Mechanisms of Polypeptide Self-Assembly and Their Applications in Biomedical Fields

Self-assembling polypeptides refer to peptide molecules or a combination of multiple peptide molecules that spontaneously or triggerly bind and connect to form nanostructured aggregates through intermolecular interactions. These nanostructures can take on various forms, including spherical micelles, vesicles, fibrils, ribbons, layers, cylinders, tubes, spheres, and networks. These aggregates possess advantages such as good biocompatibility and high stability, exhibiting unique properties and benefits that distinguish them from individual peptide molecules. As a result, they hold promising applications in fields such as drug delivery, tissue engineering, biomedical detection, and engineered vaccines.

Peptide Self-Assembly Mechanism

Peptides can achieve self-assembly spontaneously or triggered by specific conditions, further forming structurally specific nano-architectures. Peptides primarily rely on non-covalent interactions such as hydrogen bonding, hydrophilic/hydrophobic interactions, electrostatic interactions, and π-π stacking for self-assembly. Although the strength of non-covalent bonds is weaker than covalent bonds, when present in sufficient numbers and acting in concert, these non-covalent driving forces enable molecules to form stable nano-assemblies.

1. Hydrophilic/Hydrophobic Interactions: Hydrophobic interactions are the forces that drive the non-polar parts of molecules to cluster together to avoid water, while hydrophilic forces work in opposition. Together, they are the primary driving forces for the self-assembly of amphiphilic peptides, playing a crucial role in stabilizing secondary and tertiary conformations. Similar to the phospholipid bilayer structure of biological membranes, the hydrophilic and hydrophobic segments of amphiphilic peptides spontaneously arrange close to or away from each other based on these forces. By manipulating the structure of these segments, self-assembling peptide molecules with different functions and aggregate morphologies can be designed. Hydrophilic and hydrophobic interactions regulate the periodicity, morphology, and structure of self-assembly.

2.Hydrogen Bonding: Hydrogen bonding arises from the attraction between a hydrogen atom attached to a highly polar X–H group and a highly electronegative atom Y (such as O, N, F). Hydrogen bonds exhibit directionality (i.e., the angle between X and Y approaches 180°) and saturation (i.e., typically, X–H can only bind to one Y). They can form both intermolecularly and intramolecularly. Hydrogen bonding is a major contributor to the stability of higher-order structures like proteins.

3.Electrostatic Interactions: The positively and negatively charged amino acid residues within peptide molecules interact through electrostatic attraction and repulsion, influencing the self-assembly behavior of peptides. Electrostatic interactions are non-directional and susceptible to disturbances by ionic strength and pH.

4.π-π Stacking: π-π stacking occurs in self-assembling peptides containing aromatic amino acids. This weak interaction exists between relatively electron-rich and electron-deficient molecules, allowing them to stack in face-to-face or edge-to-face configurations. This interaction is directional, and introducing π-π conjugated double bonds into peptides can significantly alter the molecular environment, thereby driving the formation of specific assembly morphologies.

Applications of Self-Assembling Peptides in Biomedical Fields

1. Self-Assembling Peptides in Drug Delivery

In recent years, self-assembling peptides have made significant progress as nanomaterials for drug delivery. As drug carriers, self-assembling peptides offer numerous advantages. Apart from their rapid and tunable synthesis, they can enhance cellular uptake by providing specific ligands that bind to target cell receptors, ensuring responsive drug release at specific targets without toxic effects on other cells and eventual free excretion from the body. Clinical applications of free-form drugs are often limited by undesirable properties such as low water solubility, poor biodistribution, and high degradation rates. Additionally, some drugs cannot be administered directly or pose potential risks of inducing immune reactions. To overcome these drawbacks and improve therapeutic efficiency, it is necessary to combine drugs with suitable drug carriers. For example, the multifunctional peptide P51 has been studied for the delivery of the hydrophobic chemotherapeutic drug pirarubicin. P51 consists of a specifically targeted RGD sequence, a negatively charged amino acid sequence (enzyme-responsive), a disulfide bond linker (reduction-responsive), and an α-helical 41-peptide (acid-responsive). The α-helical portion self-assembles through hydrophobic and electrostatic interactions, forming stable spherical nanoparticles upon loading with pirarubicin. Acids, the reducing agent dithiothreitol (DTT), and enzymes can stimulate the release of the hydrophobic drug from the self-assembled nanoparticles. Notably, when all three triggering factors are present, drug release efficiency is significantly enhanced. This self-assembly has been demonstrated to have tumor targeting ability and low toxicity, providing important insights into the application of self-assembling peptides in drug delivery.

Furthermore, hydrogels possess the ability to absorb and retain water within their porous, swollen structures, making them an excellent drug delivery system due to their extensive physical and chemical adaptability. Peptide-based hydrogels, characterized by their good biocompatibility, hydrophilicity, and ease of processing, have been widely utilized as therapeutic agents and carriers for controlled drug delivery. They are formed by the stacking of nanofibers created through physical crosslinking of oligopeptide molecules, offering several advantages. Firstly, the preparation of responsive hydrogels based on external stimuli can be used to regulate drug release processes in vitro. Secondly, the chemical reaction process of peptide coupling with modifying groups facilitates the complexation of drugs with hydrogels. Thirdly, hydrogels co-assembled from two peptides with opposite charges can not only effectively control drug loading but also synergistically inhibit tumors in immune organs. Lastly, targeted drug delivery is achievable for cancer treatment.

Despite the numerous advantages of self-assembling peptides as drug carriers, the mechanical and rheological properties of assembled peptide hydrogels are often poor, leading to difficulties in injection and thereby limiting their further applications. To address this issue, research has focused on coupling positively charged peptides such as PLL with self-assembling dipeptides like Fmoc-FF. The electrostatic interactions between these two components enable the fibrous hydrogels to exhibit shear-thinning and self-healing properties, resulting in hydrogels with rheological characteristics suitable for injection.

2. Applications of Self-Assembling Peptides in Tissue Engineering

Tissue engineering methods consist of three crucial elements: scaffolds, cells, and growth factors. Among these, scaffolds are the key to providing a biomimetic environment for cells. Self-assembling peptide scaffolds have been utilized in neural, spinal cord, and bone tissue engineering due to their excellent biocompatibility and tunable biodegradability.

In neural tissue engineering, scaffold materials demonstrate good compatibility with neuronal cells, inducing axonogenesis and elongation while inhibiting scar tissue formation. N-cadherin, expressed by neuronal cells, controls axon guidance in neural networks, synapse formation, regulation of synaptic structures, and astrocyte-synapse contacts.

For spinal cord tissue engineering, scaffolds provide an environment and space for the growth of adjacent spinal cord cells, guiding the extension of neuronal axons, preventing interference from external components and scar formation, and enabling the regenerated tissue to approximate normal tissue structure. After spinal cord injury, the disruption of the blood-spinal cord barrier leads to inflammation and glial scar formation, inhibiting axon growth and reducing the effectiveness of scaffolds transplanted into the injured site. Studies on the repair of rat spinal cord injuries using RADA self-assembling peptide nanofiber scaffolds have shown that they can reduce inflammation and glial scar formation, increase axon density at the injury site, and reduce the area of spinal cord injury, demonstrating that cell-laden self-assembling peptide scaffolds can be used in spinal cord injury repair.

In bone tissue regeneration, self-assembling peptides exhibit promising therapeutic effects for bone tissue diseases such as osteoporosis and cartilage defects. Research has found that coating polycaprolactone (PCL) scaffolds with self-assembling peptide hydrogels (SAPH) provides both SAPH nanofiber and microfiber networks, creating a biomimetic ECM microenvironment conducive to bone and cartilage cell growth. Additionally, SAPH significantly improves the hydrophobicity of PCL surfaces, facilitating cell adhesion and ECM deposition, achieving simultaneous repair of bone and cartilage defects in rabbit joint cavities.

3. Applications of Self-Assembling Peptides in Biomedical Detection

Self-assembling peptides possess the ability to specifically recognize biomarkers that are highly expressed in disease sites, enabling precise diagnosis and effective treatment of diseases. By incorporating various imaging agents, such as radioactive isotopes and fluorescent chromophores, into their structures either in situ or ex situ, various highly specific self-assembling peptide probes have been developed, becoming essential tools in medical detection and imaging. For instance, inspired by the green fluorescent protein chromophore, researchers have designed a tyrosine (Y)-based peptide, Fc-YYGCGPGRC, which self-assembles into nanoparticles stabilized by intermolecular hydrogen bonds. These nanoparticles are pH-sensitive, emitting intense green fluorescence when the phenolic group of tyrosine becomes deprotonated. Furthermore, upon binding to the GPGR motif of the human immunodeficiency virus, this peptide can effectively penetrate cellular membrane barriers. Due to its exceptional photostability, pH sensitivity, and biocompatibility, it holds promise as a fluorescent nanomaterial for stable cellular imaging and has the potential to become a valuable tool in biomedical applications.

4. Applications of Self-Assembling Peptides in Vaccine Engineering

Employing self-assembling nanomaterials as carriers provides a microenvironment conducive to interactions with antigen-presenting cells (APCs), facilitating disease prevention and the development of immunotherapeutic vaccines. Peptide vaccine design primarily focuses on utilizing specific modifications of antigen proteins to enhance selective immune responses and improve immunogenicity. Q11 (Ac-QQKFQFQFEQQ-NH2) is a commonly used self-assembling peptide in vaccine engineering that can self-assemble into β-sheet nanofibers through non-covalent interactions in saline solutions.

Studies have explored the use of self-assembling nanocarriers to improve the immunogenicity of peptide-based antigen vaccines. By covalently linking an HPV16 E744-62 antigenic peptide, recognized by cytotoxic T lymphocytes, to the amino terminus of Q11, an efficient nanofiber vaccine, E744-62-Q11, was formed through self-assembly and folding in saline solutions, thereby enhancing the immunogenicity of the antigenic peptide vaccine. In a mouse model with subcutaneous heterotopic transplantation of TC-1 tumor cells, mice vaccinated with E744-62-Q11 exhibited significantly lower tumor weights compared to those vaccinated with Q11 alone. Additionally, mice vaccinated with E744-62-Q11 had higher levels of E7-specific interferon-γ (IFN-γ)-secreting T cells in their splenic lymphocytes, indicating a robust antitumor cellular immune response in vivo. Furthermore, E744-62-Q11 demonstrated good safety in mice, making it a promising candidate for the development of novel nanovaccines for HPV-associated tumors.

References

[1] Liu Xu, Yin Caiyun, Chen Haiyan, et al. Research Progress of Self-Assembling Peptides in the Biomedical Field [J]. Pharmacy and Clinical Research, 2022, 30(06): 523-528.

[2] Wang Yue, Ding Xiufang, Zhang Sida, et al. Applications of Self-Assembly in Peptide Drugs [J]. Chinese Journal of Biotechnology, 2023, 39(01): 177-191.

[3] Yu Weikang, Zhang Shanshan, Yang Zhanyi, et al. Applications of Supramolecular Peptide Self-Assembly in Biomedicine [J]. Chinese Journal of Biotechnology, 2021, 37(07): 2240-2255.

About the Author

Xiaonisha, a food technology professional holding a Master's degree in Food Science, is currently employed at a prominent domestic pharmaceutical research and development company. Her primary focus lies in the development and research of nutritional foods, where she contributes her expertise and passion to create innovative products.