and internal stimuli. Upon exposure to single or multiple stimuli, SRPs can assemble into nanostructures of varying sizes and shapes. These SRPs may self-assemble, disassemble, or undergo size and shape transformations based on chemical bonding interactions. From a preparation standpoint, single-stimulus-responsive peptide nanomaterials (SSRPN) are characterized by their simple fabrication process, allowing the design of nanomaterials that respond to a single environmental trigger. However, the complexity of the human physiological environment often necessitates SRPN to respond to multiple stimuli. Dual- or multi-stimulus-responsive nanomaterials can integrate endogenous and exogenous triggers, offering enhanced precision and controllability for targeted delivery. Furthermore, SRPNs designed to respond to multiple stimuli not only react to a single trigger but also exhibit synergistic effects when multiple stimuli are activated simultaneously. Therefore, this section discusses the impact of single-, dual-, and multi-stimulus responsiveness on peptide nanostructures.

Single Stimulus-Responsive Peptide Nanomaterials (SSRPNs)

SSRPNs can be prepared by cross-linking peptides with functional linkers or by simple coupling reactions or ring-opening polymerization (ROP) reactions. When affected by pH, GSH, and ROS, the SRPN of the design changes.

pH-Responsive Peptide Systems

The acidity of pathological tissues, such as inflammatory and tumor sites, is often lower than that of healthy tissues and blood. pH-sensitive nanomaterials can be designed to carry therapeutic agents that dissolve or degrade upon environmental pH changes, enabling targeted drug delivery to affected tissues. Additionally, hydrogel-based SRPs can be specifically modified to enhance biocompatibility for specialized applications.

For instance, Qian and colleagues designed a self-assembling peptide (SAP) nanomaterial loaded with a tetraphenylethylene-based drug conjugated to a peptide sequence (SKDEEWHKNNFPLSP) for cancer therapy. The designed peptide specifically recognized vascular endothelial growth factor receptor 2 (VEGFR2) and self-assembled into a reversible α-helix structure in acidic environments. In neutral conditions, the peptide conjugate self-assembled into peptide nanoparticles (PNP). Upon exposure to the tumor microenvironment (TME) with low pH, a morphological transformation from PNP to peptide nanofibers (PNF) was triggered, enhancing drug delivery and therapeutic outcomes.

Another example involves a pH-responsive peptide (C16-VVAEEE) designed to self-assemble into an SAP hydrogel within acidic cellular microenvironments. The C16 alkyl chain enhances self-assembly via hydrophobic interactions, while the tripeptide VVA forms a β-peptide sequence. The EEE sequence interacts with the alkyl chain via hydrogen bonding, forming a layered structure. The carboxyl groups on the side chains and C-terminus serve as the pH-responsive component, enabling C16-VVAEEE to accumulate in pathological tissues with active blood circulation. Upon exposure to mildly acidic conditions, C16-VVAEEE undergoes self-assembly into nanofibers, which further intertwine to form a hydrogel.

Additionally, L-Phe-L-Phe (FF) and its derivatives can self-assemble into different nanostructures under various conditions. Considering the acidic environment of lysosomes, Jin et al. selected an iridium (III) (Ir) complex with aggregation-induced emission (AIE) properties as the core and linked it with two carboxyl groups to form an Ir complex (Irc). The naphthalene-phenylalanine-phenylalanine-lysine (Nap-FFK) sequence was conjugated to the Ir (III) core, facilitating π–π interactions and forming a peptide–Ir complex (Irpc) for long-term lysosomal imaging. The Irpc complex exists as large nanostructures at pH 7.0–8.0 but dissociates into smaller nanoparticles at pH 6.0. In the lysosomal microenvironment (pH 4.0–5.0), these small nanoparticles further interconnect into a nanonetwork, facilitating lysosomal uptake. This peptide-based nanoprobes' excellent biocompatibility allows for long-term lysosomal imaging.

The formation and dissociation of pH-dependent peptide self-assemblies (PSA) exhibit remarkable reversibility, providing the driving force for volumetric expansion and contraction in peptide-based hydrogels.

Enzyme-Responsive Peptide Systems

Enzymes are essential biomolecules that sustain normal life processes, characterized by their high efficiency, mild reaction conditions, specificity, and sensitivity. Many pathological microenvironments exhibit abnormal overexpression of enzymes, including matrix metalloproteinases (MMPs) and hyaluronidases (HAases). Due to their mild reaction conditions and minimal side effects, enzyme-responsive systems have attracted increasing attention, enabling applications in targeted drug delivery, disease diagnosis, controlled release, and enhanced cell penetration by incorporating them into polymers, proteins, or drugs.

A simple and effective strategy to promote bone healing involves the localized delivery of osteoinductive growth factors (GFs). However, this approach faces significant challenges when treating complex clinical fractures. To overcome this, a peptide nanocapsule delivery platform for bone morphogenetic protein-2 (BMP-2) GF was developed. The BMP-2 nanocapsules were synthesized through in situ free radical polymerization, using 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) as a monomer and a bisacrylated VPLGVRTK peptide as a degradable crosslinker. Upon injection, the n(BMP-2) nanocapsules passively target the fracture site via blood circulation. The outer shell of the nanocapsules is degraded by MMPs (primarily MMP-2 and MMP-9), releasing BMP-2 to accelerate bone healing. Animal studies showed that this enzyme-responsive peptide-based n(BMP-2) delivery system exhibited low immunogenicity, excellent bone repair efficiency, and high in vivo stability.

Table.1 Matrix metalloproteinases (MMPs) related peptides at Creative Peptides.

Integrating drugs with MMP-responsive peptides for enzyme-triggered drug release is a promising approach for disease treatment. A simple and flexible strategy has been developed to create an enzyme-responsive carrier, referred to as WINNER, for the precise delivery of extracellular functional protein drugs. The outer shell of WINNER consists of phosphatidylcholine (PC), making up 50.5%–58.3% of the composition, and an MMP-2-responsive peptide (VPLGVRTK) that protects the encapsulated protein drugs. The WINNER shell prevents uptake by normal cells, but when it accumulates at tumor sites, the overexpressed MMP-2 specifically recognizes the enzymatic cleavage site in the shell. This triggers the release of the functional protein drugs into the extracellular space, where they bind to tumor cell surface receptors and effectively inhibit tumor growth.

In addition, wrapping the cells in a thin, stable membrane or semi-permeable hydrogel can reduce the immune response while enhancing the permeability of the cells. Compared to other encapsulation techniques, nanoshell-based encapsulation offers higher efficiency. For instance, enzyme-responsive peptide nanoshells have been synthesized to encapsulate HeLa cells and human bone marrow mesenchymal stem cells (hMSCs), thereby improving cancer treatment efficacy. These highly stable enzyme-responsive nanoshells are formed through the layer-by-layer (LBL) self-assembly of oppositely charged polyethylene glycol (PEG)-gelatin and peptide linkers capped with cysteine (sequence: CGGPLGLAGGC). Upon reaching tumor sites, the high concentration of MMP-7 induces the cleavage of the enzyme-responsive peptide, leading to the disassembly of the PEG-gelatin shell and subsequent release of the HeLa and hMSC cells.  

ROS-Responsive Peptide Systems

Reactive oxygen species (ROS) are a class of oxygen-derived chemical substances, including hydroxyl radicals, hydrogen peroxide, superoxide, etc. ROS are important molecules produced in the body that play an irreplaceable role in both pathological processes and physiological metabolism. When ROS are excessively produced in cells or tissues, it typically leads to oxidative stress, disrupting cellular homeostasis and affecting a range of pathological conditions.

Due to the elevated levels of ROS in pathological tissue cells, many ROS-responsive peptide systems have been designed and synthesized. One such system involves a novel peptide-based block copolymer (mPEG-b-PDTG) that contains thioether groups for drug delivery. The amphiphilic peptide-polymer complex utilizes amino-terminated PEG methyl ether (MPEG-NH₂) as an initiator in an aqueous medium. When exposed to hydrogen peroxide (H₂O₂), the thioether groups on the side chains of mPEG-b-PDTG undergo oxidation, leading to the degradation of the peptide-polymer micelles. As a result, when these micelles enter pathological tissue cells, the excessive ROS oxidize the micelles, causing them to disintegrate and release the drug for therapeutic purposes.  

Temperature-Responsive Peptide Systems

In addition to many endogenous stimuli, exogenous stimuli can also exert different effects on peptide self-assembly (PSA). Temperature, as a factor influencing the structural changes and self-assembly of peptides, has been widely studied. For example, Barker's team designed two elastin-like peptides (ELPs) (sequence (VPGXG)n, where X is any amino acid except proline) as diblock copolymers (A80-I60, P40–I60). As the temperature gradually increases to the transition temperatures of I60, A80, and P40, I60 collapses first to form a micelle shell. Then, the A80 peptide detaches from the fibrinogen, and finally, P40 contracts to form micron-scale clusters. This peptide system can trigger in situ release behavior in response to temperature stimulation.

A peptide platform was developed to create temperature-responsive elastin-like peptide amphiphiles (ELPAs). The ELP sequence was incorporated into building blocks, with the N-terminal coupled to a linear RGD sequence to facilitate self-assembly. When the temperature rises above the transition temperature (Tt), the peptide conjugates rapidly aggregate. However, as the temperature drops below Tt, the conjugates undergo a slow dissolution. Additionally, α-helix peptides are attached to the N-terminal to form ELPA for self-assembly. When the temperature exceeds Tt, the ELPA disassembles quickly. This platform shows significant promise for developing temperature-responsive applications.

Light-Responsive Peptide Systems

Among various exogenous stimuli, light stimulation has become a major focus of research in recent years due to its non-invasive nature and strong spatiotemporal selectivity. Many light-responsive molecules react to light by forming or breaking bonds, which triggers changes in their geometric shape. For instance, ultraviolet (UV) light has been used to induce the self-assembly of a peptide (NVFFAC), transforming it from one-dimensional nanofibers to two-dimensional nanosheets. An antibacterial system has also been developed using β-cyclodextrin (β-CD) and a cationic peptide with an azobenzene side chain. Both systems undergo dynamic self-assembly, forming microscale layered structures with high surface potential. Under UV light, the azobenzene and β-CD interact weakly to form small and stable nanospheres. This system combines controllable light-responsive PSA with antibacterial material preparation, offering new strategies for creating antibacterial materials.  

Other Stimulus-Responsive Peptide Systems

As mentioned earlier, factors such as pH, enzymes, ROS, temperature, and light stimulation can all influence PSA. In addition to these factors that can regulate peptide behavior, other factors can also trigger changes in PSA behavior, such as ion reactions and solvent reactions.

Many polypeptides can self-assemble into nanostructures with different morphologies through ligand binding with metal ions. Introducing metal ions into the polypeptide system can induce conformational changes in the polypeptide, leading to significant changes in the properties and performance of polypeptide nanomaterials. Ulijn's research group designed co-assembled nanostructures of the tripeptides FFD and GHK, demonstrating that PSA behavior can be influenced by metal ions. Due to electrostatic interactions, the co-assembled FFD-GHK conjugate forms a nanoribbon structure in water. After adding Cu²⁺, the peptide stimulates coordination with Cu²⁺, causing the SAP structure to change from nanoribbons to nanofibers, which then entangle to form a hydrogel. Additionally, to study the effects of different metal ions on PSA, Cienfuegos et al. added Cs⁺ and Ca²⁺ to Fmoc-FF peptide solutions. In the solution with Cs⁺, the peptides self-assemble into uniformly sized nanorods, which then aggregate into fibers. In both cases, peptide hydrogels can be obtained, but the hydrogel formed with Cs⁺ has much weaker mechanical strength than the one formed with Ca²⁺. This is because Cs⁺ tends to stabilize the water-solution interface, weakening hydrophobic interactions and promoting the formation of metastable intermediates. In contrast, Ca²⁺ disrupts the stability of the water-solution interface and enhances hydrophobic interactions, leading to larger fiber aggregates and forming a hydrogel with higher mechanical strength.

Dual and Multiple Stimulus-Responsive Peptide Systems

Clearly, SSRPNs have significant advantages in controlled and specific applications. However, due to the complexity of target site microenvironments, a single stimulus-response may not accurately achieve the expected results. Therefore, many researchers have been dedicated to developing dual or multiple stimulus-responsive peptide nanomaterials (DSRPN or MSRPN) for more precise therapy.

Among all DSRPNs and MSRPNs, pH/ROS-responsive systems are a major focus of research. For instance, a hydrophilic PEG-based copolymer containing l-cysteine peptides with thiol side chains (PEG-D-PC) was designed to form mixed nanoparticles (NPs). These nanoparticles feature a detachable PEG crown and a dynamic disulfide bond-crosslinked core. They can load the anticancer drug camptothecin (CPT) for cancer treatment. In the tumor microenvironment (TME), the nanoparticles shrink significantly in size and increase in potential. A high concentration of GSH breaks the disulfide bonds, allowing for the precise and effective release and accumulation of CPT, thus enhancing anticancer efficacy. In another example, a pH/ROS dual-responsive DOX-loaded carboxymethyl chitosan nanogel (CMCSN) was designed, modified with an angiogenic peptide-2 (ANG) for targeted glioblastoma (GBM) treatment. ANG specifically binds to low-density lipoprotein receptor-related protein 1 (LRP-1), which is highly expressed on the blood-brain barrier (BBB) and in GBM. After the DOX-ANG-CMCSN is taken up by tumor cells, the nanogel is cleaved, and due to its sensitivity to pH and GSH, DOX is released for anticancer treatment.

In addition to pH/ROS-based DSRPNs, pH/enzyme-based DSRPNs also play an important role in biomedical applications. For example, a dual-responsive nanocarrier was developed to load an anti-PD-1 antibody (aPD-1) and the chemotherapy drug paclitaxel (PTX). This carrier, with an azido-PEGPAsp shell, encapsulates PTX in the core of the micelle. In tumor tissues, overexpressed MMP-2 recognizes and cleaves the MMP-2-sensitive peptide, releasing aPD-1 into the tumor cells. Additionally, low pH stimulates the release of PTX from the nanocarriers. Therefore, the pH/enzyme-based DSRPN enables the sequential release of both drugs, fully utilizing their synergistic anticancer effects.

Beyond the aforementioned combinations of endogenous stimuli, combinations of internal and external stimuli have also been widely explored, particularly focusing on peptides responsive to both pH/temperature dual stimuli. A series of peptide amphiphiles (PPCAs) with excellent gelation ability were prepared using PEG and poly (l-glutamic acid) derivatives as temperature and pH-sensitive segments. The results showed that when pH was the only variable, PPCAs exhibited a reversible sol-gel transition temperature. At pH 7.4, the sol-gel transition temperature of the hydrogel ranged from 10–70 °C, with the gelation temperature significantly affected by pH 6.5–7.4. PPCAs gelled at 37°C. This study provides valuable insights for developing physiologically relevant dual pH/temperature-responsive peptide hydrogels.

Inspired by the advantages of dual stimulus-response systems, researchers have further developed MSRPNs. Currently, chemical immunotherapy is limited to a small subset of patients. To overcome this limitation, a prodrug nanovesicle was developed that can be stimulated in the TME by integrating oxaliplatin (OXA) and a PEG-modified photosensitizer (PS). The prodrug nanovesicle circulates to the tumor site via the bloodstream, where the MMP-2-targeted peptide removes the PEG crown layer. Under the acidic conditions of the tumor microenvironment, the surface charge of the nanovesicle changes from negative to positive, facilitating aggregation in tumor cells. Under laser irradiation and the presence of abundant GSH, OXA is precisely released. These findings indicate that MSRPNs are a more precise and promising class of materials.

Table.2 Structure, responsiveness and application of representative SRPs.

Biomedical Applications of SRPN

Compared to non-peptide nanomaterials, peptide-based materials exhibit superior biocompatibility, bioactivity, and biodegradability while also reducing immunogenicity. Specific interactions between amino acids lead to highly ordered and stable assemblies, enabling peptides to self-assemble into various nanostructures. Through rational amino acid sequence design, different functionalities, such as stimulus responsiveness, can be incorporated within a single molecule. This feature allows peptides to self-assemble or co-assemble with other materials into multifunctional nanomaterials, providing a significant advantage for nanotechnology applications. Among these peptide-based nanomaterials, stimulus-responsive peptide nanomaterials (SRPN) have demonstrated great potential in biomedical fields, including drug delivery, bioimaging, gene therapy, photothermal therapy (PTT) and photodynamic therapy (PDT), antimicrobial biomaterials, wound healing, and wound dressings. 

Drug Delivery

SRPN have been widely explored as drug carriers due to their ability to undergo conformational and chemical changes in response to stimuli such as pH, temperature, enzymes, or redox conditions. These properties enable precise drug release at targeted sites, improving therapeutic efficiency while minimizing systemic toxicity. Additionally, the self-assembling nature of SRPN allows for the encapsulation of hydrophobic and hydrophilic drugs, enhancing their stability and bioavailability. Furthermore, their biodegradability ensures controlled degradation and clearance from the body after drug delivery, making them an attractive alternative to synthetic polymer-based carriers. 

Bioimaging

The inherent bioactivity and tunable optical properties of SRPN make them promising candidates for bioimaging applications. By modifying peptide sequences, SRPN can be designed to enhance contrast in fluorescence imaging, magnetic resonance imaging (MRI), and photoacoustic imaging. Additionally, SRPN can respond to specific physiological changes in diseased tissues, enabling real-time imaging of pathological conditions. Their excellent biocompatibility and reduced cytotoxicity provide a safer alternative to traditional contrast agents, making them highly suitable for in vivo imaging applications. 

Gene Therapy

The ability of SRPN to self-assemble into stable nanostructures provides an efficient platform for gene therapy applications. These nanomaterials can serve as non-viral gene delivery vectors, facilitating the transport of nucleic acids such as DNA, RNA, or siRNA into target cells. SRPN-based carriers offer advantages such as reduced immunogenicity, high transfection efficiency, and controlled gene release in response to specific stimuli. This makes them a promising alternative to viral vectors, which often pose risks of immune reactions and insertional mutagenesis. 

Photothermal and Photodynamic Therapy

SRPN has shown great potential in cancer therapy through photothermal and photodynamic approaches. In photothermal therapy (PTT), SRPN can be designed to absorb near-infrared (NIR) light and generate localized heat, effectively killing cancer cells with minimal damage to surrounding healthy tissues. Similarly, in photodynamic therapy (PDT), SRPN can act as carriers for photosensitizers, which, upon light activation, produce reactive oxygen species (ROS) to induce tumor cell apoptosis. The responsiveness of SRPN to environmental stimuli enhances their therapeutic efficiency while ensuring targeted treatment, reducing off-target effects. 

Antimicrobial Biomaterials

The antimicrobial properties of SRPN make them valuable for preventing infections, especially in biomedical implants and wound care applications. SRPN can be engineered to release antimicrobial peptides (AMPs) in response to bacterial infections, providing a controlled and targeted antimicrobial effect. These peptide-based nanomaterials can disrupt bacterial membranes, inhibit biofilm formation, and enhance immune responses. Additionally, their biodegradability minimizes long-term accumulation in the body, making them a safer alternative to conventional antibiotics, which often lead to antimicrobial resistance. 

Table.3 Antimicrobial peptides at Creative Peptides.

Wound Healing

SRPN plays a crucial role in wound healing by promoting cellular regeneration and tissue repair. These materials can be designed to respond to wound microenvironments, releasing bioactive molecules that enhance fibroblast proliferation, angiogenesis, and extracellular matrix remodeling. Furthermore, SRPN can protect wounds from bacterial infections while providing a moist and supportive environment for tissue regeneration. Their tunable mechanical properties make them suitable for use in various wound types, from chronic wounds to surgical incisions. 

Wound Dressings

SRPN-based wound dressings offer several advantages over traditional wound care materials. Their self-assembling nature allows for the formation of hydrogels or nanofibrous scaffolds that provide an optimal environment for wound healing. These dressings can be engineered to release growth factors, antimicrobial agents, or anti-inflammatory molecules in response to specific wound conditions. Moreover, their biodegradability eliminates the need for frequent dressing changes, reducing patient discomfort and improving overall healing outcomes.

Summary

Stimulus-responsive peptide nanomaterials (SRPNs) have shown remarkable potential in various biomedical applications due to their ability to self-assemble and respond to external stimuli such as temperature, pH, metal ions, and light. By modifying the amino acid side chains, SRPNs can be tailored for specific biomedical needs, including drug delivery, tumor diagnostics, photothermal therapy, gene therapy, and wound healing. The adaptability of SRPNs in response to environmental changes enhances their therapeutic efficacy and targeting capabilities, making them a versatile tool in tissue engineering and biomedicine. The significance of SRPN technology lies in its ability to offer highly targeted, responsive treatments that can minimize side effects and improve patient outcomes. As research progresses, SRPNs hold great promise for advancing personalized medicine and therapeutic strategies, making them a valuable asset in the development of next-generation biomedical technologies.

References

1. Jin, Chengzhi, et al., Robust Packing of a Self-Assembling Iridium Complex via Endocytic Trafficking for Long‐Term Lysosome Tracking. Angewandte Chemie International Edition 60.14 (2021): 7597-7601.
2. Mañas-Torres, Mari C., et al, In situ real-time monitoring of the mechanism of self-assembly of short peptide supramolecular polymers. Materials Chemistry Frontiers 5.14 (2021): 5452-5462.