Functional peptides can perform different biological and engineering functions through sequence and structural design. Cell-penetrating peptides are primarily used to improve the cellular uptake and intracellular delivery of macromolecular and hydrophilic cargoes; targeting peptides increase local accumulation by recognizing specific receptors or tissue markers; self-assembling peptides construct local delivery systems and tissue scaffolds by forming supramolecular structures such as nanofibers and hydrogels. These three functions can also be combined through sequence fusion, chemical conjugation, or carrier assembly to achieve targeted recognition, cellular internalization, cargo delivery, and material construction.

Keywords: cell-penetrating peptides; targeting peptides; self-assembling peptides; TAT; RGD; iRGD; RADA16; peptide-drug conjugates; drug delivery

1 Structural Basis and Classification of Functional Peptides

1.1 Peptide Structure, Physicochemical Properties, and Functional Basis

Peptides are formed by the sequential linkage of amino acids through amide-type peptide bonds and contain an N terminus and a C terminus. Compared with proteins, peptide chains are generally shorter, although no universal chain-length boundary separates the two. Short peptides can form local structures such as α-helices, β-sheets, and β-turns. Under conditions close to physiological pH, the side chains of Arg and Lys are generally positively charged, whereas those of Asp and Glu are generally negatively charged; residues such as Leu, Ile, and Val are strongly hydrophobic, while Phe, Tyr, and Trp contain aromatic side chains. Amino acid composition, sequence, and spatial conformation jointly determine a peptide’s net charge, solubility, membrane affinity, and molecular-binding interface, while cyclization, terminal modification, D-amino acid substitution, and conjugation with hydrophobic groups can further alter peptide-chain flexibility, protease stability, and receptor-binding properties.

1.2 Functional Differences Among Cell-Penetrating, Targeting, and Self-Assembling Peptides

Cell-penetrating peptides (CPPs) promote cellular internalization or membrane translocation through peptide–membrane interactions; targeting peptides recognize cell-surface receptors, vascular markers, or extracellular-matrix components through specific sequences; self-assembling peptides (SAPs) depend on noncovalent interactions among multiple peptide molecules to form ordered supramolecular structures. Receptor binding by a targeting peptide does not necessarily result in membrane translocation, increased cellular uptake mediated by a CPP does not imply tissue selectivity, and self-assembly describes the process by which peptide molecules form higher-order structures.

2 Cell-Penetrating Peptides: Membrane Interactions and Cellular Internalization

2.1 Cell-Membrane Barriers and the Structural Basis of CPPs

The phospholipid bilayer forms the basic structure of the cell membrane, with hydrophilic phospholipid head groups facing the aqueous environment and hydrophobic fatty-acid chains forming the low-polarity membrane interior; charged molecules, hydrophilic macromolecules, proteins, and nucleic acids generally cannot readily cross this barrier through simple diffusion. CPPs can be classified according to their physicochemical properties as cationic, amphiphilic, or hydrophobic. TAT and polyarginine are typical cationic CPPs, whereas Penetratin contains both cationic and hydrophobic residues. Different CPPs vary in their dependence on electrostatic interactions, hydrophobic interactions, and local membrane reorganization.

2.2 Membrane Binding Mediated by Positive Charge and Amphiphilicity

The guanidinium group of the Arg side chain can form electrostatic interactions and multidentate hydrogen bonds with negatively charged cell-surface components such as phospholipid head groups and heparan sulfate proteoglycans, promoting the accumulation of Arg-rich CPPs on the cell surface; hydrophobic residues can enhance peptide contact with lipid regions, while amphiphilic CPPs can form relatively distinct hydrophilic and hydrophobic surfaces, allowing membrane-surface adsorption and lipid interactions to occur cooperatively. Excessive positive charge or hydrophobicity may also increase nonspecific cell binding and membrane disruption.

2.3 Endocytosis and Direct Membrane Translocation

(1) Endocytic Pathways

CPPs can enter cells through energy-dependent endocytosis, during which the cell membrane encloses the CPP or CPP–cargo complex to form a membrane-bound vesicle; this process can involve macropinocytosis, clathrin-mediated endocytosis, caveolin-associated endocytosis, and other pathways. Large cargoes such as proteins, nucleic acid complexes, and nanoparticles generally depend more strongly on endocytosis.

(2) Direct Membrane Translocation

Direct membrane translocation does not involve the formation of a complete endocytic vesicle. Once a CPP reaches a sufficient local concentration on the membrane surface, it may induce lipid rearrangement. The transient-pore model involves the temporary formation of an aqueous channel, the carpet model involves disruption of lipid organization after peptide accumulation on the membrane surface, and the inverted-micelle model involves rearrangement of lipid head groups around the peptide molecule. The entry pathway used by the same CPP can be influenced by peptide concentration, cell type, membrane composition, and the conjugated cargo.

2.4 Endosomal Entrapment and Cytosolic Release

CPP–cargo complexes internalized through endocytosis are initially located within the endosomal lumen and do not directly enter the cytosol. As endosomes acidify and mature into late endosomes and lysosomes, failure of the cargo to cross the endosomal membrane can result in an effective cytosolic concentration substantially lower than the total cellular uptake. Some membrane-active peptides can undergo protonation, conformational changes, or enhanced membrane affinity in acidic environments, thereby promoting endosomal-membrane disruption and cargo release. For siRNA, mRNA, and proteins that act in the cytosol, endosomal escape is generally an important determinant of effective cytosolic delivery.

2.5 Modes of CPP–Cargo Association

CPPs can be covalently conjugated to small molecules, peptides, or proteins and can also form noncovalent complexes through electrostatic or hydrophobic interactions. DNA and RNA contain negatively charged phosphate backbones and can undergo electrostatic condensation with Arg- or Lys-rich CPPs to form complex particles; after cargo conjugation, particle size, net charge, hydrophobicity, and the degree of CPP exposure at the particle surface may all change. The membrane-translocation behavior of a free CPP therefore cannot directly represent the delivery characteristics of a CPP–cargo complex.

3 Targeting Peptides: Receptor Recognition and Tissue Homing

3.1 Molecular Basis of Target Recognition

Targeting peptides use specific amino acid side chains to form receptor-binding interfaces that recognize cell-surface proteins, vascular markers, or extracellular-matrix components, and the binding process is generally maintained through a combination of spatial shape complementarity, hydrogen bonding, electrostatic interactions, and hydrophobic interactions. Targeting sequences may originate from functional regions of natural ligands or be identified through methods such as phage display; changes in the positions of critical residues, cyclization, and side-chain modifications can all alter receptor-binding ability.

3.2 RGD Recognition by Integrins

RGD consists of Arg-Gly-Asp and is a classical cell-adhesion motif found in extracellular-matrix proteins such as fibronectin and vitronectin. Its primary receptors are integrins, which consist of α and β transmembrane subunits, with their extracellular regions binding extracellular-matrix components and their intracellular regions connecting to cytoskeletal and signaling proteins. Several integrins, including αvβ3, αvβ5, and α5β1, can recognize RGD, but different RGD derivatives vary in their affinity and selectivity for individual subtypes; by restricting peptide-chain conformation, cyclic RGD peptides can maintain critical residues in a relatively fixed spatial arrangement. Additional neighboring residues or functional groups can also be introduced to produce derivatives such as RGDS, GRGDS, and cyclo(RGDfK), thereby altering conformational stability, receptor binding, and material-conjugation properties.

3.3 NGR Recognition by CD13

NGR consists of Asn-Gly-Arg, and certain NGR peptides can recognize aminopeptidase N (APN/CD13) associated with tumor neovasculature. CD13 is also expressed by various normal cells, so NGR homing is additionally influenced by the molecular state of CD13, the vascular environment, and target accessibility. The Asn residue in NGR can undergo deamidation to form isoDGR, changing the peptide-backbone linkage and spatial conformation, and isoDGR can also interact with certain integrins.

3.4 Sequential Tissue-Penetration Mechanism of iRGD

iRGD contains an RGD motif and a potential C-end Rule (CendR) motif. Intact iRGD first binds tumor-associated αv integrins through its RGD region, after which proteolysis exposes an R/KXXR/K-type CendR motif, originally located within the peptide chain, at the newly generated C terminus; the exposed CendR motif then binds neuropilin-1 (NRP1), initiating internalization and tissue penetration. This process sequentially involves integrin-mediated homing, proteolysis, CendR-motif exposure, and NRP1-mediated penetration, and this sequential receptor recognition is an important feature distinguishing iRGD from conventional RGD-targeting peptides.

3.5 RVG-Mediated Targeted Delivery to the Nervous System

Peptides derived from rabies virus glycoprotein (RVG) exhibit nervous-system-associated targeting and delivery properties. RVG29 can be used to construct brain-targeted delivery systems, and the addition of a terminal Cys residue provides a thiol conjugation site; RVG-9R further incorporates a polyarginine segment, allowing it to bind negatively charged nucleic acids through electrostatic interactions while enhancing cellular uptake. When RVG-derived peptides are combined with nucleic acid cargoes such as siRNA, target recognition, nucleic acid binding, and nervous-system delivery can be integrated within the same system.

3.6 Receptor Affinity and Tissue-Targeting Efficiency

(1) Target Accessibility

Targeting efficiency is influenced not only by binding affinity but also by receptor density, spatial location, and internalization properties. Receptors located on the luminal surface of blood vessels can directly interact with circulating peptides, whereas receptors on cells deep within a tissue require the peptide to first cross the vascular endothelium and interstitial space.

(2) Multivalent Binding

Conjugation of peptides to nanocarriers can generate multivalent effects, allowing multiple ligands on the surface of the same particle to participate simultaneously or sequentially in receptor binding. Ligand density, spatial interval, and motif exposure all influence this interaction, so the affinity of a free peptide cannot directly substitute for the tissue-targeting performance of a peptide-modified carrier.

4 Self-Assembling Peptides: Molecular Assembly and Supramolecular Structure Formation

4.1 Self-Assembly and Noncovalent Driving Forces

Peptide self-assembly is the process by which multiple peptide molecules form ordered higher-order structures through noncovalent interactions and may proceed through different organizational levels, including peptide monomers, secondary structures or primary aggregates, nanostructures, and three-dimensional networks. Hydrogen bonds can form between peptide-backbone carbonyl groups and N–H groups, hydrophobic side chains tend to aggregate, oppositely charged residues can undergo electrostatic attraction, and aromatic residues such as Phe, Tyr, and Trp can also participate in aromatic interactions. Together, these forces influence the formation of nanofibers, nanotubes, micelles, hydrogels, and other structures.

4.2 β-Sheets and Nanofiber Formation

β-Sheets are stabilized by regularly arranged backbone hydrogen bonds between adjacent peptide chains or chain segments. When peptide chains adopt extended conformations, carbonyl and N–H groups pair continuously in specific directions to form sheet-like structures; when these arrangements continue to extend and undergo lateral stacking, they can form one-dimensional nanofibers. β-Sheets provide an important structural basis for many fibrous self-assembling peptides, although not all self-assembling systems depend on the same β-sheet pathway.

4.3 Ionic-Complementary Assembly of RADA16

RADA16 consists of repeating Arg-Ala-Asp-Ala units. Arg is positively charged, Asp is negatively charged, and Ala is hydrophobic, allowing the peptide chain to form a β-sheet structure with hydrophilic and hydrophobic surfaces. Peptide-backbone hydrogen bonding, hydrophobic interactions, and charge interactions promote ordered molecular organization and nanofiber formation, and further entanglement and physical crosslinking of the nanofibers generate a highly hydrated three-dimensional network. Macroscopic hydrogel properties also depend on fiber length, connection density, and network organization; formation of β-sheets or nanofibers therefore does not necessarily indicate that a hydrogel with stable material properties has already formed.

4.4 Ordered Assembly of FF and Other Aromatic Short Peptides

Diphenylalanine (Phe-Phe, FF) consists of two Phe residues. Aromatic interactions between the Phe side chains and hydrogen bonding within the peptide backbone jointly promote ordered molecular arrangement, allowing nanotubes, nanofibers, and other structures to form under different conditions. Introduction of aromatic protecting groups such as Z or Fmoc at the N terminus can further alter hydrophobic interfaces and aromatic interactions, thereby regulating aggregate morphology and supramolecular organization.

4.5 Assembly of Peptide Amphiphiles

Peptide amphiphiles (PAs) generally consist of a hydrophobic alkyl tail and a hydrophilic peptide segment. In aqueous environments, the hydrophobic tails aggregate within the structural interior, while the hydrophilic peptide segments remain exposed to the aqueous phase, and the peptide segments can further form hydrogen bonds or β-sheets that promote nanofiber formation; functional motifs involved in receptor binding or cell adhesion can also be incorporated into the hydrophilic terminus and displayed on the fiber surface.

4.6 Environmental Regulation of Assembly Equilibria

pH can alter the protonation states of ionizable residues such as Asp, Glu, and Lys, thereby affecting electrostatic attraction or repulsion; salt ions can screen electrostatic interactions; peptide concentration influences nucleation, fiber elongation, and interfiber entanglement. The same peptide sequence may therefore form aggregates with different sizes, morphologies, or network densities under different pH, ionic-strength, and concentration conditions.

5 Engineering Integration of the Three Functional Peptide Classes

5.1 Tandem Combination of Targeting Peptides and CPPs

Targeting peptides mediate receptor recognition and local accumulation, while CPPs promote membrane binding and cellular internalization. Connecting these two modules can integrate target recognition and intracellular delivery within the same molecular system. Continuous exposure of a strongly cationic CPP may increase binding to nontarget cell membranes, so some designs incorporate protease-sensitive linker sequences, pH-responsive structures, or removable shielding groups to restore CPP membrane activity within a specific microenvironment.

5.2 Multivalent Display of Targeting Peptides on Nanocarriers

When targeting peptides are conjugated to the surfaces of liposomes, lipid nanoparticles, or other nanocarriers, a single particle can display multiple ligands simultaneously and generate multivalent binding. The actual effect depends on ligand density, PEG-spacer length, and whether the targeting motif remains exposed on the outer particle surface; if the peptide becomes buried within the carrier during assembly or is affected by steric hindrance, its high affinity in the free state may not translate directly into effective cellular targeting.

5.3 Formation of CPP–Nucleic Acid Complexes

The phosphate backbones of DNA, siRNA, and mRNA carry high-density negative charges, and CPPs enriched in Arg or Lys can interact electrostatically with these molecules to form nanocomplexes. CPPs simultaneously perform nucleic acid-condensation and membrane-binding functions during this process, but excessive condensation may reduce nucleic acid-release efficiency. Effective delivery therefore requires simultaneous consideration of complex stability, cellular uptake, and intracellular dissociation.

6 Delivery and Biomaterial Applications of Functional Peptides

6.1 Targeted Therapeutic Cyclic Peptide: Cilengitide

Cilengitide is a cyclic pentapeptide containing an RGD motif that binds αvβ3 and αvβ5 integrins and inhibits integrin-mediated cell adhesion and related signaling. Unlike peptides used exclusively as carrier-targeting modules, the cyclic structure of Cilengitide itself performs both target-recognition and pharmacological-inhibition functions. The molecule entered clinical studies for glioblastoma, and early studies observed some antitumor activity; however, a subsequent phase III study did not show an improvement in survival when Cilengitide was added to standard therapy, and it ultimately did not achieve marketing approval. Cilengitide is therefore a representative example of an RGD-targeting peptide developed directly as a pharmacologically active molecule and also illustrates that receptor affinity does not necessarily correspond directly to final clinical efficacy.

6.2 Peptide-Drug Conjugate: ANG1005 for Blood–Brain Barrier Delivery

ANG1005 is a representative peptide-drug conjugate (PDC) formed by covalently connecting the brain-targeting peptide Angiopep-2 to multiple paclitaxel molecules. Angiopep-2 uses a low-density lipoprotein receptor-related protein 1 (LRP1)-associated transport system to facilitate transport across the blood–brain barrier and entry into brain-tumor tissue, paclitaxel acts as the drug payload to stabilize microtubules and produce cytotoxic effects, and the linker structure maintains peptide–drug conjugation while allowing payload release. ANG1005 has entered clinical studies, and intracranial antitumor activity has been observed in patients with breast-cancer brain metastases and leptomeningeal metastases, making it a representative example of combining a brain-targeting peptide, linker structure, and cytotoxic drug within a PDC.

6.3 Peptide–Radionuclide Conjugate: ¹⁷⁷Lu-DOTATATE

¹⁷⁷Lu-DOTATATE is a clinically translated peptide receptor radionuclide therapy (PRRT) system. Its targeting component is the somatostatin analog octreotate, which recognizes somatostatin receptors highly expressed by certain neuroendocrine tumors, particularly SSTR2; DOTA serves as a chelator that stably coordinates the radionuclide ¹⁷⁷Lu. After the complex accumulates in receptor-positive tumor tissue and undergoes receptor-mediated internalization, β radiation emitted by ¹⁷⁷Lu causes radiation damage to tumor cells. This system has been approved for selected somatostatin-receptor-positive neuroendocrine tumors and represents a major clinical application of targeting-peptide-mediated radionuclide localization and therapy.

6.4 Construction of RVG-Cys-Modified Lipid Carriers

The thiol group of the terminal Cys residue in RVG-Cys can selectively react with a maleimide group. For example, RVG-Cys can be conjugated to DSPE-PEG-maleimide to form RVG-PEG-DSPE, which can then be incorporated into liposomes through coassembly or post-insertion so that the RVG sequence is displayed on the particle surface. In this system, RVG supports nervous-system-associated targeting and cellular recognition, the lipid bilayer encapsulates and protects small-molecule drugs, proteins, or nucleic acids, and the PEG spacer helps improve the surface accessibility of the peptide motif. For cytosolically active cargoes such as siRNA and mRNA, the particles must still achieve endosomal escape and nucleic acid release after targeting and endocytosis, so this system specifically illustrates the distinct functional roles of the targeting peptide, conjugation structure, and nanocarrier.

6.5 RADA16 Self-Assembling Hydrogels for Local Delivery and Tissue Repair

Under suitable conditions, RADA16 can form β-sheet nanofibers, which subsequently become entangled and physically crosslinked to generate a three-dimensional hydrogel network. This network creates a local three-dimensional environment resembling the extracellular matrix (ECM), providing a scaffold for cell migration and tissue repair, while also encapsulating proteins, growth factors, or small molecules; cargo release is jointly regulated by network pore size, peptide–cargo interactions, and changes in hydrogel structure. RADA16-related self-assembling peptide materials have entered practical hemostatic applications and have also been investigated in wound repair, tissue engineering, and local drug delivery, extending self-assembling peptides from molecular-assembly research into functional biomaterial systems.

7 Products

7.1 Cell-Penetrating, Targeting, and Delivery-Related Functional Peptides

7.2 Self-Assembly-Related Short Peptides and Aggregation-Model Products

7.3 Target Validation and Functionalization Products

References

[1] Guidotti G, Brambilla L, Rossi D. Cell-Penetrating Peptides: From Basic Research to Clinics. Trends Pharmacol Sci. 2017;38(4):406-424.

[2] Hynes RO. Integrins: Bidirectional, Allosteric Signaling Machines. Cell. 2002;110(6):673-687.

[3] Corti A, Curnis F, Arap W, Pasqualini R. The Neovasculature Homing Motif NGR: More than Meets the Eye. Blood. 2008;112(7):2628-2635.

[4] Sugahara KN, Teesalu T, Karmali PP, et al. Tissue-Penetrating Delivery of Compounds and Nanoparticles into Tumors. Cancer Cell. 2009;16(6):510-520.

[5] Kumar P, Wu H, McBride JL, et al. Transvascular Delivery of Small Interfering RNA to the Central Nervous System. Nature. 2007;448(7149):39-43.

[6] Stupp R, Hegi ME, Gorlia T, et al. Cilengitide Combined with Standard Treatment for Patients with Newly Diagnosed Glioblastoma with Methylated MGMT Promoter. Lancet Oncol. 2014;15(10):1100-1108.

[7] Kumthekar P, Tang SC, Brenner AJ, et al. ANG1005, a Brain-Penetrating Peptide-Drug Conjugate, Shows Activity in Patients with Breast Cancer with Leptomeningeal Carcinomatosis and Recurrent Brain Metastases. Clin Cancer Res. 2020;26(12):2789-2799.

[8] Strosberg J, El-Haddad G, Wolin E, et al. Phase 3 Trial of ¹⁷⁷Lu-Dotatate for Midgut Neuroendocrine Tumors. N Engl J Med. 2017;376(2):125-135.

[9] Sankar S, O'Neill K, Bagot D'Arc M, et al. Clinical Use of the Self-Assembling Peptide RADA16: A Review of Current and Future Trends in Biomedicine. Front Bioeng Biotechnol. 2021;9:679525.

Major Abbreviations

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[4] Peptide Screening and Optimization

[5] PEGylation in Drug Modification and Delivery: Applications to Proteins, Peptides, Small Molecules and Liposomes

[6] The application of click chemistry in chemical ligation and peptide modification

[7] Nanoparticle-Based Small Molecule Drug Delivery

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[9] What are the differences between encapsulation efficiency, loading capacity, and yield?