Self-assembling biomaterials have attracted widespread attention due to their potential applications in biomedical imaging, drug delivery, and disease diagnosis and treatment, making them one of the frontier research areas in recent years. The self-assembly processes of biomolecules such as proteins and peptides are widely present in the physiological activities of living organisms, and their assemblies exhibit good biocompatibility and controllable assembly functions in vivo, enabling aggregation and retention at lesion sites. Over the past few decades, researchers have been inspired by the understanding of amino termini, natural peptides, and proteins to construct self-assembling peptides.
A typical self-assembling peptide is KLVFF, whose sequence is derived from the β-amyloid protein associated with Alzheimer's disease. In addition, many peptide sequences with self-assembly potential have been screened from natural proteins. The driving forces of peptide self-assembly mainly include π-π stacking, electrostatic interactions, hydrogen bonding, and hydrophobic interactions, which facilitate the formation of specific nanostructures. Researchers studying natural self-assembling peptides have discovered that the dipeptide sequence FF exhibits excellent self-assembly ability due to the π-π stacking interactions of the side-chain benzene rings. Based on FF as the assembly core, various functional self-assembling molecules have been designed. The secondary structures of peptides in assemblies are usually β-sheets and α-helices, while the assembled morphologies are diverse, leading to different applications.
Moreover, self-assembling peptides exhibit the Assembly-Induced Retention (AIR) effect, meaning that peptides undergo self-assembly due to endogenous factors in vivo, allowing long-term retention at lesion sites, enhancing aggregation and efficacy, and reducing toxicity to normal tissues. Additionally, some small molecules also have a tendency to self-aggregate, such as chlorophyll molecules containing large conjugated π-systems. By modifying these molecules onto self-assembling peptides, they can not only enhance imaging capabilities but also improve the assembly ability of target molecules.
In recent years, interdisciplinary developments have led to increased applications of self-assembling peptides in biological and medical fields. The characteristics of self-assembling peptides inspire us to use peptide self-assembly strategies to address current challenges faced by small-molecule drugs, such as poor recognition and enrichment at lesion sites, susceptibility to degradation in systemic circulation, and significant toxicity to normal cells and tissues. Therefore, how to modify these functional molecules onto peptides is a valuable research question. To enable the broader application of self-assembling peptides, the selection of modification methods needs to fully consider factors such as the active groups of functional molecules, steric hindrance, synthesis efficiency, and molecular functionality. Choosing an appropriate synthesis method can significantly enhance peptide modification efficiency. This paper summarizes the current research on self-assembling peptides, particularly those based on FF dipeptide as the assembly core, to provide methodologies and ideas for future studies.
The main functional modification sites of self-assembling peptides include the main-chain amino and carboxyl groups, as well as the side-chain amino, carboxyl, hydroxyl, and thiol groups. The modification methods are mainly divided into two types: direct modification and indirect modification.
Direct modification involves the covalent conjugation of functional molecules to peptides, either directly or after activating reactive groups. The main types of functional molecules involved in this method include:
1. Drug molecules, which can be directly modified onto peptides to achieve precise drug delivery and disease treatment.
2. Probe molecules, which utilize the AIR effect of self-assembling peptides to achieve efficient probe enrichment at lesion sites, enhancing imaging signal-to-noise ratio.
3. Alkyl chains, which regulate hydrophilic-hydrophobic balance and enhance assembly ability.
4. Polymers, which, due to their large steric hindrance, may hinder peptide assembly but are required in specific strategies.
5. Carbohydrate, which participate in various physiological activities in vivo. Glycopeptide assemblies can be involved in physiological activities through multivalent coordination effects and enzymatic cleavage.
6. Other small molecules, as there are many additional small molecules valuable for peptide assembly beyond those mentioned above, and we will discuss some of the most commonly applied ones.
Indirect modification involves using linker units to connect functional molecules with peptides. Some molecules have large steric hindrance or lack active groups that efficiently conjugate with peptides, necessitating the introduction of other molecules as linkers.
Table.1 Custom conjugation service at Creative Peptides.
Direct Modification
Drug Molecular Modifications
Utilizing the recognition, therapeutic properties, and hydrophilic-hydrophobic characteristics of small-molecule drugs, covalently conjugating them with self-assembling peptide materials can prolong drug retention, enhance efficacy, and reduce toxic side effects. To facilitate the attachment of drug molecules to peptides, certain functional groups of the drug molecules need to be activated.
For example, chlorambucil (CRB), also known as Leukeran, is a commonly used anticancer drug. The positive butyric acid group of CRB can be covalently linked to the N-terminus of peptides through standard solid-phase peptide synthesis (SPPS). Specifically, using 1H-benzotriazolium-1-[bis(dimethylamino)methylene]-5-chloride-hexafluorophosphate (HBTU) as a coupling reagent, the carboxyl group of CRB is activated and reacts with the free amino terminus of the peptide to form an amide bond. In 2020, a new self-assembling short peptide, CRB-YpYY, was designed, in which CRB functioned as both a drug delivery agent and a capping group. It was covalently conjugated to the amino terminus of the phosphorylated short peptide YpYY via amide condensation, replacing commonly used naphthalene and fluorene-based capping groups. CRB-YpYY is cleaved by alkaline phosphatase overexpressed in tumor regions, converting into CRB-YYY and undergoing in situ self-assembly to form a gel. Compared to free CRB molecules, this gel exhibits better tumor inhibition effects. This strategy of replacing conventional capping groups with drug molecules provides a new approach for designing functionalized short peptide gelators.
Camptothecin (CPT) is an anticancer drug with strong therapeutic effects on gastrointestinal cancer, liver cancer, and head and neck cancer. The hydroxyl group of camptothecin can be activated to facilitate its covalent conjugation to the exposed amino terminus of peptides through solid-phase synthesis. In an alkaline environment, camptothecin reacts with p-nitrophenyl chloroformate (npc) to form CPT-npc, allowing camptothecin to be more easily linked to peptides. The activated hydroxyl group on the camptothecin molecule is further conjugated to the exposed amino terminus of the peptide. After drug molecules are modified onto peptides, the entire molecule can form self-assembled structures. These assemblies exhibit more controllable hydrophilicity and can undergo enzyme-responsive drug release at specific locations, achieving precise drug delivery and sustained release.
Probe Molecular Modifications
Small-molecule probes often face challenges such as rapid metabolism and low signal-to-noise ratio. Self-assembling peptide materials modified with probe molecules can utilize the in vivo assembly strategy, where the microenvironment of the lesion site induces the formation of nano-assemblies of peptide probes. The AIR effect of peptide self-assembly can slow down the metabolism of probe molecules in the lesion area, prolong their retention time, and thereby enhance the probe signal and signal-to-noise ratio.
4-Nitro-2,1,3-benzoxadiazole (NBD) is an environment-sensitive fluorescent group that exhibits enhanced fluorescence in hydrophobic environments, making it suitable for self-assembly-based imaging in specific regions. The k(NBD) module, NBD-ɛ-D-lysine, was synthesized using NBD-Cl, which can be directly covalently linked to the exposed amino terminus of peptides via solid-phase synthesis. The synthesis process involves a nucleophilic substitution reaction between Boc-protected lysine and NBD, followed by deprotection to obtain the intermediate k(NBD), which is then used for peptide modification through a coupling reaction.
In 2019, a peptide modification method was applied to incorporate NBD, leading to the design of a peptide derivative, D-2, covalently linked to thymine. This derivative contains a phenylalanine sequence (FF) that facilitates self-assembly and an aspartic acid sequence (KKFKLKL) that specifically binds to adenosine triphosphate (ATP). D-2 undergoes self-assembly to form oligomers, which further assemble into complexes upon binding to RNA. The thymine residues in the fibers trigger DNA damage during DNA replication, inducing apoptosis. The entire process can be monitored by observing NBD fluorescence.
Near-Infrared Fluorescent Probes
Since cellular and tissue autofluorescence is minimal in the near-infrared (NIR) region, using NIR probes for imaging can significantly enhance tissue penetration while providing higher specificity and sensitivity. A chlorine-substituted NIR cyanine dye, Cy-Cl, was synthesized using a method that allows it to undergo a nucleophilic substitution reaction with the thiol side chains of peptides in Tris-Cl buffer (pH = 7.5). This reaction produces Cy-modified self-assembling peptides, which can be purified and utilized for detecting various diseases.
In 2019, an in situ self-assembling fluorescent probe was developed for detecting small tumors. When this molecule circulates through the bloodstream and reaches tumor-associated fibroblasts, it is cleaved by fibroblast activation protein-α (FAP-α), which is overexpressed in these cells. This cleavage triggers in situ self-assembly, leading to efficient accumulation at the tumor site, enhancing probe sensitivity and imaging capabilities, and enabling effective imaging of tumors as small as 2 mm.
Aggregation-Induced Emission (AIE) Fluorescent Probes
Fluorescent probes with aggregation-induced emission (AIE) properties have gained widespread attention in recent years. Unlike conventional fluorophores, which tend to undergo fluorescence quenching upon aggregation, AIE molecules exhibit enhanced fluorescence when in an aggregated state while showing minimal emission in solution. Due to this characteristic, combining AIE units with self-assembling peptides results in highly efficient fluorescent probes.
First, AIE molecules typically have large conjugated systems, and their strong hydrophobicity provides a driving force for self-assembly. The π-π interactions between molecules further stabilize the assembled structures, reinforcing the assembly ability of peptide probes. Second, the AIE effect increases the fluorescence contrast between the assembled structures and free molecules, significantly improving the signal-to-noise ratio. Meanwhile, peptide self-assembly promotes AIE molecule aggregation. As a result, compared to other fluorescent probe modifications, AIE fluorophore-modified self-assembling peptides offer distinct advantages in medical applications such as imaging and diagnostics.
A biperylene molecule (BP) with AIE properties was first synthesized, featuring a biperylene structure that forms J-type nanoaggregates. The synthesis process involves a Friedel-Crafts acylation reaction between perylene and alkyl (aryl) dichlorides in the presence of aluminum chloride, followed by carboxyl derivatization. The resulting product can be covalently linked to the free amino terminus of peptides via amide bonds through solid-phase synthesis.
In 2016, BP was incorporated into self-assembling peptide materials, resulting in the supramolecular molecule BKP. BKP consists of three components: BP, the KLVFF self-assembling peptide sequence, and polyethylene glycol (PEG). The hydrophobicity and π-π stacking interactions of BP promote peptide self-assembly, while the PEG chain balances the overall hydrophilicity and hydrophobicity of the molecule. This study explored the effect of PEG chain length on the self-assembly process and the morphology of the final assembled structures.
In 2017, a similar self-assembling peptide molecule, BP-KLVFF-His6-PEG, was designed to construct a nested structure at tumor sites. The His6 module, composed of six histidine residues, is positively charged and undergoes protonation in the mildly acidic tumor microenvironment, increasing hydrophobicity and promoting self-assembly. Upon injection into tumor-bearing mice, this molecule responds to the acidic tumor environment by triggering self-assembly into fibrous structures. These assembled structures can capture free small molecules such as Nile red (NR), indocyanine green (ICG), and doxorubicin (DOX), embedding them within β-sheets, which enhances fluorescence intensity at the tumor site by 7.5-fold.
In 2020, a similar strategy was applied to develop BP-modified self-assembling peptide nanomaterials that mimic human defensin-6, laminin, and platelets, further enabling imaging of lesion sites.
Alkyl Chains Modification
Alkyl chains are structurally simple hydrophobic units that can be used to regulate the hydrophilic-hydrophobic balance of self-assembling peptides, enabling the formation of more controllable assemblies. Alkyl chains modification is a straightforward process, typically accomplished via solid-phase synthesis, allowing for the facile conjugation of fatty acids to peptides.
In 2013, an extracellular matrix was designed to influence neuronal differentiation and maturation. This matrix consists of fibers formed by the self-assembly of a peptide amphiphile (PA), composed of a hexadecyl chain and a hydrophilic peptide sequence KKKGKDD. In an aqueous environment, the hexadecyl chain tends to aggregate internally to form a hydrophobic core, while the hydrophilic peptide remains exposed. Due to the presence of charged amino acids in the peptide sequence, the combination of the hydrophobic core and electrostatic interactions drives the formation of β-sheet structures, leading to the orderly assembly of fibers. The study demonstrated that this PA-assembled matrix effectively regulates hippocampal neuron development.
In 2019, the effect of peptide chirality on cytotoxicity and cell membrane binding was investigated using alkylated D- and L-type V3A3K3 self-assembling peptides.
The modification of alkyl chains is not solely for regulating the hydrophilic-hydrophobic balance. it also serves other functions. In 2018, a diacetylene (DA)-modified peptide, DA-EGGGGH, was designed for the naked-eye detection of metal ions. The EGG peptide sequence enhances the hydrophilicity of the peptide terminus, while GGH coordinates with zinc and copper ions to trigger self-assembly. A diacetylene unit, 10,12-pentacosadiynoic acid, was conjugated to the amino terminus of the peptide. Upon self-assembly, UV irradiation induces polymerization between the diacetylenes, forming blue polydiacetylene. Since the molecule only assembles into a gel in the presence of metal ions and exhibits a blue color upon UV exposure, this property enables rapid and sensitive naked-eye detection of metal ions.
Polymer Modification
Polyethylene glycol (PEG) is a low-toxicity, low-immunogenicity compound commonly used to modify amphiphilic self-assembling molecules. PEG enhances the hydrophilicity of the modified molecules, reduces material loss during delivery, and prolongs their circulation time in the body. PEG-NHS can be conveniently conjugated to peptides via solid-phase synthesis to obtain PEGylated peptides.
In 2017, a self-assembling peptide-polymer conjugate (P-S-H) was designed to generate toxicity in response to the tumor microenvironment. This conjugate consists of a hydrophobic polymer backbone (P), a PEG-modified matrix metalloproteinase-2 (MMP-2)-responsive sequence GPLGIAGQC (S), and a pH-responsive peptide HLAH (H). In a neutral environment, P-S-H forms nanoparticles with P as the hydrophobic core and S as the shell. Upon entry into the tumor microenvironment (pH ≤ 6.5) and exposure to overexpressed MMP-2, the acidic pH and enzyme-cleavable sequence trigger the removal of the PEG protective layer. The resulting hydrogen ion-encapsulated positively charged nanoparticles efficiently internalize into cells, disrupt mitochondria, and induce apoptosis.
That same year, an endogenous stimulus-induced aggregation (eSIA) strategy was reported. The thermosensitive polymer used in the study exhibits a phase transition below 37 ℃, and each unit contains a terminal alkyne that undergoes a Michael addition reaction with the thiol group on an enzyme-responsive peptide side chain. The polymer reacts with 1.3 equivalents of peptide and 2.1 equivalents of triethylamine in dimethyl sulfoxide, stirred at 37 °C for 12 hours, and purified via dialysis. The resulting peptide-modified polymer exhibits a phase transition temperature above 37 °C. When exposed to endogenous enzymes in vivo, the peptide is cleaved from the polymer backbone, reducing the phase transition temperature below 37 °C, allowing body temperature to trigger nanoparticle assembly. This innovative strategy can be applied for tumor imaging.
Glycosylation Modification
Carbohydrates participate in various biological processes in the human body. For instance, mannose can target macrophages, and the hydroxyl groups on carbohydrates significantly enhance molecular hydrophilicity. Thus, chemically modifying peptides by conjugating glycosyl groups enables diverse applications.
A glycosylated tyrosine derivative, Fmoc-Tyr- [β-D-Glc (OAc)4]-OPfp, was synthesized as an amino acid module for solid-phase glycopeptide synthesis. The synthesis process involves protecting the carboxyl group of Fmoc-Tyr-OH with pentafluorophenol to obtain Fmoc-Tyr-OPfp. This protected amino acid is then reacted with β-D-galactose pentaacetate, where the hydroxyl group on the tyrosine side chain reacts with the acetyl groups on the sugar, yielding a glycosylated amino acid. The final product is conjugated to peptides via solid-phase synthesis. After synthesis, the acetyl protecting groups on the sugar and the pentafluorophenyl group are removed, ultimately linking the glyco-amino acid to the peptide. The glycosidic bond in this glycopeptide can be enzymatically cleaved, altering the molecule's hydrophilicity and leading to the formation of nanostructures from the peptide cleavage residues.
Other Small Molecules Modification
In addition to the aforementioned molecules, the introduction of other small molecular units can also significantly impact the self-assembly of peptides. Below is a summary of some common and widely used units.
Phosphorylation Modification
Alkaline phosphatase (ALP) exhibits dephosphorylation activity and is overexpressed on the surface of various cancer cells, making it widely used to control the self-assembly of amphiphilic peptides. Phosphate molecular groups are small in volume but highly hydrophilic. The introduction of phosphate groups into peptides increases the overall hydrophilicity of the molecule.
In 2018, a self-assembling material, NBD-GFFpY-ss-ERGD, was designed for selective uptake by liver cancer cells. This molecule consists of three modules: (1) The fluorescent molecule NBD serves as a probe group. (2) The phosphorylated peptide segment GFFpY is cleaved by ALP overexpressed on the liver cancer cell surface, triggering nanoparticle assembly. (3) The ERGD targeting peptide module is linked to the assembly sequence via a disulfide bond. Once the nanoparticles enter the cell, intracellular overexpression of glutathione cleaves the disulfide bond, leading to fiber assembly. Through multiple recognition steps by liver cancer cells, this molecule achieves selective uptake of materials by liver cancer cells, providing a useful strategy for the design of supramolecular nanomaterials for liver cancer diagnosis and therapy.
In 2020, phosphorylated tryptophan-rich short peptides were designed to create a lysosome-accumulating molecule. Compared with hydrophobic phenylalanine-rich peptides, tryptophan-rich peptides more easily accumulate in lysosomes and exhibit lower cytotoxicity. This study provides a basis for substituting strong hydrophobic amino acids with tryptophan in self-assembling peptide research.
A carrier molecule was designed for delivering the drug bortezomib (BTZ) specifically within tumor cells, which releases the phosphorylated carrier upon cleavage. The molecular composition consists of two parts: (1) The AVPI peptide segment, which targets inhibitor of apoptosis proteins (IAPs) to induce apoptosis, and (2) Nap-pYFF, which triggers self-assembly in response to alkaline phosphatase (ALP). The carrier molecule forms micellar particles in vitro, encapsulating BTZ in the hydrophobic core. Phosphate groups on the micellar surface recognize ALP on tumor cell surfaces, enabling endocytosis. After enzymatic cleavage, the particle morphology changes to fibers, releasing BTZ and allowing AVPI on the fiber surface to bind to IAPs in cancer cells, promoting apoptosis. This delivery method significantly reduces BTZ toxicity to normal cells, providing a novel low-toxicity drug delivery strategy.
Naphthalene Group Modification
Naphthalene-containing units (Nap) can serve as capping groups for peptides, utilizing π-π interactions of naphthalene to enhance the self-assembly and gelation capacity of short peptides. 2-Naphthylacetic acid can be conveniently coupled to peptides via solid-phase synthesis.
In 2017, a Nap-capped self-assembling D-peptide, Nap-GFFY, was designed to form a three-dimensional nanofiber network capable of encapsulating ovalbumin (OVA) for use as a vaccine adjuvant. Compared with free OVA, the adjuvant significantly enhances immune effects. The study also verified that substituting Nap with biotin, Fmoc, or phenothiazine (PTZ) reduces the vaccine adjuvant effect. This finding demonstrates that an appropriate aromatic capping group is crucial for the vaccine adjuvant efficacy of GFFY.
In 2019, a novel peptide-based supramolecular protein gel (Nap-GFFYK(γE)2-NH2) was designed. Upon binding with proteins, this compound rapidly folds into a β-sheet structure and co-assembles with proteins into nanofibers and hydrogels. This supramolecular protein gel can co-assemble with glucose oxidase/horseradish peroxidase (GOx/HRP) and GOx/cytochrome c (cyt c) to form nanofibers, significantly enhancing the catalytic activity of cascade enzyme reactions. These findings demonstrate the great potential of supramolecular protein hydrogels in constructing various protein complexes and advancing supramolecular therapeutics.
Indirect Peptide Modification
When functional molecular units need to be conjugated to peptides through linker groups, this modification method is referred to as indirect modification. To achieve specific biological functions or molecular design, different types of functional molecules need to be attached to peptides. However, due to factors such as steric hindrance of some functional molecules or the lack of functional groups in the molecular units that can react with peptides, other molecular units must be introduced—one end connecting to the peptide and the other end to the functional molecular unit—to achieve the desired outcome. There are many methods for indirect peptide modification, and some of the more common ones are introduced below.
Table.2 Peptide modification service at Creative Peptides.
Modification via Alkynyl and Azido Groups
Click chemistry is a simple and efficient reaction approach, with the azide-alkyne Huisgen cycloaddition reaction being a representative example. This method can be applied in peptide modification. Solid-phase synthesis was employed to attach hexynoic acid to the amino terminus of a peptide, followed by modification of small molecules using azidoacetic acid. The azide-modified peptide then underwent a click reaction with alkynyl-containing small molecules, forming a triazole at the connection site to yield the final product. The advantages of this peptide modification method include high molecular utilization, mild reaction conditions, and stability of the final product in complex physiological environments. Additionally, the carbon chain length of the linker group can be adjusted according to molecular design, making it suitable for small molecules that are otherwise difficult to efficiently conjugate to peptides.
A peptide molecule was designed and synthesized, incorporating chlorophyll derivatives and mannose for eliminating intracellular infections. This molecule utilized click chemistry between alkynyl and azido groups, with hexynoic acid serving as the linker unit to connect mannose to the peptide. The chlorophyll unit enabled the molecule to assemble into a "sandwich" dimer structure, exposing mannose on the peptide surface. Mannose then targeted macrophages and entered cells via receptor-mediated endocytosis, effectively killing intracellular Staphylococcus aureus.
In 2020, a similar method was used to conjugate furan-based nitric oxide donors to peptides. The nitric oxide donor was attached to the peptide through click chemistry between azide and alkyne groups, offering flexibility in modification, as the positions of the azide and alkyne groups could be interchanged. That same year, another approach employed click chemistry to link a peptide sequence at both ends of an aggregation-induced emission (AIE) molecule, tetraphenylethylene. The FF sequence in the peptide segment exhibited strong self-assembly capability, leading to molecular aggregation and fluorescence activation upon assembly, which was used for pancreatic tumor imaging.
In 2015, a near-infrared fluorescence-activatable probe [DBT-2(EEGK-maleimide)] was reported, consisting of three parts: (1) a central environmentally sensitive unit, 4,7-di(2-thienyl)-2,1,3-benzothiadiazole (DBT), (2) peptide sequences EEGK flanking the DBT unit for hydrophilic balance, and (3) a maleimide group as the capping group. Using solid-phase synthesis, azidoacetic acid was conjugated to the peptide, followed by maleic anhydride coupling to alanine and further modification with NHS to activate the carboxyl terminus for attachment to the lysine side-chain amino group. Finally, click chemistry between alkyne and azide groups was used to connect the components, yielding the final product. This molecule exhibited weak fluorescence in solution but assembled into nanoparticles with thiol-containing proteins, forming a hydrophobic region that activated DBT fluorescence. This method was used to detect protein thiols in cells, demonstrating its broad applicability.
In complex biological environments, reaction and assembly conditions can be stringent, necessitating highly efficient and rapid chemical reactions. Click chemistry meets these requirements. In 2019, a recognition-reaction-aggregation (RRA) cascade strategy was proposed for kidney cancer treatment. The molecule P1-DBCD consisted of a targeting peptide and a DBCD unit containing an alkyne group. After entering the body, it specifically targeted renal carcinoma cells. The molecule P2-N3 was a peptide containing the assembly sequence KLVFF, with an amino-terminal modification of hexynoic acid as a linker unit and a fluorescent Cy dye attached to its side chain. When P1-DBCD localized on the cell surface, P2-N3 reached the tumor region and rapidly reacted with P1-DBCD via click chemistry, expanding the hydrophobic unit. This led to the formation of fibrils on the cell surface, disrupting the cancer cell membrane and enhancing the sensitivity of cancer cells to the chemotherapy drug DOX. These findings demonstrate that this peptide modification approach is viable in complex biological environments, providing new insights into the application of self-assembling peptides.
Modification via Anhydrides
When molecules contain hydroxyl, amino, or other groups that can react with carboxyl groups, cyclic anhydrides can be used as linker units for peptide modification. Under certain conditions, the hydroxyl group attacks the carbonyl carbon of the anhydride, undergoing a nucleophilic ring-opening substitution reaction to form an ester group. The other end of the anhydride exposes a carboxyl group, which can be linked to peptides through solid-phase synthesis.
Curcumin is a phenolic antioxidant with significant inhibitory effects on liver cancer cells, but its poor water solubility limits its widespread application. In 2017, a peptide derivative capable of forming a hydrogel was synthesized for liver cancer treatment. The structure GA-GFFYK(Cur)E-ss-ERGDd consists of an assembly sequence GFFYK and a hydrophilic sequence ERGD, connected by a disulfide bond. The amino terminus of the peptide is conjugated with glycyrrhetinic acid (GA) as a targeting moiety for liver cancer cells. Curcumin first reacts with glutaric anhydride, and the carboxyl group of the resulting product is activated using dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) before being linked to the peptide through solid-phase synthesis. The disulfide bond in this molecule can be recognized and reduced by glutathione (GSH) in cancer cells, leading to bond cleavage, peptide assembly, and the controlled release of curcumin.
In 2018, a self-assembling peptide molecule, Curcumin-FFE-CS-EE, was first reported for tumor treatment. This molecule consists of two peptide segments and curcumin, where the self-assembling short peptide and hydrophilic short peptide are connected by a disulfide bond, and curcumin is conjugated to the peptide through glutaric anhydride. In the presence of glutathione (GSH), the disulfide bond is cleaved, disrupting the amphiphilic balance and allowing the peptides to self-assemble into nanofibers and hydrogels. This hydrogel acts as a sensitizer, significantly reducing colorectal tumor volume when used in combination with radiotherapy.
Modification via Polyethylene Glycol (PEG)
Polyethylene glycol (PEG) is a hydrophilic polymer with excellent biocompatibility, making it widely used in biomaterials. Short PEG chains can serve as linker units to conjugate functional molecules with peptides. A research group utilized Fmoc-NH-(PEG)2-COOH as a linker unit and synthesized PEG-linked peptides through solid-phase synthesis. Due to the flexibility of PEG chains, the small molecular units or peptide units at both ends are less likely to interfere with each other, allowing them to retain their biological functions.
In 2017, a molecule, Pam3CSK4-K5, capable of self-assembling into nanoparticles was designed. This amphiphilic molecule features three alkyl chains connected to the amino terminus and side chain of the peptide, serving as the hydrophobic segment. The hydrophilic segment consists of oligolysines K4 and K5, linked through a PEG unit. Through electrostatic and hydrophobic interactions, the molecule self-assembles into nanoparticles. When combined with three types of immune stimulants, it forms self-assembling nano immune stimulators (SANIs), which can be used in tumor immunotherapy to inhibit tumor growth.
Summary
In recent years, self-assembling peptides have been widely applied in biological and medical research fields. Their advantages lie in their ability to arrange in an orderly manner through intermolecular interactions such as hydrogen bonding, forming well-structured and controllable assemblies. Additionally, self-assembling peptides can be driven to assemble in situ by endogenous factors in the body. Therefore, they can serve as excellent carriers that combine with functional molecules via covalent bonding or encapsulation, enhancing the efficacy of drugs, probes, and other molecules.
When functionalizing peptides, selecting an appropriate modification method can significantly improve efficiency. The choice of modification approach depends on factors such as the function of the modifying molecule, steric hindrance, and the presence of suitable functional groups for peptide conjugation. Direct modification offers advantages such as fewer synthesis steps and the feasibility of solid-phase synthesis, which greatly simplifies the process. However, some functional molecules lack reactive groups necessary for direct conjugation with peptides, making direct modification inefficient in terms of reaction yield and product purity. In contrast, indirect modification provides higher reaction efficiency, mild reaction conditions, and broad applicability. It is particularly suitable for molecules that are difficult to react directly with peptides or need to maintain a certain distance from them. However, this method requires the introduction of linker units, resulting in a greater spatial separation between the functional molecule and the peptide, as well as additional synthesis steps.
In summary, selecting an appropriate modification strategy based on the characteristics and functional requirements of the functional molecule is crucial for self-assembling peptide modification. It also presents a challenge in the development of new functionalized derivatives of self-assembling peptides. In recent years, an increasing number of researchers have been exploring the biological applications of self-assembling peptides, which has raised the demand for more advanced functionalization strategies. Therefore, continuous expansion of novel functionalization methods is necessary to provide new options and directions for the synthesis and application of self-assembling peptide derivatives.
References
- Jiang, Qiaochu, et al., Self-assembly of peptide nanofibers for imaging applications. Nanoscale 13.36 (2021): 15142-15150.
- Yang, Shihua, et al., Self-assembled short peptides: Recent advances and strategies for potential pharmaceutical applications. Materials Today Bio 20 (2023): 100644.
- La Manna, Sara, et al., Self-assembling peptides: From design to biomedical applications. International journal of molecular sciences 22.23 (2021): 12662.
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