Self-assembling peptides refer to a class of peptidic materials that can form assemblies under certain conditions through intermolecular forces such as π-π stacking and electrostatic interactions. These peptides exhibit excellent biocompatibility and controllability, enabling them to form nanostructures like particles, fibers, and gels, thereby performing specific morphological functions. They have found widespread applications in biomedical fields and beyond. Given the unique assembly characteristics and inherent biological functions of peptides, along with the need to fulfill demands in drug delivery, disease diagnosis, and treatment, modifications to peptides are necessary. The modified self-assembling peptides demonstrate even greater advantages in terms of their assembly capabilities and biomedical applications.

Types of Self-Assembling Peptides

RADA16-I is a classic example of an ion-complementary peptide that can spontaneously form fibrous hydrogels in aqueous solutions. It has found applications in biomedical and clinical fields. Ion-complementary peptides are characterized by alternating sequences of negatively and positively charged amino acid residues, which initiate molecular self-assembly through electrostatic interactions, hydrogen bonds, and van der Waals forces. The hydrophilic and hydrophobic regions are arranged alternately, forming two ordered domains. The hydrophobic amino acid residues fold to shield water molecules, while the hydrophilic region exhibits a regular and ordered attraction between positive and negative charges. The formation of intermolecular hydrogen bonds accelerates peptide self-assembly, and the interlocking of ionic bonds enhances the strength of the self-assembled structure. However, a significant drawback of these peptides is their poor stability at low pH levels.

To address the shortcomings of ion-complementary peptides, researchers have focused on altering hydrophobic interactions and further exploring the ratio of hydrophilic to hydrophobic amino acids. This led to the development of Surfactant-like Peptides (SLPs). SLPs mimic the properties of polypeptide aggregation and surfactant molecules, consisting of hydrophobic and hydrophilic regions. The hydrophilic head typically comprises 1-2 charged amino acid residues (such as His, Asp, Glu), while the hydrophobic tail is made up of 3-9 non-polar amino acids (like Ala, Phe, Ile, Val). SLPs can assemble into nanostructures like nanotubes, nanocapsules, and nanofibers.

In recent years, scientists have gone beyond modifying natural amino acids to design self-assembling peptides. They have utilized highly hydrophobic alkyl chains, lipid groups, and sugars to modify peptides. Chemically modified peptides exhibit increased secondary structures, leading to more stable nanostructures. These chemical groups can be strategically placed within the peptide chain to perform specific functions. Currently, there has been extensive research on self-assembly of amphiphilic peptides with hydrophobic alkyl chains. By incorporating alkyl carbon chains at the amino termini, the functions and properties of these peptides can be altered, with hydrophobic interactions serving as the core driving force for molecular self-assembly.

Factors Affecting Peptide Self-Assembly

Peptide self-assembly is a dynamic equilibrium process where hydrogen bonds, hydrophobic forces, electrostatic attractions, and other interactions play crucial roles in organizing small molecules into ordered nanostructures. Similarly, changes in environmental factors can also induce variations in the morphology and properties of the self-assembled structures.

① pH Value: The primary driving force for the self-assembly of dipeptides and polypeptides lies in the formation of hydrogen bonds between molecules. However, the formation of hydrogen bonds is susceptible to changes in pH. Altering the pH of the solution can lead to the positive or negative electrification of the C- and N-terminals of the peptide chain or certain chemical groups. This, in turn, results in the formation of polypeptides with positive and negative charges, exhibiting distinct self-assembly trends, nanoscale structures, and structural-functional characteristics. The pH value is particularly crucial for peptide sequences rich in charged amino acids (such as Glu, Asp, Lys, His, and Arg), significantly impacting the formation of hydrogen bonds and the electrification of the peptide's terminal groups. Consequently, by controlling the pH, one can rationally design self-assembling peptides with applications in drug delivery, sustained release, and other purposes tailored to specific acidity or alkalinity levels.

② Peptide Concentration: Concentration is a vital parameter in the self-assembly and aggregation of oligopeptides. Studies on concentration can determine the critical aggregation concentration (CAC) at which oligopeptides begin to aggregate. Below the CAC, oligopeptides exist as individual molecules, while above this threshold, they start to aggregate. When the peptide concentration exceeds the critical micelle concentration (CMC), peptide molecules undergo association, forming micelles. Variations in concentration lead to changes in the content of non-covalent bonds such as hydrogen bonds, which can also trigger electronic cloud rearrangement among peptide molecules, subsequently altering the nanoscale morphology. Furthermore, the dense surface of the fibrous network may interact synergistically with solvents like water, enhancing the stability of the nanostructures.

③ Ion Concentration: Ion concentration is a significant factor influencing the packing of peptide molecules and the structural and functional properties of proteins. The presence of salt ions can shield charged groups, thereby weakening the electrostatic interactions between molecules. This charge shielding effect also enhances the hydrophobic interactions between molecules, making peptide molecules more prone to aggregation and self-assembly. Ions can also specifically recognize and interact with individual amino acid sequences, forming salt bridges in polar amino acids, which promote the formation of self-assembled structures through physical crosslinking between molecules. Given that various ions play critical roles in regulating cellular metabolism, maintaining ion balance across blood vessels, and promoting bone development in the body, self-assembling peptides responsive to ion concentration have broad application potential in the medical field.

④ Temperature: An increase in temperature can disrupt the hydrogen bonds within the system, reducing the stability of the self-assembled structure and leading to conformational changes. For instance, a de novo designed peptide molecule (KIGAKI)3-NH2 connected to a central tetrapeptide Thr-DPro-Pro-Gly, due to the presence of Pro, exhibits a random coil conformation in an aqueous solution between 20–50°C. However, when the system temperature is raised to 60°C, the conjugate initially displays β-sheet formation. Further increasing the temperature to 70°C significantly enhances β-sheet formation, leading to the formation of nanofibers and eventually rigid hydrogels. This temperature rise enhances the solubility of hydrophobic groups, affecting the balance between hydrophilic and hydrophobic groups, and such behavior can be reversibly altered with changes in temperature. As the temperature increases, the hydrogen bonds within the peptide break, altering the secondary structure, and hydrophobic forces and π-π stacking become the primary driving forces for molecular self-assembly, leading to the transformation of nanostructures. When the temperature decreases, hydrogen bonds reform, restoring the secondary structure and, subsequently, the nanoscale morphology of the self-assembled peptides.

⑤ Chirality: Natural amino acids in nature are all of the L-form, and their corresponding D-isomers have been designed to exhibit superior advantages and properties. Chiral amino acids play a pivotal role in controlling the folding and supramolecular assembly of peptides or proteins. Studies have shown that different chiralities of peptides can lead to variations in their self-assembly tendencies and molecular structures in solution. Substituting L-amino acids with D-isomers can result in changes in key parameters (such as amphiphilicity) that govern the formation of assemblies.

Modification of Self-Assembling Peptides

The functional modification sites of self-assembling peptides primarily encompass the amino and carboxyl groups on the main chain, as well as amino, carboxyl, hydroxyl, and thiol groups on the side chains. There are two main types of modification methods: direct modification and indirect modification. Direct modification involves the direct covalent coupling of functional molecules with peptides, either directly or after activating the reactive groups to facilitate covalent bonding with the peptides. In contrast, indirect modification utilizes linking units to connect functional molecules with peptides. This approach is necessary when certain molecules possess significant steric hindrance or lack reactive groups that can efficiently couple with peptides, necessitating the introduction of other molecules as linking units.

1. Direct Modification

The functional molecules used in direct modification methods primarily encompass the following categories:

(1) Drug Molecule Modification: Drug molecules are directly modified onto peptides to achieve precise drug delivery and disease treatment. By leveraging the recognition and therapeutic capabilities of small molecule drugs towards the disease site, along with their hydrophilic/hydrophobic properties, they can be covalently coupled with self-assembling peptide materials. This approach can prolong drug retention, enhance efficacy, and reduce toxic side effects. To facilitate the attachment of drug molecules to peptides, certain functional groups on the drug molecules need to be activated. For instance, chlorambucil (CRB), also known as Leukeran, is a commonly used anticancer drug. The n-butylbenzoic acid group of CRB can be covalently attached to the N-terminus of a peptide using standard solid-phase peptide synthesis (SPPS) methods. Specifically, benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) is employed as a coupling agent to activate the carboxyl group of CRB, which then reacts with the free amino terminus of the peptide to form an amide bond. Researchers have designed a novel self-assembling short peptide, CRB-YpYY, where CRB serves as both the drug delivery vehicle and the capping group. It is covalently bound to the amino terminus of the phosphorylated short peptide YpYY through amide condensation, replacing the commonly used naphthalene and fluorene capping groups. When CRB-YpYY is cleaved by overexpressed alkaline phosphatase in tumor regions, it transforms into CRB-YYY, which self-assembles in situ to form a gel. Compared to free CRB molecules, this gel exhibits superior tumor suppression effects.

(2) Probe Molecule Modification: By harnessing the aggregation-induced retention (AIR) effect of self-assembling peptides, efficient enrichment of probes at the disease site can be achieved, enhancing the signal-to-noise ratio of imaging. Small molecule probes often suffer from issues such as easy metabolism and poor signal-to-noise ratios. Probe molecule-modified self-assembling peptide materials can utilize in vivo assembly strategies, where the microenvironment at the disease site prompts the formation of nano-assemblies from the peptide-probe molecules. The AIR effect of peptide assembly can slow down the metabolism of probe molecules in the disease region, prolonging their retention time and thereby enhancing probe signals and the signal-to-noise ratio.

(3) Alkyl Chain Modification: Alkyl chains are utilized to adjust the hydrophilic-hydrophobic balance and enhance assembly capabilities. As simple hydrophobic units, fatty chains can be employed to regulate the hydrophilic-hydrophobic balance of self-assembling peptides, enabling the formation of more controllable assemblies. The modification with fatty chains is straightforward and convenient, typically achieved by coupling fatty acids to peptides using solid-phase synthesis. Additionally, lipid-like modifications of peptides can enhance the ability of the assemblies to form particles, making them suitable for drug delivery applications.

(4) Polymer Modification: Polymers possess large steric hindrance, which can hinder peptide assembly, but their unique properties can be harnessed in specific strategies. Polyethylene glycol (PEG) is a low-toxicity, low-immunogenic compound that is commonly used as a modification unit for amphiphilic assembly molecules. It enhances the hydrophilic properties of the modified molecule's hydrophilic end, reduces material delivery losses, and prolongs the molecule's circulation time in the body. PEG-NHS (N-hydroxysuccinimide ester of PEG) can be conveniently coupled to peptides through solid-phase synthesis to obtain PEGylated peptides.

(5) Glycosylation Modification: Sugars participate in various physiological activities within organisms, and glycopeptide self-assemblies can engage in these activities through multivalent coordination effects, enzymatic cleavage, and other mechanisms. For instance, mannose can target macrophages, and the hydroxyl groups on sugars significantly enhance the hydrophilicity of the entire molecule. Therefore, through chemical modification, the coupling of glycans to peptides enables a wide range of applications.

(6) Other Molecular Modifications: In addition to the molecules mentioned above, the introduction of other small molecular units can also significantly impact self-assembling peptides. For instance, alkaline phosphatase (ALP), which exhibits dephosphorylation activity and is overexpressed on the surface of various cancer cells, is widely used to control the self-assembly of amphiphilic peptides. Phosphate groups, though small in size, possess strong hydrophilic properties, and their introduction into peptides can increase the overall hydrophilicity of the molecule. Naphthalene units (Nap) can serve as capping groups for peptides, leveraging the π-π interactions of naphthalene to enhance the self-assembly and gelation capabilities of short peptides. 2-Naphthylacetic acid can be conveniently coupled to peptides through solid-phase synthesis.

2.Indirect Modification of Polypeptides

To achieve certain biological functions or design molecules, it is necessary to attach various functional molecules to polypeptides. However, due to factors such as large steric hindrance of some functional molecules or the absence of functional groups that can react with polypeptides, it may be necessary to introduce other molecular units, where one end connects to the polypeptide and the other end connects to the functional molecule unit. There are numerous methods for indirect modification of polypeptides, including modifications with alkyne and azide groups, anhydride modifications, and polyethylene glycol (PEG) modifications. The Husigen cycloaddition reaction between azide and alkyne groups can be used for the modification of polypeptides. When molecules possess hydroxyl, amino, or other groups that can react with carboxyl groups, cyclic anhydrides can be selected as linking units for modifying polypeptides. Under certain conditions, a hydroxyl group attacks the carbonyl carbon of the anhydride, resulting in a ring-opening nucleophilic substitution reaction to form an ester group, while the other end exposes a carboxyl group that can be connected to a polypeptide through solid-phase synthesis. PEG, a hydrophilic polymer with excellent biocompatibility, is often used in biomaterials. Short PEG chains can serve as linking units to connect functional molecules to polypeptides.

Related Reading: "Mechanisms of Polypeptide Self-Assembly and Their Applications in Biomedical Fields"

References:

[1] Ren Han, Li Ruxiang, Chen Zhijian, et al. "Modification Methods and Applications of Self-Assembling Polypeptides." Journal of Organic Chemistry, 2021, 41(10): 3983-3994.

[2] Yu Weikang, Zhang Shanshan, Yang Zhanyi, et al. "Applications of Supramolecular Polypeptide Self-Assembly in Biomedicine." 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.