Drug delivery systems (DDS) aim to target specific sites in the body, minimizing toxicity and enhancing the sensitivity of the drug to elimination during transport. DDS can control the slow release of drugs within the body, intelligently adjusting the drug's distribution in terms of time, space, and dosage, further improving drug concentration and efficacy at therapeutic sites. Peptides are active site and component fragments of enzymes, antibodies, and protein hormones. The U.S. Food and Drug Administration (FDA) defines peptides as polymers composed of ≤40 amino acid molecules. Due to their good biocompatibility, ease of synthesis and purification, high modifiability, and stability, peptides have been widely used and developed. Their side chains can carry various active functional groups (carboxyl, hydroxyl, amino, and thiol groups), allowing for a variety of chemical modifications to achieve the desired functions in drug delivery systems.
Peptides possess characteristics such as high affinity, low immunogenicity, simple amino acid composition, and adjustable molecular size. Therefore, most peptides can be conjugated with drugs via linkers to form peptide-drug conjugates, acting as prodrugs with enhanced bioactivity. These properties have demonstrated significant potential in drug delivery applications.
Peptide-based DDS is commonly used for delivering therapeutic agents such as cytotoxic molecules for tumors, nucleic acid drugs, and radioactive isotopes. These systems can improve drug biocompatibility and physiological stability, reduce side effects, maintain stable and effective drug concentrations in the bloodstream, and enhance drug concentration at targeted areas. Additionally, they can slow the degradation rate of drugs within the body and ensure that drugs maintain their half-life at target sites.
Drug delivery systems (DDS) generally target highly expressed receptors at the site of action, delivering therapeutic drugs directly to target cells or tissues, thereby enhancing the therapeutic effects of the drug and reducing its toxic side effects on the body. Cell-targeting peptides (CTPs) typically consist of 3-25 amino acids and are commonly used as targeting agents for specific binding to their targets. These peptides show specific affinity to receptors that are overexpressed on certain cells or tissues, promoting the targeted delivery of drugs to therapeutic sites. CTPs conjugated with drugs to form DDS have garnered considerable attention, especially in the treatment of cancer and other diseases.
Broadly speaking, peptide-drug conjugates (PDCs) are equivalent to peptide-based drug delivery systems. In drug development, the term PDC typically refers to drugs with cytotoxic payloads that are conjugated to targeting peptides via linkers. The mechanism of action for PDCs is as follows: initially, the peptide ligand specifically binds to certain receptors that are overexpressed on tumor cell surfaces, facilitating receptor-mediated internalization, which delivers the cytotoxic agent into the tumor cell. Then, the linker breaks down within the cell or under specific conditions in the tumor environment, releasing the free drug, which fully exerts its tumor-killing effect.
Targeted peptides, as affinity ligands, offer several advantages over antibodies. They are small in size, aiding better tissue and cell penetration, and have lower immunogenicity. Additionally, peptides often interact with conserved and biologically meaningful binding sites on target molecules, generating functional activity. To enhance stability, target binding, and introduce functional groups for site-specific conjugation, targeted peptides can be engineered via non-natural modifications. For example, Bicycle Therapeutics, one of the fastest-growing and most advanced companies in the PDC field, has developed bicyclic peptide ligands based on its proprietary phage display platform. Several of their targeted drugs have entered pivotal clinical stages, with BT8009 having entered the worlds first Phase III clinical trial. Clinical data shows that BT8009 demonstrates significant efficacy in treating metastatic urothelial carcinoma, with an objective response rate (ORR) of 38%. It also shows excellent safety, with significantly lower rates and severity of side effects, such as ocular reactions, peripheral neuropathy, and skin reactions, compared to similar antibody-drug conjugates like Padcev.
Schematic representation of a peptide–drug conjugate (PDC), comprising three key components: a homing peptide for targeted delivery, a cleavable or stable linker, and a therapeutic payload.(Jadhav, Krishna, et al. 2025)
Peptide Receptor Radionuclide Therapy (PRRT) is a targeted radionuclide therapy where therapeutic peptides are administered systemically and labeled with radioactive isotopes to selectively target cancer cells. High-affinity, radio-labeled targeting peptides are the preferred PRRT ligands because the receptor-peptide complex is internalized through endocytosis or tightly binds to the cell surface, allowing radioactive isotopes to be preferentially retained by the tumor cells expressing the target receptor. Subsequently, β-particles induce single-strand DNA breaks, or α-particles cause double-strand DNA breaks, leading to cell death. Furthermore, besides the direct effects of radiation on treated cells, internal radiation can affect neighboring cells through crossfire and bystander effects, enhancing the efficacy of PRRT.
PRRT has now become a mature therapeutic strategy for neuroendocrine tumors (NETs). This treatment began over 30 years ago but only gained widespread adoption in recent years after demonstrating proven efficacy and safety in clinical trials. In 2017, based on the results of the Phase III NETTER-1 trial, the radiopharmaceutical Lutathera® was approved for use in G1/G2 NET patients. Long-term results from the Phase III NETTER-2 trial may provide a new first-line treatment option for G2/G3 advanced patients.
Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are oligonucleotide (ON) drugs that primarily work by recruiting functional enzymes (ASOs recruit RNase H1, and siRNAs recruit Ago2) to induce the cleavage of complementary RNA. Several ASOs and siRNAs have already been approved for the treatment of human diseases. However, ASOs and siRNAs, which exert their effects through RNA cleavage mechanisms, have only been approved for the treatment of liver diseases (via systemic administration), eye diseases, and central nervous system disorders (via local administration). In animal studies, administered ASOs are widely distributed throughout the body and show activity in most tissues, while the bioavailability of siRNAs is low, resulting in lower activity across all tissues. However, receptor-mediated uptake can enhance activity, as demonstrated by GalNAc-conjugated ASOs/siRNAs targeting liver hepatocytes, which has shown improved performance compared to unconjugated ASOs/siRNAs. Peptide-based targeting for the delivery of nucleic acids outside the liver is a promising direction in this field.
Compared to current antibody targeting, the affinity of peptide-based ligands can reach nanomolar (nM) levels after modification and cyclization. Furthermore, the efficiency of endocytosis is not solely dependent on affinity; peptides need to dissociate after internalization. Peptides have inherent advantages over antibodies, including smaller molecular weight and the ability to modify with non-natural amino acids to extend their half-life. With a molecular weight nearly 150 times smaller than that of antibodies, peptides can be delivered in smaller injection doses and are more suitable for subcutaneous administration.
The cell membrane, as a natural physiological barrier, not only obstructs the transport and delivery of molecules such as proteins and nucleic acids into the cell but also limits drug permeability. Cell-penetrating peptides (CPPs) are small molecular peptides composed of approximately 8-30 amino acid residues, which have cell membrane permeability and can cross the cell membrane without disrupting its integrity. Therefore, CPPs can be used as carriers to deliver therapeutic materials such as nucleic acids, proteins, and small molecule drugs to the appropriate locations.
CPPs primarily enter cells via two pathways:
Direct Pathway: This pathway relies on the polarity of positively charged CPPs to disrupt the membrane's stability and form pores, allowing CPPs to cross the cell membrane without energy consumption. This includes:
Endocytosis Pathway: This energy-consuming process involves the inward bending of the cell membrane's phospholipid bilayer, which wraps the extracellular CPPs into the cell. The energy for endocytosis is mainly consumed through three mechanisms: macropinocytosis, caveolin-mediated endocytosis (CvME), and clathrin-mediated endocytosis (CME).
For example, the cationic peptide R3V6, composed of 3 arginines and 6 valines, forms nanomicelles that can load siRNA and carmustine (BCNU), creating a siRNA/R3V6-BCNU complex. Through charge interactions, the size of this complex is reduced to approximately 400 nm and it exhibits the ability to penetrate C6 glioblastoma cell membranes, enabling the co-delivery of siRNA and carmustine to therapeutic targets. Drug delivery systems (DDS) utilizing cationic peptides to carry chemotherapeutic drugs enhance the permeability of drugs into tumor cells. Chablani et al. reported a selective cationic peptide-functionalized polymer (SCPP-PS) with the sequence RLWMRWYSPRTRAYGC. The small size (63-65 nm) of the cationic peptide enables deep penetration into A549 cells. SCPP-PS achieved a 19.4 wt% loading of the anticancer drug methotrexate (MTX) and completely inhibited cancer cell survival.
The tumor microenvironment refers to the internal and external surroundings that promote tumor growth and metastasis. It is influenced not only by surrounding tissue structures but also by the internal environment of the cancer itself. Responsive drug delivery systems (DDS) can change in charge and size in response to various endogenous stimuli (such as pH, enzymes, and redox potential) or exogenous stimuli (such as temperature, light, and heat). These changes increase epithelial cell uptake and internalization in cancer cells, improving the targeted and penetrative delivery of drugs and enhancing controlled drug release. Such peptides can be used in linker design or undergo self-assembly due to their conformational changes after response. In recent years, antibody-drug conjugates (ADCs) have increasingly utilized responsive peptides to facilitate drug release.
Enzymes overexpressed in different types of disease tissues can trigger the activation of peptides. When extracellular enzymes (such as MMPs and proteases) are elevated in tumor tissues, they can trigger peptide-based drug delivery systems (DDS) to control the release of anticancer drugs. For example, cathepsin B (CTS B), which is highly expressed in the cytoplasm of bladder tumor cells, specifically recognizes the GFLG peptide and cleaves the peptide sequence between the F and L sites. The YSA (YSAYPDSVPMMS) peptide targets the EphA2 protein, which is overexpressed on the membrane of T24 human bladder cancer cells, and enters tumor tissues in mice. When exposed to CTS B, the HCPT–FF–GFLG-EEYSA complex undergoes cleavage and transforms into fibrous structures. Liquid chromatography-mass spectrometry (LC-MS) analysis showed that the CTS B-induced fibrous structure delayed the release of hydroxycamptothecin (HCPT), extended its therapeutic effect, and demonstrated the ability to prevent bladder tumor recurrence after surgery.
pH-responsive peptides are carrier molecules sensitive to acidic environments, where lactic acid produced by high glycolysis and CO2 metabolism in tumor cells lead to a pH range of 6.5 to 7.2 in the tumor microenvironment. This allows DDS composed of pH-responsive peptides to efficiently deliver and precisely release drugs in the tumor's acidic microenvironment, increasing the drug concentration at the targeted site. For example, poly(L-lysine) (PLL) can form pH-responsive peptide nanocarriers with varying structures. In this regard, Nguyen et al. prepared a system with succinylated ε-PLL (SPL) nanoparticles functionalized with 3-aminopropyl mesoporous silica (MCM-NH2). This system demonstrated that SPL could release significant amounts of prednisone in colon epithelial cancer cells (LS 174T and Caco-2) at low pH (pH 5.5-7.4) and was used for the diagnosis and treatment of colon cancer, with a loading capacity of approximately 34% w/w. Zhang et al. encapsulated the P-glycoprotein (P-gp) inhibitor disulfiram (DSF) in a pH-responsive conjugate of dimaleic anhydride (DMMA) and paclitaxel (PTX), linked to the L-lysine side chain of poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLL) micelles. Studies showed that at pH = 7.4, the zeta potential of the DA-NPs remained at -1.5 ± 0.5 mV, but at pH = 6.6, the zeta potential increased from -13.3 mV to +10.5 mV within 1.15 hours. The surface charge of the co-loaded DSF and PTX micelles changed from negative to positive, enhancing tumor cell uptake. This change in surface charge facilitated sequential drug release in the tumor microenvironment, thus enhancing the inactivation of paclitaxel in doxorubicin-resistant human breast cancer cells (MCF-7/ADR). DSF played a key role in inhibiting P-gp transporter activity, thereby enhancing paclitaxel's ability to overcome drug resistance. Another application of pH-responsive peptides is in nucleic acid drug delivery. Although oligonucleotides can be successfully internalized into cells through various delivery methods, they often remain in vesicular structures. Only a few oligonucleotide molecules successfully escape from the endosome to the cytoplasm and exert their therapeutic effects, while the majority are either degraded by lysosomes or expelled from the cell. After internalization, oligonucleotides gradually move from early endosomes (EEs) to late endosomes (LEs), then to multivesicular bodies (MVBs), and finally are degraded in lysosomes (Lys). During this process, oligonucleotides experience a significant pH change from 7.4 to 4.5. Many endosomal disruptors are non-biodegradable, non-biocompatible, and highly toxic to cells, limiting their use in gene/drug delivery systems. In recent years, pH-sensitive endosomal disruptors have been widely used in gene/drug delivery systems because they are biodegradable and biocompatible. These disruptors are inert at neutral/physiological pH but exhibit high toxicity at the low pH typical of endosomes, allowing them to escape from endosomes to the cytosol. By utilizing the pH gradient during endosomal maturation, certain segments that easily protonate at low pH are incorporated into oligonucleotide carriers, facilitating enhanced endosomal escape in acidic environments. In responsive peptide nanocarrier systems, Nishimura et al. reported a peptide called GALA (WEAALAEALAEALAEHLAEALAEALEALAA), which can promote lysosomal escape. This amphipathic, pH-sensitive peptide undergoes a conformational change from an unordered coil at pH 7 (due to repulsion of negative ions) to an α-helix at pH 5 (when glutamic acid residues are protonated). When conjugated to the surface of biogenic nanocapsules, GALA peptide facilitates lysosomal escape, improving the intracellular transport efficiency of drug-loaded nanoparticles.
Redox-stimuli-responsive peptides are designed to respond to the redox potential in the tumor microenvironment (TME), which is determined by the levels of reactive oxygen species (ROS) and the concentration of glutathione disulfide bonds. These peptides can be used to construct nanodrug delivery systems that are triggered by differential changes in ROS and glutathione (GSH), enabling localized drug release and accumulation while maintaining significant colloidal stability. ROS mainly include hydrogen peroxide (H2O2), superoxide anion (O2-), and hydroxyl radicals (•OH). When peptides containing thioether groups undergo redox reactions with ROS, the hydrophobic thioether group is oxidized into a hydrophilic sulfonyl group, leading to the cleavage of the thioether-based drug delivery system (DDS) and the subsequent release of therapeutic drugs.
Several factors hinder the development of peptide DDS, including a limited selection of peptides, limited linker options, a lack of effective payload scoring models, the absence of in vivo prediction systems, and insufficient database platforms. With the empowerment of AI, rationally designed peptide-drug conjugates (PDCs) can achieve better therapeutic efficacy with lower toxicity.
Since the early 1920s, when insulin (composed of 51 amino acids) was isolated and commercialized, the peptide drug industry has experienced profound development. Thousands of peptides have been discovered, typically derived from plants, animals, microorganisms, and other biological sources, possessing various significant biological functions. Although advancements in proteomics, solid-phase peptide synthesis (SPPS), DNA-encoded chemical libraries (DELs), mRNA display, and phage display technologies have accelerated the discovery of novel peptides, as reported recently, these methods remain resource-intensive, requiring substantial time and financial investment. Moreover, identifying effective druggable peptides, particularly peptide mimetics (linear/cyclic) with optimized pharmacological properties, remains a critical goal for overcoming common limitations of most reported peptides, such as short circulation half-life, rapid renal clearance, and poor targeting. These characteristics present major challenges when developing peptide-drug conjugates with enhanced targeting capability and therapeutic potency.
AI has made significant achievements in the development and structural validation of proteins and peptides. The 2024 Nobel Prize in Chemistry was awarded for breakthroughs in AI and de novo protein design, highlighting the increasing importance of these technologies in drug discovery and development. In the development of peptide ligands for drug delivery, AI has revolutionized traditional design paradigms. For instance, deep learning frameworks like RFdiffusion can now generate cyclic cell-targeting peptides with 60% higher tumor affinity than phage display sequences (RMSD<1.5 Å). The integration of AI in the design and evaluation of peptide-based delivery systems represents a paradigm shift, providing innovative solutions to challenges such as ligand selection, linker optimization, payload identification, and ADME prediction modeling-an aspect emphasized by the recent Nobel Prize.
An overview of AI applications in promoting the research and development of PDCs.(Zhang, Dong-E., et al. 2025)
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