Progress in peptide and protein therapeutics: Challenges and strategies.

Mengyang Liu and Jingyuan Wen designed the research. Mengyang Liu wrote the manuscript. Mengyang Liu, Darren Svirskis, Thomas Proft, Jacelyn Loh, Naibo Yin, Hao Li, Danhui Li, Yongzhi Zhou, Shuo Chen, Lizhuo Song, Guanyu Chen, Wei-Yue Lu, Zhiwen Zhang, Zhou Zhou, Lian Li, Yuan Huang, Craig Bunt, Guiju Sun, Paul W.R. Harris, Margaret A. Brimble, and Jingyuan Wen revised the manuscript. All of the authors have read and approved the final manuscript.

Peptides and proteins face considerable hurdles in their development as therapeutic agents, primarily because of inherent physical and chemical instabilities. Key challenges include rapid clearance from the bloodstream, vulnerability to enzymatic degradation, potential immunogenic responses, and loss of therapeutic efficacy resulting from structural conformational changes 9 . The instability of peptides and proteins can result from multiple degradation pathways, including oxidation, deamidation, conformational unfolding, aggregation, and denaturation at interfaces 10 , 11 , 12 . Additionally, peptides and proteins are characterized by intricate secondary and tertiary structures, which are essential for maintaining their biological activity and are particularly vulnerable to disruption. Even subtle perturbations to their native structures can lead to a loss of bioactivity, complicating their therapeutic potential 13 . Therefore, the main intrinsic challenges of peptides and proteins are their physicochemical barriers in terms of molecular weight, structure, solubility, stability, immunogenicity, and so on. Peptides and proteins possess significantly larger molecular sizes compared to conventional small-molecule drugs (<500 Da), which greatly limits their ability to cross biological membranes via passive diffusion 14 . Their high molecular weight, bulky structure, and hydrophilicity hinder absorption through non-invasive routes such as oral, nasal, and transdermal pathways. In general, passive transcellular diffusion is most favorable for compounds under 500 Da with high lipophilicity and balanced hydrophilic–lipophilic properties (log P between 1 and 3) 15 . Therefore, the inherent size and polarity of peptides and proteins remain major barriers in designing effective non-invasive delivery systems. Proteins exhibit complex secondary, tertiary, and often quaternary structures that are essential for their biological function 16 . These higher-order conformations are inherently sensitive to physicochemical stressors such as pH variation, thermal fluctuations, shear forces, and enzymatic degradation 17 . Exposure to such conditions can result in denaturation, aggregation, or misfolding, which not only reduces therapeutic efficacy but may also provoke undesired immunogenic responses 18 . Peptides, while structurally simpler, remain vulnerable to chemical degradation pathways including deamidation, oxidation, and hydrolysis, particularly at asparagine, methionine, or cysteine residues 19 . To maintain therapeutic activity and safety, peptide and protein formulations must preserve conformational integrity across manufacturing, storage, and delivery processes. Peptides and proteins are highly hydrophilic macromolecules, which severely limit their membrane permeability and ability to cross epithelial barriers via passive diffusion 20 . Their solubility is governed by factors such as amino acid composition, net charge, isoelectric point (pI), and the presence of hydrophobic or amphiphilic domains 20 . Proteins are particularly prone to aggregation and precipitation when the formulation pH approaches their pI, which is commonly between pH 4 and 8, and where the net charge is minimized 21 . To optimize solubility, formulation pH is typically adjusted at least 1 to 2 units close to the pI, thereby increasing repulsive electrostatic interactions and reducing aggregation risk 21 . Peptide and protein therapeutics face substantial stability challenges, particularly within biological environments. They are highly susceptible to enzymatic degradation by proteases such as pepsin (active in the stomach), trypsin, and chymotrypsin (active in the intestine and systemic circulation), which rapidly cleave peptide bonds and inactivate the molecule 22 . Additionally, peptides and proteins with molecular weights typically below 60 kDa are subject to rapid renal clearance, contributing to short plasma half-lives and often in the range of min to a few hours 23 . For example, unmodified insulin has a half-life of less than 5 min 23 . These rapid clearance and degradation processes significantly limit therapeutic exposure and necessitate frequent or high-dose administration, which can negatively impact patient adherence and increase treatment burden. Although therapeutic peptides and proteins are often derived from or structurally similar to endogenous human biomolecules, they can still elicit undesired immune responses, especially when their structure or presentation is altered during production, formulation, or delivery 24 . Modifications such as non-native glycosylation patterns, oxidation of methionine or tryptophan residues, deamidation, or protein aggregation can introduce neoepitopes or expose normally hidden regions of the molecule, triggering recognition by the immune system 24 , 25 . Aggregated proteins, in particular, have a high potential to activate antigen-presenting cells and initiate adaptive immune responses. Immunogenicity may lead to the production of neutralizing antibodies, which can reduce or abolish the therapeutic effect, and in some cases result in hypersensitivity reactions or autoimmunity 24 , 26 . Risk factors include the degree of sequence homology to endogenous proteins, route of administration (with subcutaneous and intramuscular routes typically more immunogenic than intravenous), dose frequency, and patient-specific factors such as genetic background and immune status 25 , 26 . External challenges, including formulation stability, handling requirements, cold-chain storage, and transport logistics, pose significant hurdles to the successful deployment of peptide- and protein-based therapeutics 27 . These issues are further compounded by the distinct physicochemical sensitivities of such biopharmaceuticals, necessitating route-specific considerations. Various routes, such as topical/transdermal, oral, and parenteral administration, each present unique challenges in terms of peptide/protein stability and bioavailability. Examples of specific peptides/proteins and their challenges across different routes of administration are summarized and provided in Table 1 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 . These challenges are further compounded by the complexity of ensuring effective delivery without compromising the peptide/protein's therapeutic efficacy. For instance, peptides and proteins administered orally must contend with harsh gastrointestinal conditions, while those administered topically or transdermally must overcome skin barriers to achieve systemic absorption. To address these challenges, an extensive and deep understanding of the physical, chemical, pharmaceutical, and biological factors influencing peptide/protein stability and absorption is essential. This knowledge is essential for the rational design of therapeutic peptides and proteins. In this article, we examine the key obstacles and limitations involved in delivering peptide/protein drugs via various routes, providing insights into strategies for overcoming these obstacles. The discussion highlights the importance of tailored approaches for each route of administration to ensure the successful application of peptide/protein therapeutics. Table 1 Examples of peptides/proteins and their administration challenges across various routes 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 . Table 1 Route Example Formulation Clinical application Delivery strategy Challenges in delivery Ref. Skin Insulin Microneedles, hydrogel, liposomes Diabetes management Microneedle arrays, iontophoresis, ultrasound Low permeability of large molecules, enzymatic degradation, and stability in skin layers 28 , 29 , 30 Calcitonin Liposomes, patches Osteoporosis treatment Iontophoresis, encapsulation Limited absorption through the stratum corneum, stability during formulation 31 , 32 Collagen Hydrogel Wound healing, anti-aging Passive diffusion, crosslinking hydrogel networks Degradation by skin proteases, inefficient transdermal delivery 33 Leuprolide acetate Polymeric microspheres Prostate cancer, endometriosis Controlled-release microneedles Pain with larger dosages, difficulty maintaining sustained release in microenvironments 34 Argireline Molecular modification Reduce wrinkle Chemical modification to increase lipophilicity Low permeation of argireline through skin due to its large molecular weight and hydrophilicity 41 Oral Insulin Penetration enhancer salcaprozate sodium (SNAC) Diabetes Permeation enhancer to open the tight junctions Intestinal epithelium barrier, high molecular weight of insulin 35 , 36 , 37 GLP-1 analogs Lipid nanoparticles Type 2 diabetes, obesity Lipase protection, polymer nanoparticle encapsulation Low intestinal permeability, susceptibility to first-pass metabolism 38 Linaclotide Capsules Chronic constipation Enteric coating protection Swallow issues and low bioavailability 47 Exendin-4 pH-triggered hydrogel Anti-diabetes Large pore size and surface area of nanoparticles enhance drug loading, and protection of nanoparticles from the acidic environment of the stomach Acidic environment and pepsin-mediated degradation 42 S.C. Insulin Injection vials Diabetes management Subcutaneous injections Patient compliance issues (needle phobia), frequent administration, and risk of infections 39 Erythropoietin Hydrogel Anemia treatment Thermosensitive hydrogel containing nanoparticles to avoid protein aggregation and to achieve sustained release Frequent injection, no other patient-friendly administration routes 43 I.V. Interferon- α PEGylation solutions Hepatitis B/C, oncology PEGylation for extended half-life Immune reactions, injection site pain, and protein aggregation during storage 40 Mucosal Recombinant growth hormone (RGH) Cell penetrating peptide (CPP) together with nanoparticle Growth hormone deficiency NPs improve the stability of CPP and therapeutic proteins and peptides Poor permeation across intestinal mucosal barriers limits the oral delivery of prospective therapeutic proteins and peptides 44 Sublingual Ovalbumin (OVA) Mucoadhesive patch (chitosan and hyaluronic acid) Preclinical: immunization, anti-cancer Mucoadhesive patch allowed an extended time of contact between the protein and the mucosa The dilution of bioactive compounds in saliva 45 Nasal Insulin Liposomes functionalized with cell-penetrating peptides Anti-diabetes CPPs are able to penetrate cell membranes, and liposomes are capable of protecting the drug from rapid elimination or degradation Mucociliary clearance mechanism, low permeability of the nasal epithelium 46 Examples of peptides/proteins and their administration challenges across various routes 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 . The skin, being the largest organ in the body, acts as the outermost protective barrier and has an average surface area of approximately 1.8 m 2 48 . Structurally, the skin consists of three primary layers: the epidermis, dermis, and subcutaneous tissue, along with various appendages such as apocrine glands, sweat glands, hair follicles, and sebaceous glands 49 . The thickness of these layers varies across different anatomical sites; for example, the skin on the forehead averages 1.57 ± 0.2 mm, whereas it measures around 0.95 ± 0.11 mm on the forearm 50 . These layers, particularly the stratum corneum, play an essential part in safeguarding the body from microbial invasion and environmental insults, but also pose a significant barrier to the transdermal delivery of exogenous peptides and proteins 51 . Among these layers, the epidermis is the initial and primary obstacle that should be addressed to enable efficient drug absorption 52 . Although it comprises only about 5% of the total skin thickness, the epidermis performs vital functions such as acting as a physical barrier, producing keratin, and facilitating the renewal of corneocytes 53 . The structure of epidermis can be categorized into four parts, as shown in Fig. 1 A, which are stratum corneum, granular layer, spinous layer, and basal layer. Of these four parts, the first barrier to penetration is the physical barriers imposed by the stratum corneum and tight junctions. Figure 1 (A) Schematic representation of the skin epidermis, illustrating its distinct layers and key cellular components; (B) Overview of the three primary pathways for the delivery of active compounds through the skin: (i) intracellular (through the cells), (ii) transappendageal ( via hair follicles and sweat glands), and (iii) intercellular (between the cells). Figure 1 (A) Schematic representation of the skin epidermis, illustrating its distinct layers and key cellular components; (B) Overview of the three primary pathways for the delivery of active compounds through the skin: (i) intracellular (through the cells), (ii) transappendageal ( via hair follicles and sweat glands), and (iii) intercellular (between the cells). The stratum corneum (SC) is the skin's primary barrier, constituting the outermost, non-living layer of the epidermis, and has an approximate thickness of 15 μm 55 . It consists of around 15 layers of flattened, non-viable keratinocytes—referred to as corneocytes—which have undergone terminal differentiation, losing their nuclei and metabolic activity 55 . The SC contains roughly 5%–20% water, 40% keratinized protein, and 15% lipid content 56 . The interaction between lipids and corneocytes forms a cohesive, water-resistant barrier that limits transepidermal water loss (TEWL), maintaining skin hydration and flexibility while also protecting against the entry of pathogens and chemical substances 57 , 58 . This layer is highly selective and significantly impedes the penetration of hydrophilic compounds and macromolecules 59 . Only drug candidates possessing optimal physicochemical characteristics are capable of traversing this barrier to reach deeper skin layers. Another physical barrier in dermal or transdermal delivery is tight junctions (TJs), which are specialized cell-to-cell connections found in both simple and stratified epithelial tissues, and also in endothelial layers 60 . These structures are composed of various transmembrane proteins, including members of the claudin family and TJ-associated MARVEL proteins such as occludin and junctional adhesion molecules. Additionally, intracellular scaffold proteins—commonly referred to as TJ plaque proteins—such as zonula occludens (ZO-1, ZO-2, ZO-3), MUPP-1, and cingulin, anchor these complexes to the actin cytoskeleton 61 . The principal role of TJs is to seal the paracellular space, thereby limiting the diffusion of substances between adjacent cells 62 . Beyond their barrier function, TJs also participate in cellular processes such as polarization, differentiation, proliferation, and signal transduction 63 . These junctions form a selective barrier that prevents the passage of molecules and ions of various sizes; for instance, tracer molecules with molecular weights of 557 Da, 1500 Da, 5 kDa, and 31/32 kDa, as well as ion tracers like La 3+ , are impeded by TJs in the granular layer following intradermal administration 64 . Computational models further support the role of TJs in restricting calcium ion permeability 65 . According to the pore and leak pathway model, which distinguishes between diffusion routes for small ions and larger macromolecules, epidermal TJs serve as a restrictive barrier to both classes of molecules 64 . Permeation of compounds crossing the SC is based on a passive diffusion-driven transport. The main difficulty for the rate of diffusion is that these layers of skin have the highest resistance to penetration for most chemicals compared to other routes of administration. As for the diffusion in the layers of epidermis and dermis, the rate of penetration can be influenced by the nature of the drug (hydrophilic or lipophilic). According to the nature of the drug candidates, there are typically three pathways ( Fig. 1 B) in which compounds are transported into the dermis: intracellular, appendageal, and intercellular routes 54 , 66 . Representative examples of peptides/proteins and other compounds associated with various transdermal transport pathways are shown in Table 2 28 , 29 , 31 , 32 , 67 , 68 , 69 , 70 , 71 , 72 , 73 . Table 2 Representative examples of peptides/proteins and other compounds associated with various transdermal transport pathways 28 , 29 , 31 , 32 , 67 , 68 , 69 . Table 2 Pathway Chemical example Type Transport/mechanism notes Ref. Intracellular (transcellular via transcytosis) GHRP-6 IGF-I Proapoptotic peptide KLA Platelet-derived growth factor-BB Insulin Cyclosporin A SNAC + semaglutide Lipophilic peptides/proteins Endocytosis-mediated lipophilic peptides/proteins 67 , 68 , 69 Intercellular Acyclovir Tacrolimus Vitamin C Caffeine Curcumin analogues Low molecular weight compounds Diffusion is modulated by molecular weight, partition coefficient, and vehicle composition. 70 , 71 , 72 Appendageal (transfollicular/sweat glands) des(1–3)IGF-I + TD-1 Nanoparticle-encapsulated peptides/proteins Nanoparticle systems Through follicular openings and sweat glands by particle size (<500 nm). 73 Representative examples of peptides/proteins and other compounds associated with various transdermal transport pathways 28 , 29 , 31 , 32 , 67 , 68 , 69 . The intracellular route, also known as the transcellular pathway, refers to the movement of solutes across a cell membrane, which includes transcellular active carrier-mediated transport, transcellular diffusion, and transcytosis 74 . Because the cell membrane is hydrophobic in nature, it may be resistant to the passive diffusion of charged or hydrophilic compounds. Active carrier-mediated transport assists certain molecules in moving across the skin barriers and against the concentration gradient by the use of energy, unlike transcellular diffusion 75 . Intracellular is the major transport pathway for macromolecules across the cell membranes 76 . These macromolecules are typically internalized into vesicles on one side of the cell, transported via the cytoplasm, and released on the opposite side through a process known as transcytosis 76 . Nevertheless, the majority of experimental evidence indicates that the primary route for molecular diffusion across the stratum corneum (SC) is the intercellular pathway, as outlined below. The more common transportation mechanism penetrating the SC is via the intercellular pathway because of the fact that cornified cells have an impermeable feature, where the external lipids of cells are important for the skin's barrier function 77 , 78 . These external lipids mainly consist of fatty acids, ceramides, triglycerides, and cholesterol 79 . Drugs crossing the skin via this route must pass these surrounding lipids, increasing their difficulty 79 . This makes the pathway of drug diffusion many times longer (450 μm) than the actual thickness of the SC (30 μm), thereby reducing the penetration rate of chemicals 80 . Studies from Matsuzaki suggested that polar compounds of molecular weight less than 500 Da have similar permeability to potassium ions, and these molecules are almost constant in permeability 54 , 81 . Some compounds, depending on the formulation, can cross through the SC faster than the intercellular route by using the appendageal route 82 , 83 . Appendageal transport primarily involves the utilization of skin appendages such as hair follicles, sweat glands, and sebaceous glands for the delivery of therapeutic agents 84 . The contribution of the appendageal route to overall transdermal drug diffusion is generally minimal, primarily due to its relatively small surface area compared to other pathways such as the intercellular route 85 , 86 . Moreover, the structural complexity of hair follicles presents an additional challenge, requiring a two-step process for effective delivery: initial penetration into the follicular canal, followed by trans -follicular transport into the viable tissue surrounding the appendages 87 . Nevertheless, research by Patzelt et al. 88 has demonstrated that enhancing drug delivery via this route often necessitates modifications to the physicochemical characteristics of the therapeutic agent or the incorporation of follicular penetration enhancers 88 . This is important as nanocarrier systems have been shown to be a beneficial way to overcome some of these barriers and transport drugs via this route 88 . The primary chemical barrier of the skin includes antimicrobial peptides (AMPs), which are synthesized by keratinocytes and immune cells. These peptides play crucial roles in host defense, inflammation, and wound healing 89 . AMPs display broad-spectrum activity, effectively targeting bacteria, fungi, and viruses 90 . While some AMPs are constitutively expressed at low levels in healthy skin, their production can be markedly upregulated in response to infection or tissue injury 91 . Others are inducible and expressed only upon pathogenic challenge 91 . In addition to AMPs, the acidic pH of the skin surface constitutes a vital component of the chemical barrier, as it inhibits microbial proliferation 92 . Notably, AMPs may also contribute to the formation of a protein corona around nanoparticles, potentially influencing their interaction with skin tissue. Moreover, pH is a key factor influencing the function of specific drug formulations that exploit local pH variations within the skin to trigger sustained drug release 93 . It is significant to highlight that skin disorders, such as atopic dermatitis, are often associated with an elevated skin surface pH, which may compromise the effectiveness of this natural defense mechanism 94 . Foreign substances that bypass the initial skin barrier may undergo biotransformation within the viable layers of skin 95 . This metabolic processing typically enhances the water solubility of xenobiotics, thereby facilitating their excretion and reducing systemic bioavailability 96 . Such metabolic activity may partly explain the reduced or negligible pharmacological efficacy of certain potent drugs following dermal application 97 . The primary function of cutaneous biotransformation is to detoxify and convert lipophilic molecules into more hydrophilic and less active forms for easier elimination 98 . This occurs through a two-phase enzymatic process: the functionalization phase (phase I) and the conjugation phase (phase II) 99 . During phase I, oxidative, reductive, or hydrolytic reactions introduce or expose polar functional groups on the substrate 100 . In phase II, these metabolites undergo covalent conjugation with endogenous hydrophilic molecules such as glucuronic acid, sulfate, or glycine, resulting in compounds with increased molecular weight and hydrophilicity 101 . Although this modification enhances solubility and promotes excretion, it often leads to reduced biological activity of the compound. While hepatic metabolism remains the most efficient and well-characterized system for biotransformation in the human body, the skin also expresses several enzymatic families capable of metabolizing topically applied substances 102 . However, the extent and implications of cutaneous metabolism are not yet fully understood. Table 3 97 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 summarizes the key enzyme families identified in human skin that are involved in the degradation of transdermally delivered drugs 97 . Table 3 Enzymes associated with skin and their cleavage functions 97 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 . Table 3 Name Cleavage specificity Example Ref. Alcohol dehydrogenase Catalyzes the oxidation of aliphatic alcohols to their corresponding aldehydes Retinol 97 Aldehyde dehydrogenase Oxidizes these aldehydes to form carboxylic acids Acetaldehyde 103 Carboxylesterase Mediates the hydrolysis of ester compounds such as 4-methylumbelliferone heptanoate and acetate, yielding the fluorescent compound 4-methylumbelliferone 2-Arachidonoylgylcerol 104 Cytochrome P450 Facilitate the monooxygenation of substrates through the insertion of one oxygen atom, typically producing alcohols and water as byproducts Testosterone 105 Flavin-dependent monooxygenase Involved in the N -oxidation of secondary and tertiary amines, contributing to phase I metabolic biotransformation Tertiary amines 106 Hydroxylases Aromatic ring cleavage in phase I oxidation reactions of skin aging Phenylalanine 107 5 α -Reductase C=C double bond cleavage in phase I reduction reactions of skin aging Testosterone 108 Esterase Ester bond cleavage in phase I hydrolysis reactions of skin aging Butyrylcholine 109 Glutathione- S -transferases Glutathione oxidation in phase II conjugation reactions of skin aging Cisplatin, glutathione 110 , 111 Enzymes associated with skin and their cleavage functions 97 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 . Immunological factors involved in dermal and transdermal drug delivery encompass elements of both the cellular and humoral branches of the immune system. Key cellular components include Langerhans cells, dermal dendritic cells, mast cells, basophils, and T lymphocytes, while humoral factors primarily involve cytokines 112 . Pathogen recognition is facilitated through conserved pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors, which detect pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides and peptidoglycans 113 . These immune components may impact transdermal drug delivery outcomes, as drug delivery systems can interact with and modulate immune cells, which in turn may affect other skin barrier mechanisms—for instance, through the release of pro-inflammatory or regulatory cytokines 97 , 114 . Oral administration is widely favored owing to its ease of use and high patient adherence; however, it encounters multiple physiological and structural barriers within the gastrointestinal tract (GIT), as illustrated and summarized in Fig. 2 115 . Therapeutic peptides and proteins must cross multiple physical and biochemical barriers throughout the oral cavity, oropharynx, stomach, and intestines, before transiting through the intestinal epithelium into the bloodstream or lymphatic system 116 , 117 . Among these challenges for oral delivery, the most important barrier to penetration is the physical and functional barrier of the gastrointestinal mucus layer and the unstirred water layer 118 , 119 . Figure 2 Schematic depiction of the oral administration pathway and the key barriers encountered during the delivery of therapeutic peptides and proteins. Figure 2 Schematic depiction of the oral administration pathway and the key barriers encountered during the delivery of therapeutic peptides and proteins. The gastrointestinal mucus layer is a sophisticated structure that consists of cell-associated mucins and glycoproteins, positioned adjacent to absorptive enterocytes in the epithelial mucosa of the stomach and intestines. This layer functions both as a lubricant to facilitate the passage of ingested materials and as a protective barrier that captures larger molecules and pathogens 120 . Within the gastric environment, the mucus layer exhibits a pH gradient, with values dropping below two at the luminal interface and approaching neutrality at the epithelial boundary 120 . Mucin exhibits a rapid and dynamic turnover, characterized by its breakdown in the acidic and enzyme-rich gastric environment, counterbalanced by ongoing synthesis to maintain mucosal integrity. Up to 20 distinct mucin isoforms have been identified as either secreted or membrane-bound, the latter possessing short cytoplasmic domains that facilitate intracellular signaling processes 120 . Secreted mucins are cross-linked through disulfide bonds to form macromolecules that are heavily glycosylated, providing structural stability and resistance to enzymatic degradation 120 , 121 . The size, structure, and intermolecular interactions of mucus influence the diffusion rate of peptides, proteins, and larger molecules through this layer 120 , 122 . Furthermore, the mucus layer serves as a reservoir for antimicrobial peptides such as defensins and entraps microorganisms within its outer regions, thereby limiting microbial access to and interaction with the underlying enterocytes 120 , 123 . Certain mucins, such as MUC2, also regulate luminal bacterial colonization by modulating dendritic cell activity, which suppresses inflammatory responses and enhances tolerogenic responses within the gut mucosa 120 , 124 . In-depth research using both cell-based and cell-free models has elucidated key factors affecting hydrophobic drug–mucus interactions, emphasizing the importance of peptide/protein molecular weight, charge, and mucus pore size in determining mobility and penetration across experimental mucus systems 120 , 125 . Nevertheless, the further ex vivo experimental models of peptide/protein permeation through mucus accurately replicate the complexity of the in vivo mucosal layer remains unclear. The unstirred water layer (UWL) in the intestine refers to a slow-moving layer composed of water, mucus, and glycocalyx, situated between the apical surface of intestinal epithelial cells and the lumen 126 , 127 . The UWL also exhibits a lower pH of 0.5–1.0 when compared to the equivalent region of the GI lumen 126 , 127 . The UWL provides a hydrophilic and viscous barrier for the intestinal absorption of all substances in addition to the aqueous-lipid interface and the lipophilic cell membrane itself 128 . The UWL over intestinal villi also decreases the potential surface area for absorption 129 . The overall charge of the UWL is negative due to the secretion of mucins 130 . This can cause repulsion towards negatively charged compounds, hence decreased absorption. The permeability of substances across the UWL is associated with influencers such as the rate of stirring, effective surface area, and thickness of the UWL 131 , 132 . The absorption of peptides and proteins administered orally across the intestinal membrane can occur through various pathways, including the transcellular route via enterocyte membranes, the paracellular route through tight junctions between epithelial cells, active carrier-mediated transport, and endocytosis 133 . Paracellular passive diffusion is mainly restricted by TJs. Composed of claudins and occludins, TJs are important for intercellular adhesion and maintenance of apical and basolateral polarity 134 , 135 . Relative to the transcellular surface area, the paracellular surface area accounts for up to 1% of the total surface area for absorption 136 . However, the paracellular pathway for transport of peptides and proteins, especially hydrophilic bioactive compounds, is important, as it can bypass the hydrophobic phospholipid bilayer. Pore size of tight junctions of rat intestinal epithelium has been reported to be between 8 and 13 Å and can allow transport of rigid spherical compounds below this limit 137 . Paracellular uptake of compounds, therefore, favors small compounds of high conformational freedom, such as peptides and proteins. Leakiness of tight junctions can be influenced by endogenous and exogenous factors such as permeation enhancers 138 . The transcellular passive diffusion route across the cell membrane is dependent on the permeability of both the apical and basolateral membranes as well as the physical properties of peptides and proteins 139 , 140 . Transcellular diffusion favors more hydrophobic compounds, as the lipid bilayer of cell membranes is hydrophobic 1 . Small peptides are generally hydrophilic, so uptake of peptides via the transcellular route is proposed to occur through water channels formed in the cell membrane, allowing passage of solutes with minimum interaction with the phospholipid bilayer 141 , 142 . In addition to the concentration gradient and membrane permeability, blood flow, GI transit time, and surface area are other physiological factors that can also affect the rate of uptake for the transcellular passive diffusion route 141 , 143 . Carrier-mediated transport is the main route of uptake for small di-/tri/tetra/pentapeptides such as GSH and thymopentin (TP5) through the mammalian intestine 144 . Utilizing a pre-established proton gradient and membrane potential set up by the Na + /H + exchanger, peptides are able to be absorbed into cells along with protons using peptide carriers such as the peptide transporter located on apical membranes 145 , 146 . However, due to the lack of specific transporter carriers on the basolateral side and the abundance of cytosolic peptidases in intestinal epithelium cells, 90% of absorbed peptides are broken down into amino acids before leaving the basolateral membrane 147 . Endocytosis is an active transport mechanism for the absorption of material into cells 148 . Endocytosis is involved in the formation of enclosed vesicles, entrapping extracellular material with extracellular fluid, and can be either receptor-mediated or non-receptor-mediated 149 . Substances taken up by endocytosis can optionally interact with the endosome membrane. This allows a lot of flexibility with the compounds it can uptake 150 . Pinocytosis is a form of non-receptor-mediated endocytosis, which can indiscriminately incorporate extracellular fluids and solutes, making it a viable strategy for the absorption of small hydrophilic peptides 149 , 150 . Phagocytosis is another form of endocytosis, which may incorporate extracellular solid debris, as well as microorganisms 151 . Finally, receptor-mediated endocytosis is another pathway for absorption of peptides and larger proteins, but is only initiated if membrane-bound surface proteins are triggered 152 . Common membrane receptors that can trigger endocytosis are caveolin and clathrin 153 . Specific moieties that trigger these receptors can be conjugated to peptides and proteins to trigger receptor-mediated endocytosis, such as the cell-penetrating peptide polyarginine chain 154 . Upon ingestion, peptide formulations are first exposed to digestive enzymes such as amylase and lipase in the saliva. Once they enter the stomach, they encounter a highly acidic environment and enzymes like pepsin and cathepsin, which are efficient in breaking down peptides. In the small intestine, additional proteolytic enzymes, including those secreted by the pancreas and brush border membrane peptidases expressed by enterocytes, continue the digestion process. These enzymes include trypsin, chymotrypsin, carboxypeptidase, and various dipeptidases and aminopeptidases 155 . Models commonly used to study drug transport across epithelial cells may not fully replicate the complex enzymatic conditions, including regional pH variations within the gut lumen, potentially leading to an overestimation of peptide bioavailability 120 . Table 4 shows a summary of the activity of endo and exopeptidases associated with the cleavage of peptides via the oral route of administration 152 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 . Table 4 Associated enzymes in the oral route of administration and their cleavage functions 152 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 . Table 4 Name Cleavage specificity Example Ref. Pepsin Low specificity and preference for before non-terminal Leu, Phe, Trp, or Tyr, except adjacent to pro Casein, collagen 156 , 157 , 158 Trypsin After non-terminal Arg or Lys except adjacent to pro Bovine serum albumin, insulin 157 , 159 Elastase Carboxylesterase catalyzes the ester hydrolysis of 4-methylumbelliferone heptanoate and acetate, converting them into 4-methylumbelliferone Elastin peptides 152 , 157 Endopeptidase Low specificity before non-terminal amino acids Enkephalins 160 Dipeptidyl peptidase I Low specificity and preference for after the second amino acid from the N-terminus, except for N-terminal Arg or Lys Proinsulin 161 Dipeptidyl peptidase IV Low specificity and preference for after the second amino acid from the N-terminus if Ala or pro Glucagon-like peptide-1 (GLP-1) 161 N -Acyl-amino acid releasing enzyme High specificity and cleaves N-terminal N -acetyl amino acids N -Acetylaspartate 162 Pyroglutamate aminopeptidase High specificity and cleaves N-terminal cyclic lactam amino acids Pyrrolidone carboxylate peptides 167 Methionine aminopeptidase High specificity and cleaves N-terminal Met Adenylate cyclase 157 , 163 Aminopeptidase (A, B, W, and II) High specificity and cleaves N-terminal amino acids Leukotrienes 157 , 164 Aminopeptidase (N and P) Low specificity and cleaves N-terminal amino acids Neuropeptide Y 157 , 164 Cytosol alanyl aminopeptidase Low specificity and preference for N-terminal Ala Ala-derivatives of peptides 157 , 164 Tripeptide aminopeptidase After the third amino acid from the N-terminus Glutathione, bradykinin 157 , 164 Angiotensin-converting enzyme After the second amino acid from the C-terminus Angiotensin I 165 Carboxypeptidase (A, B, M, and P) High specificity and cleaves C-terminal amino acids Insulin 166 Associated enzymes in the oral route of administration and their cleavage functions 152 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 . The sulfur barrier in oral drug delivery refers to the influence of sulfur-containing biological components on drug absorption, metabolism, and bioavailability within the gastrointestinal (GI) tract 168 . This barrier is primarily attributed to sulfur-rich macromolecules such as mucin glycoproteins, which form a protective mucus layer that hinders drug diffusion, and enzymatic systems including sulfotransferases (SULTs) and glutathione (GSH)-mediated metabolism, which contribute to drug biotransformation and clearance 169 . Sulfation, a key phase II metabolic pathway, often leads to rapid drug inactivation and elimination, particularly for hydrophilic and peptide-based drugs, thereby reducing their systemic availability 170 . Furthermore, interactions with sulfur-containing amino acids such as cysteine and methionine can impact drug stability and absorption 171 . Strategies to overcome the sulfur barrier include prodrug design, which enhances drug stability and transport before metabolic activation, nanocarrier systems that shield drugs from premature metabolism, and enzyme inhibitors that modulate sulfur-mediated metabolism. Understanding and addressing the sulfur barrier is crucial for optimizing the oral bioavailability of peptide drugs, biologics, and small-molecule therapeutics, particularly those prone to extensive sulfation or glutathione conjugation. A critical factor in formulating orally delivered peptide and protein therapeutics lies in the substantial variability in gastrointestinal tract physiology among individuals. This includes differences in mucus and enzyme production, as well as variations in gastric emptying and gut motility 120 , 172 . The rate of gastrointestinal transit is a key determinant of epithelial exposure to ingested peptides, and it can be influenced by factors such as meal composition, meal size, meal timing, and age. Variations in gut motility and absorption rates are particularly important when developing therapeutics like insulin for diabetes. Individuals with mild dysglycaemia or diabetes often exhibit dysregulated gut motility, and impaired gastric emptying is commonly observed in individuals with type 1 diabetes, which can further complicate glycaemic control 120 , 173 . Additionally, differences in the luminal epithelial environment, such as the expression levels of digestive enzymes, peptidases, and transporters, can influence the timing and extent of transmucosal drug absorption 120 , 174 . The blood‒brain barrier (BBB) poses both physical and enzymatic barriers that restrict the passage of various compounds, including numerous peptides and proteins 175 . The BBB is formed by brain capillary endothelial cells, pericytes, astrocytes, and neuronal cells, and performs several vital functions. These involve preserving neural connectivity, maintaining ion homeostasis, preventing the entry of neurotoxic molecules, and selectively transporting essential nutrients into the brain 175 , 176 . A key feature of the BBB is the asymmetrical distribution of membrane-bound transport systems across the apical and basolateral surfaces of the endothelial cells. Notably, P-glycoprotein efflux pumps located on the apical surfaces play a crucial role in protecting the brain from potentially harmful or unwanted compounds 176 , 177 . The endothelial cells of brain capillaries are tightly joined by continuous TJs, which, together with adherens junctions on the apical side, form a robust physical barrier that restricts paracellular transport into the brain 177 , 178 . These tight junctions are associated with exceptionally high transendothelial electrical resistance, rendering the BBB highly resistant to passive diffusion 179 . As a result, approximately 100% of macromolecular drugs and 98% of small lipophilic drug candidates (500 to 1000 Da) are unable to cross the BBB, with the exception of small lipophilic molecules (<500 Da) such as certain nutrients 180 , 181 . Furthermore, an additional metabolic barrier exists within the BBB due to the presence of various proteolytic enzymes, both intracellularly and extracellularly 179 , 180 . Collectively, these characteristics make the BBB a significant challenge for drug delivery. As a result, researchers are increasingly exploring the use of ligands to target specific transporters or carrier systems, aiming to improve the uptake of therapeutic compounds penetrating the BBB, as summarized in Table 5 180 , 182 , 183 , 184 , 185 , 186 , 187 . Table 5 Brief list of peptide and protein delivery penetrating the BBB 180 , 182 , 183 , 184 , 185 , 186 , 187 . Table 5 Peptide/protein Strategies Route of administration Therapeutic effect Ref. Leucine-enkephalin N -Palmitoyl N -monomethyl, N -dimethyl, N -trimethyl-6- O -glycolchitosan ligand Oral Antinociception 182 Neurotensin Angiopep-2 ligand IV Allodynia 183 O- palmitoyl tyrosinate ester-dalarg Nanofibers IV Antinociception 180 Dalargin Polymeric nanoparticles IV Antinociception 184 Opioid peptide VH0445 peptide conjugates IV Antinociception 185 4-Demethyl penclomedine Derivative of 4-demethyl penclomedine conjugation IV Brain neoplasms 186 Aurimmune (TNF- α conjugate) Cytokine protein chemical conjugation IV Solid tumor 187 Brief list of peptide and protein delivery penetrating the BBB 180 , 182 , 183 , 184 , 185 , 186 , 187 . The main mechanisms that enable drug transport penetrating the BBB include the receptor-mediated transcytosis, transcellular pathway, adsorptive transcytosis, and transporter protein-mediated transport, as depicted in Fig. 3 180 . Any ligand or chemical modification aimed at improving drug delivery across the BBB is integrated into the transport mechanisms presented in Fig. 3 . The efficiency of the paracellular and transcellular lipophilic pathways, as well as adsorptive transcytosis, is affected by the physical and chemical properties of the drug or ligand, particularly its lipophilicity and ionization state, which determine its interaction with the BBB. In contrast, transport via receptor-mediated transcytosis, transporter proteins, and efflux pumps is primarily governed by the affinity between the receptor and the drug or ligand, facilitating its uptake across the barrier, as depicted in Fig. 3 . The enzymatic barrier further complicates drug delivery to the brain due to the presence of numerous proteolytic enzymes on the endothelial surface, which can degrade therapeutic molecules before they reach their target site. However, researchers have achieved some success in BBB penetration by exploiting specific receptors that are overexpressed on the endothelial cells of the BBB. Notably, various receptors, including those for transferrin, insulin, low-density lipoprotein-related proteins, diphtheria toxin, heparin-binding EGF-like growth factors, and leptin, have been investigated as potential targets for facilitating drug delivery across the blood–brain barrier 175 , 176 . Additionally, transport proteins for glutathione and choline, which are highly expressed at the BBB, have also been investigated as potential transport mechanisms 176 , 177 . To improve BBB penetration, two primary strategies have been developed: (1) the use of active targeting ligands and (2) the application of cell-penetrating peptides (CPPs) 188 , 189 . To further improve brain uptake and specificity, scientists have increasingly designed multifunctional delivery systems that incorporate multiple ligands within a single formulation, allowing for enhanced targeting and transport efficiency. Figure 3 Schematic representation of the various drug transport routes across the blood–brain barrier. Figure 3 Schematic representation of the various drug transport routes across the blood–brain barrier. A promising non-invasive approach for improving oral drug delivery to the brain is the use of active targeting ligands that specifically interact with the BBB, as outlined in Table 6 180 , 190 , 191 , 192 , 193 , 194 , 195 , 196 , 197 , 198 , 199 . These ligands are equipped with functional groups that selectively bind to receptors on brain endothelial cells, thereby promoting transport across the BBB. This transport occurs through mechanisms including receptor-mediated transcytosis, carrier-mediated transport, or adsorptive-mediated endocytosis. Despite their potential, several challenges hinder the effective use of brain-targeting ligands. Studies have shown that dual-ligand functionalization of drug delivery systems significantly improves BBB penetration compared to single-ligand approaches. However, optimizing ligand selection remains critical, as the protective role of drug carriers reduces the necessity for enzyme inhibitors to prevent peptide degradation. A major limitation is the distinct physiological differences between the GIT and the BBB. A single ligand or transport mechanism is typically insufficient for facilitating absorption across both barriers due to their structural and functional disparities. Even when a receptor is expressed on both the GIT and BBB, its utilization for both pathways via a single ligand is often suboptimal due to variations in receptor density, localization, and function. Furthermore, even with direct intravenous administration, brain uptake remains minimal, underscoring the difficulty of bypassing the BBB. Future oral drug delivery strategies must therefore incorporate tailored approaches that address the challenges of both the GIT and the BBB independently to achieve efficient brain-targeted therapy. Table 6 Examples of different types of ligands for receptor-mediated transport in the BBB 180 , 190 , 191 , 192 , 193 , 194 , 195 , 196 , 197 , 198 , 199 . Table 6 Ligand Target receptor Transport mechanism Advantages Challenges Key findings Ref. Transferrin Transferrin receptor (TfR1, TfR2) Receptor-mediated transcytosis Well-studied; involved in iron homeostasis High endogenous transferrin competes for receptor sites Transferrin-PEGylated nanoparticles delivered ∼20% dose to the brain 190 Monoclonal antibodies (OX26, 8D3, RI7) Transferrin receptor (TfR) Receptor-mediated transcytosis High affinity to receptor; effective brain uptake Species-specificity (OX26 for rats, RI7 for humans) RI7 ligand showed the highest brain selectivity (∼3.1% uptake) 191 Lactoferrin Lactoferrin receptor Receptor-mediated and adsorptive transcytosis Lower plasma competition; unidirectional transport Receptor variability across species and tissues Lactoferrin-modified nanoparticles showed superior BBB penetration 192 Insulin Insulin receptor (IR) Receptor-mediated transcytosis High brain uptake potential Short half-life; risk of hypoglycemia MAb 83-14 delivered ∼4% dose to brain in primates; humanized MAbs reduce immunogenicity 193 Low-density lipoprotein (LDL) & angiopep-2 LDL receptor (LDL-R, LRP-1) Endocytosis High transcytosis and brain accumulation Cargo-dependent efficacy; variable receptor expression Angiopep-2 demonstrated superior transport vs . transferrin and lactoferrin 194 Glucose GLUT1 (glucose transporter 1) Carrier-mediated transport (CMT) Essential nutrient transport system High endogenous glucose competes for transport GLUT1-conjugated nanoparticles improved brain uptake 195 Leptin Leptin receptor (OB-R) Receptor-mediated transcytosis Naturally crosses the BBB; hormone-mediated pathway Receptor saturation limits dose delivery Leptin-functionalized nanocarriers showed increased brain permeability 196 Apolipoprotein E (ApoE) LDL receptor (LDL–R, LRP–1) Receptor-mediated transcytosis Endogenous brain transport system; high targeting efficiency Competitive inhibition with natural ApoE ApoE-coated nanoparticles significantly enhanced BBB penetration 197 Tetanus toxin C fragment (TTCF) Gangliosides (GT1b, GD1b) Adsorptive-mediated transcytosis (AMT) High neuronal affinity; retrograde transport Potential immunogenicity TTCF-modified nanocarriers improved neuron-specific delivery 198 Rabies virus glycoprotein (RVG29) Nicotinic acetylcholine receptor (nAChR) Receptor-mediated transcytosis High specificity for neurons Limited receptor expression in non-neuronal cells RVG29-decorated nanoparticles showed enhanced brain targeting 199 Examples of different types of ligands for receptor-mediated transport in the BBB 180 , 190 , 191 , 192 , 193 , 194 , 195 , 196 , 197 , 198 , 199 . Traditionally, the parental route has been the most practical and efficient way of delivering peptide therapeutics. However, parental drug delivery is painful and often requires repeated injections because of the short plasma half-life of peptides, enzymatic degradation in the blood or tissue fluid, and immunogenicity, which results in poor patient compliance. Parenteral administration of peptides is usually achieved via intravenous (IV), subcutaneous (SC), or intramuscular (IM) injections, as shown in Fig. 4 200 . Figure 4 Illustration of the parenteral delivery including intramuscular (IM), subcutaneous, and intravenous (IV) injections. Figure 4 Illustration of the parenteral delivery including intramuscular (IM), subcutaneous, and intravenous (IV) injections. The main challenges of IV injection can be displayed in terms of physical barriers, sterilization requirements, frequent applications, and low patient compliance 201 . Therapeutic peptides administered intravenously have to cross capillary endothelial barriers to reach various organs for local or systemic applications if the target of the drug is not in the blood 202 . The structural properties and permeability of the capillary endothelium vary in different organs and are classified as continuous, fenestrated, and sinusoidal 203 , 204 , 205 . Continuous endothelium is present in muscles, lungs, heart, the central nervous system, and connective tissues 203 , 204 , 205 . The capillary endothelium in the brain (BBB) is characterized by tight junctions between cells and the absence of any gaps or fenestrations 206 . This makes brain capillaries impermeable to polar molecules. However, there are abundant mitochondria and enzymes that facilitate active transport of molecules in the brain 206 . Most of the continuous capillaries in the peripheral tissues (other than the brain) are permeable to peptides and proteins up to 70-kDa molecular weight 207 . Fenestrated endothelium is present in intestinal mucosa, renal glomeruli, and endocrine and exocrine glands 203 , 204 , 205 . Fenestrations are openings of 60 to 80 nm with or without diaphragm membranes and allow rapid movement of fluid and low molecular weight solutes in the respective organs 208 . Fenestrae with diaphragm membranes are impermeable to macromolecules, whereas those without diaphragms allow permeation of large macromolecules, peptides 209 . Sinusoidal capillary endothelium is found in the liver, spleen, and bone marrow 210 , 211 , 212 . These capillaries have large pores (size >100 nm) and do not offer a barrier to the passage of macromolecules, peptides, and proteins 213 . Another challenge for IV injections or even all parenteral formulations is the requirement of a higher standard of sterilization 214 . However, due to the sensitive and fragile properties of peptides, the process of sterilization is special and needs extra attention. Sterile filtration is used to ensure sterility rather than a mediated approach, which directly increases the economic burden for health researchers and patients 215 . Frequent injections derived from the short half-life property of therapeutic peptides are another issue that impede the development of IV injection 216 . Meanwhile, frequent injections not only cause pain to patients but also raise the issue of inconvenience and higher cost of customer service 216 . SC and IM injections are introduced to reduce the frequency of IV injections and favor a controlled release profile to achieve a sustained action. Similarly, the physical barrier associated with SC and IM injections is the capillary endothelial membrane for the absorption of peptides and proteins. Small molecular weight peptides are mainly absorbed through the blood capillaries, whereas large peptides (>16 kDa) are taken up by the lymphatic system 217 . However, because of the slow rate of absorption in subcutaneous and IM injections, the difference between subcutaneous and IM with IV injections is that the injected volume at subcutaneous and IM sites is up to 2 and 5 mL, respectively 218 . This small capacity, therefore, restricts the application of high doses of therapeutic peptides. It can also result in local tissue inflammation and skin irritation and can increase the risk of reactogenicity 219 , 220 . For example, blood clotting factor VIII has extremely low bioavailability via subcutaneous because of proteolytic degradation in lymphatic fluids and phagocytosis in lymph nodes 221 . Various alternative routes, including pulmonary, nasal, and buccal administration, are being explored for peptide and protein therapeutics. While these methods aim to enhance bioavailability and patient compliance, each presents significant challenges, including enzymatic degradation, limited permeability across biological membranes, and variability in absorption. Overcoming these barriers remains a critical focus in peptide drug delivery research. The pulmonary route offers several advantages for peptide delivery, including a large surface area (∼70–100 m 2 ) and a dense network of capillaries surrounding the alveoli, which facilitates rapid systemic absorption while bypassing hepatic first-pass metabolism 222 . The thin alveolar epithelium (0.1–0.2 μm) enables efficient diffusion of therapeutics into the bloodstream 223 . However, peptides and proteins delivered via this route face several challenges. Enzymatic degradation by pulmonary proteases, macrophage uptake, mucociliary clearance in the upper airway, and surfactant-mediated denaturation can significantly reduce bioavailability 222 . Furthermore, successful alveolar deposition is highly dependent on particle characteristics, especially aerodynamic diameter. Ideal deposition occurs with particles sized between 0.5 and 3 μm, while larger particles are likely to be deposited in the oropharyngeal region, and smaller particles may be exhaled 224 . Despite these favorable anatomical and physiological features, the systemic bioavailability of peptides delivered via this route remains below 50% 222 . The nasal route presents a promising non-invasive alternative for peptide and protein delivery due to its high vascularization and relatively large absorptive surface area (∼150 cm 2 ), which facilitates rapid absorption and potential brain targeting via olfactory and trigeminal pathways 225 . However, several physiological and biochemical barriers restrict its effectiveness. First, the nasal cavity's tight epithelial junctions limit the paracellular transport of hydrophilic and high-molecular-weight peptides, which must instead rely on less efficient transcytosis or endocytosis mechanisms 226 . Second, the nasal environment presents a hostile milieu: enzymatic degradation by nasal peptidases, acidic pH, and rapid mucociliary clearance (with turnover times of 15–20 min) can lead to premature drug elimination 227 , 228 . Additionally, the volume that can be administered intranasally is limited (∼150–200 μL per nostril), restricting the achievable therapeutic dose. These anatomical constraints, coupled with physicochemical challenges such as peptide hydrophilicity, instability, and limited permeability, significantly impact absorption efficiency 226 . Buccal delivery is another alternative route under investigation for peptide administration. The buccal mucosa is relatively permeable and has lower enzymatic activity compared to the gastrointestinal tract, reducing peptide degradation 229 . Its rich blood supply enables direct systemic absorption while avoiding first-pass hepatic metabolism. However, the main limitation lies in the stratified squamous epithelial barrier, which restricts passive diffusion primarily to small, lipophilic molecules 230 . Additionally, the continuous production of saliva (∼1–1.5 L/day) can dilute the peptide concentration, while involuntary swallowing may reduce drug residence time at the site of absorption. These challenges, combined with the low intrinsic permeability of large hydrophilic peptides and proteins, have limited the translation of buccal peptide formulations beyond preclinical development 230 .

To enhance the transdermal, topical, and oral delivery of peptides, overcoming physiological barriers to transport across the skin and gastrointestinal tract is crucial. Four primary strategies have been proposed to address these challenges: (1) the utilization of physicochemical penetration enhancers to increase permeability; (2) chemical modification of peptide structures to enhance lipophilicity and improve membrane interactions; (3) the incorporation of enzymatic inhibitors to improve the stability of protein and peptide drugs, particularly for oral administration; and (4) advanced formulation approaches, including nanocarrier systems, to facilitate transport and provide enzymatic protection. Chemical penetration enhancers enhance the skin permeability of molecules by interacting with the lipidic complex in the skin barriers. They achieve this by fluidizing the lipid structure and disrupting the organized lipid domains, thereby facilitating the passage of molecules. Additionally, these enhancers can influence intercellular factors, metabolic processes, and thermodynamic properties, further promoting the transdermal delivery of active agents 231 . Chemicals, including dimethylsulphoxide 232 , azone 233 , pyrrolidones 234 , fatty acids 235 , fatty alcohols 236 , and surfactants 237 are some examples of chemical enhancers currently used in the pharmaceutical field. These are used mainly to improve the penetration of the epidermis. For example, Magnusson et al. 238 reported a significant enhancement in the transdermal delivery of thyrotropin-releasing hormone in vitro , with skin penetration increasing from 0.92 ± 0.03 to 1.6 ± 0.02 μg/cm 2 ·h when penetration enhancers, such as ethanol and cineole, were used. However, the effectiveness of chemical penetration enhancers in oral drug delivery is often limited by their large molecular size, which restricts their ability to diffuse across biological membranes efficiently. Additionally, the hydrophilic nature of most peptides and proteins presents a significant challenge, as their poor lipid solubility hinders passive permeability through the intestinal epithelium. These factors collectively diminish the efficacy of penetration enhancers in enhancing the bioavailability of peptide- and protein-based drugs 238 . Additionally, skin irritation is a potential concern associated with chemical enhancers, which may signal skin toxicity due to their physicochemical properties 239 . Chitosan is a biodegradable, non-toxic polymer derived from the deacetylation of N -acetylglucosamine units in chitin, typically through hydrolysis under alkaline conditions at elevated temperatures 240 . Penetration enhancers represent a diverse group of chemical agents that facilitate the permeation or transport of molecules across biological membrane barriers 241 . These enhancers operate through various mechanisms, including altering membrane fluidity, reducing mucus viscosity, promoting protein leakage across membranes, and inducing the opening of tight junctions 242 . Chitosan is a semi-natural bioadhesive polymer. The phenomenon of bioadhesion enables a higher concentration of drug to remain at the target site, thereby enhancing the therapeutic effect 243 . Mucoadhesive polymers, which adhere to the mucin layer of the mucosal epithelium, have the potential to improve the oral bioavailability of protein and peptide-based therapeutics 244 . Therefore, chitosan, owing to its penetration enhancement, has generated significant interest. For example, the chitosan-ethylenediaminetetraacetic acid (EDTA) conjugate has been shown to protect peptide and protein drugs from enzymatic degradation in the gastrointestinal tract 245 . Additionally, novel chitosan derivatives are being explored to enhance permeation, including chitosan-thiobutylamidine 246 , N -trimethyl chitosan 247 , and methyl-pyrrolidinone chitosan 248 . Chitosan and its derivatives are generally considered non-toxic 249 However, several studies have highlighted potential risks associated with penetration enhancers, including the possibility of tissue damage or systemic absorption due to their low molecular weight, which could result in unintended systemic effects 250 , 251 , 252 . Another prominent chemical enhancer of the successful clinical translation is the use of salcaprozate sodium (SNAC), a transient permeability enhancer that facilitates the absorption of peptide drugs across the gastrointestinal epithelium 253 . SNAC acts by temporarily modulating the local environment within the stomach or intestine, increasing membrane fluidity and enabling transcellular transport without permanently disrupting epithelial integrity 254 . Importantly, SNAC also protects peptides from proteolytic degradation by modifying pH and enzyme accessibility 255 . This approach has culminated in the approval of oral semaglutide (Rybelsus®), the first oral glucagon-like peptide-1 receptor agonist (GLP-1RA) for type 2 diabetes, by the FDA and EMA 256 . Oral semaglutide combines SNAC with a high-dose peptide formulation to achieve therapeutic plasma concentrations. Clinical trials, such as PIONEER 1–12, have demonstrated that oral semaglutide is not only efficacious in glycaemic control but also shows cardiovascular and weight-reducing benefits comparable to its injectable counterpart 257 . These advances reflect a paradigm shift in peptide drug development, demonstrating that oral delivery is becoming an increasingly viable and promising approach. Iontophoresis involves the application of a mild electric current to drive charged peptides and proteins across epithelial barriers. In transdermal delivery, it enhances penetration through the stratum corneum by electrorepulsion and electroosmosis 258 . Raiman et al. 69 demonstrated effective delivery of luteinizing hormone-releasing hormone (LHRH) and Nafarelin through human skin, reporting that pulsed direct current (DC) led to 9.87 ± 4.91 μg/cm 2 ·h of LHRH delivery, while alternating current (AC) was ineffective 259 . Electroporation, which uses short high-voltage pulses, creates transient aqueous pores in lipid bilayers, facilitating the transport of macromolecules such as peptides through both skin and intestinal epithelium 260 . Its transient and localized nature makes it applicable to multiple routes, although skin remains the primary target in clinical development. Microneedles (MNs) are one of the most promising physical strategies for bypassing epithelial barriers. In transdermal delivery, solid, coated, or dissolving MNs breach the stratum corneum and deliver biomolecules into the viable epidermis or dermis 261 . For oral delivery, MN capsules function analogously, penetrating the intestinal mucosa and releasing drugs directly into underlying tissues 262 . Delivery of adalimumab using such oral MN capsules demonstrated ∼50% bioavailability in swine, indicating successful bypass of enzymatic and transport barriers 263 , 264 . Both approaches depend on microneedle design ( e.g. , material, length, dissolution profile) and are influenced by local anatomical factors such as vascular density, mucus thickness, and mechanical peristalsis (in the GI tract) or skin elasticity (transdermal) 265 . Low-frequency ultrasound (20–100 kHz) enhances drug delivery by inducing cavitation and acoustic streaming, which disrupts tight junctions and increases tissue permeability. In transdermal delivery, ultrasound (sonophoresis) can fluidize stratum corneum lipids, while in oral delivery, it promotes mucosal uptake by increasing paracellular transport 266 . Schoellhammer et al. 265 applied ultrasound to porcine colonic epithelium, demonstrating a 2- to 10-fold increase in the uptake of peptides and 3 to 70 kDa dextran molecules, which was not achievable without sonication 267 , 268 , 269 . Importantly, this enhancement was reversible and localized, indicating clinical applicability for site-specific oral delivery devices 267 , 268 , 270 . Enzymatic inhibitors, as their name implies, function by inactivating specific enzymes. The co-administration of enzyme inhibitors targeting GI peptidases, enzymes responsible for metabolizing peptide drugs, can help suppress the degradation of peptides in the GI tract, thereby enhancing the oral bioavailability of peptide therapeutics 271 . Enzyme inhibitors used in conjunction with peptide drugs can be categorized into four main types: polypeptide protease inhibitors, peptide-based inhibitors, amino acid inhibitors, and non-amino acid-based inhibitors 272 . Maianti et al. 139 were able to observe a decrease in insulin degradation when a protease inhibitor was co-administered with insulin, resulting in an improved oral bioavailability 273 . This study demonstrated an improved reduction in plasma glucose levels and a marked increase in blood insulin concentrations at 20- and 135-min after oral insulin administration, when co-administered with a peptidase or proteinase inhibitor, in both lean and obesity-induced diabetes rat models. Bacitracin, a known enzyme inhibitor, has been utilized to inhibit the breakdown of several therapeutic peptides, such as insulin and buserelin 274 , 275 . Aminoboronic acid derivatives, which are amino acid-based enzyme inhibitors, have been utilized in the past to improve the delivery of peptide and protein drugs, though their use has declined in favor of more effective alternatives 276 . In contrast, polypeptide protease inhibitors have gained significant attention as adjuncts for overcoming the enzymatic barrier in the oral administration of therapeutic proteins, owing to their low toxicity and potent inhibitory activity 277 . Aprotinin, a small protein extracted from bovine pancreatic trypsin, is commonly used as a peptidic enzyme inhibitor to enhance the bioavailability of insulin 278 . For instance, Kraeling et al. 153 achieved a 6.2% oral bioavailability of insulin using aprotinin, up from 5.0% with the same formulation without aprotinin 279 . However, a limitation of this approach is that enzyme inhibition may lead to undesirable side effects, including impaired digestion, systemic toxicity, and pancreatic hyperplasia 279 . In addition, several oral enzymatic inhibitors are currently in late-stage clinical development for insulin delivery. Notably, Oramed Pharmaceuticals has advanced oral insulin formulations incorporating protease inhibitors and absorption enhancers into phase II/III clinical trials 280 . Chemical modification involves the conjugation or integration of chemical moieties with peptide drug candidates to enhance their pharmacokinetic properties. The modifications can result in a new compound that mimics the original in the form of analogues. For peptide-based therapeutics, the incorporation of lipophilic moieties, such as myristic acid 281 mystic acid 282 , lauric acid 282 , palmitic acid 282 , stearic acid 283 , and oleic acid 283 , has been widely employed to improve permeability across the skin and GI epithelial barriers. These modifications facilitate enhanced transmembrane transport and absorption, addressing one of the primary challenges associated with peptide drug delivery 284 . Extensive research has demonstrated that chemically modified peptides exhibit improved absorption and bioavailability. For instance, the bioavailability of interferon-alpha (IFN- α ) increased from 0.4 ± 0.2 to 2.1 ± 1.1 μg/cm 2 following fatty acylation, representing a fivefold enhancement 285 . Another notable example is Matrixyl® 3000 286 , a chemically engineered compound comprising palmitoyl oligopeptide (Pal–GHK) and palmitoyl tetrapeptide-7. Pal–GHK, synthesized through the conjugation of palmitic acid with an oligopeptide sequence, enhances lipophilicity and facilitates improved dermal absorption. Palmitoyl tetrapeptide-7, a peptidic conjugate consisting of palmitic acid and a tetrapeptide sequence (glycine–glutamine–proline–arginine), has shown notable efficacy in both cosmetic and therapeutic applications 287 . Strategic chemical modifications introduced at enzymatic cleavage sites significantly enhance the in vivo stability of peptide therapeutics by preventing rapid degradation. Thus, the identification of enzymatically labile sites within the peptide sequence is critical for rational chemical modification strategies. These chemically modified peptides incorporate diverse functional groups, as detailed in the following section and summarized in Table 7 288 , 289 , 290 , 291 , 292 , 293 , 294 . Table 7 Summary of chemical modifications of peptides 288 , 289 , 290 , 291 , 292 , 293 , 294 . Table 7 Modification type Description Impact/benefits Examples/applications Ref. N - and C - terminal modifications Modification of N- and C - termini to protect from exopeptidase degradation ( e . g ., acetylation, amidation) Improves enzymatic stability and half-life. Some modifications increase lipophilicity and membrane permeability N-terminal acetylation of somatostatin, melanocyte-releasing hormone ( α -MSH), TRH. N -Linked lipidation 288 , 289 , 290 Cyclization Formation of cyclic peptides via peptide bonds or other bridges ( e . g ., disulfide, ester, ether) Reduces conformational flexibility, increases stability, potency, selectivity, and bioavailability Cyclic analogues of vasopressin, opioid peptides; disulfide bridge cyclization 291 Methylation of amide nitrogen Methylation of amide nitrogen to prevent enzymatic degradation Increases stability and resistance to proteolytic enzymes while retaining biological activity N -Methylated analogues of enkephalin, substance P, cyclosporin A 292 Side-chain modifications Modifying side-chains of amino acids involved in protease recognition Stabilizes peptide by preventing enzyme cleavage while preserving peptide activity Prosaptide peptide derivatives of prosaposine (TX14) 293 Chirality changes Replacing l -amino acids with d - amino acids to enhance resistance to proteolytic degradation and reduce immunogenicity Improves protease resistance and reduces immunogenicity. Enhances the stability and activity of the peptide d - Amino acid analogues of neurotrophic peptides, as used in peptide drug development 294 Summary of chemical modifications of peptides 288 , 289 , 290 , 291 , 292 , 293 , 294 . In serum or plasma, small peptides are primarily degraded by exopeptidases, with all peptides possessing free N - and C-termini being particularly susceptible to rapid enzymatic degradation, often within min in physiological conditions. However, certain endogenous hormones and neuropeptides exhibit natural end-protection, conferring enhanced stability. For instance, thyrotropin-releasing hormone (TRH) is stabilized by an N- terminal pyroglutamyl residue and a C-terminal proline-amide moiety, while α -melanocyte-stimulating hormone ( α -MSH) is protected by N- terminal acetylation and C-terminal amidation, rendering it susceptible only to endopeptidase-mediated degradation in vivo 295 . The acetylation process is catalyzed by acetyl-coenzyme A, whereas peptidylglycine-amidation monooxygenase facilitates C-terminal amidation 296 . These natural modification processes are important in preserving the biological activity and stability of many neuropeptides, and chemical modification methods mimic their effects 297 . End-protection strategies have been extensively employed in peptide drug development to enhance enzymatic stability. For example, N-terminal acetylation of a somatostatin analogue extended its in vivo half-life from 3 min to over 400 min 298 . While effective in delaying degradation, end-protection rarely confers complete resistance to enzymatic metabolism. Instead, it primarily slows peptide catabolism, as degradation still can continue via specific endopeptidases. Beyond enzymatic stability, N-terminal modifications such as acetylation and lipidation can modulate peptide lipophilicity, which may theoretically enhance membrane permeability, particularly across physiological barriers such as the intestinal epithelium and the blood‒brain barrier (BBB). However, permeability enhancement is not universally observed. For instance, acetylated enkephalin analogues demonstrated reduced permeability across an in vitro model of the BBB using bovine brain endothelial cells 299 . It was proposed that the acetylated peptide exhibited increased cellular uptake but remained trapped intracellularly rather than traversing the endothelial monolayer 299 . The lipidation of polypeptides with fatty acids to create N- linked lipopeptides can be a complex and labor-intensive procedure, frequently necessitating the protection of other reactive functional groups present on the amino acid side chains. Although lipidation is usually performed on N- or C-termini, lipidation can also be done on residues within a peptide chain. Recent advancements in chemical synthesis, particularly Cysteine Lipidation on a Peptide or Amino Acid (CLipPA) technology, have simplified this process 300 . CLipPA facilitates the direct lipidation of unprotected peptides that possess a free thiol group, resulting in the formation of S- lipidated lipopeptides. This method allows for the efficient and rapid synthesis of multiple lipopeptide analogs from a single thiolated polypeptide precursor 301 . Similar to N-linked lipidation, S- lipidation can enhance peptide-membrane interactions and intracellular retention, potentially improving cellular uptake while limiting transendothelial transport 302 . The impact of these modifications on in vivo BBB permeability remains to be elucidated 299 . Cyclization is a way to impose structural constraints that reduce conformational flexibility, thereby improving receptor binding affinity, selectivity, enzymatic resistance, bioavailability, and membrane permeability. This approach has been explored extensively in the design of orally administered cyclic peptides, including vasopressin and opioid analogues 303 , 304 . Cyclic peptides can be classified as homodetic or heterodetic, depending on the nature of the linkage forming the ring structure. Homodetic cyclic peptides are defined by an intramolecular peptide bond between the N- and C-termini, whereas heterodetic cyclic peptides feature alternative bridging elements such as ether, disulfide, thioether, or ester (lactone) bonds. In addition, modern synthetic strategies, including all-carbon staples ( e.g. , hydrocarbon stapling) and click chemistry-based linkages ( e.g. , triazoles), are increasingly employed to generate heterodetic macrocycles 305 , 306 . Numerous chemical modifications have been explored and applied to modify linear peptides into cyclic structures. Among these, a widely used approach for synthesizing heterodetic cyclic peptides involves the formation of a disulfide bond between two cysteine residues, which effectively stabilizes the cyclic conformation 307 . However, in cases where cysteines are absent from the native peptide sequence, their incorporation may necessitate sequence modifications, potentially altering biological activity and receptor interactions 307 . Despite these challenges, cyclization remains a promising approach for optimizing peptide therapeutics by improving pharmacokinetic and pharmacodynamic properties and potentially removing N-terminal and C-terminal degradation sites. Advances in peptide engineering continue to refine cyclization strategies, expanding the potential of cyclic peptides for clinical applications. Endopeptidases cleave peptide bonds, and as a result, N- methylation of the amide nitrogen within the peptide bond is a commonly utilized strategy to improve peptide stability. This modification enhances resistance to enzymatic degradation, particularly by endopeptidases, thereby prolonging the therapeutic efficacy of peptides 308 . This modification has been extensively investigated in the development of N- methylated enkephalin analogues, which retain their biological activity while demonstrating significantly improved metabolic stability 309 . Similarly, N- methylated derivatives of substance P, a neuropeptide involved in smooth muscle contraction and vasodilation, exhibit increased resistance to proteolytic enzymes without compromising potency 310 , 311 . A notable example of a naturally occurring N- methylated peptide is cyclosporin A, a cyclic undecapeptide with immunosuppressive properties. Cyclosporin A contains one unnatural amino acid in the form of 4-butenyl-4-methyl-threonine, and features N-methylation on seven of its eleven peptide bonds, contributing to its exceptional oral bioavailability and enzymatic stability 310 , 312 . However, N- methylation imposes structural constraints that can influence peptide conformation. Specifically, it disrupts hydrogen bonding interactions and promotes a cis -amide configuration, which may affect biological activity. For instance, reduced potency observed in N- methylated neurotensin analogues has been attributed to the loss of crucial intramolecular hydrogen bonds and conformational incompatibilities with the target receptor 313 . Therefore, while N- methylation is a powerful tool for enhancing peptide stability, its impact on structural dynamics and bioactivity must be carefully evaluated to ensure the retention of therapeutic efficacy. A rational design approach that balances stability and functional integrity is essential for optimizing peptide-based drug candidates. A straightforward strategy to enhancing peptide and protein stability involves modifying the side chains of amino acids located at protease recognition sites 314 , 315 . Substituting these residues with natural or non-natural amino acids that possess chemically similar side chains can preserve the biological activity of the peptides and proteins while preventing enzymatic cleavage 314 , 315 . Altering the enzyme recognition section of peptides effectively enhances metabolic stability without significantly compromising the peptide's native structure and function. A well-documented example of this approach is the development of prosaptide peptide derivatives of prosaposin, a neurotrophic and neuroprotective agent. One such derivative, TX14, a tyrosine-sulfated prosaptide analog, demonstrated potent activity in the peripheral nervous system but exhibited limited efficacy in the central nervous system (CNS) due to rapid enzymatic degradation in the brain 316 . Early pioneers in peptide and protein chemistry recognized the critical role of stereochemistry in dictating the structural and functional properties of these biomolecules. Their investigations revealed that stereochemical selectivity profoundly influences biological recognition, binding affinity, and functional specificity, laying the foundation for modern peptide-based drug design and protein engineering 316 , 317 , 318 , 319 . The stereochemical specificity inherent in natural proteins, containing exclusively l - enantiomer amino acids, plays a critical role in molecular recognition, folding, and biological activity 316 , 317 , 318 , 319 . This specificity is fundamental to the precise interactions between peptides and their receptors, enzymes, and other biomolecules. The strategic incorporation of d - amino acids into peptide sequences, either partially or completely replacing the natural l -amino acids, has gained recognition as an effective approach to improve peptide stability. d - Amino acid analogues are less susceptible to enzymatic degradation, as they are not readily recognized by the majority of proteolytic enzymes, thus extending their half-life in biological systems 320 . Moreover, d - amino acid-containing peptides have demonstrated significantly lower immunogenicity compared to their l -amino acid counterparts, which play a significant role in peptide and protein drug design and development. This reduction in immunogenicity not only improves the safety profile of these peptides but also enhances their potential for clinical applications, particularly in chronic treatments where long-term exposure to the peptide may otherwise elicit immune responses 316 , 317 , 318 , 319 . Consequently, the incorporation of d - amino acids represents a valuable approach in designing peptides and proteins with improved pharmacokinetic properties, reduced immunogenicity, and enhanced therapeutic efficacy. Some examples and applications of chirality changes have been listed in 321 , 322 , 323 , 324 Table 8 . Table 8 Examples of peptide drug modifications by chirality changes and their benefits 321 , 322 , 323 , 324 . Table 8 Drug Modification Benefit Ref. Desmopressin d - Arg at position 8; deamination at position 1 Enhanced T 50 10 times 321 Difelikefalin d - Phe at positions 1 & 2; D-Leu at position 3 High κ -opioid activity, protease-stable 322 Etelcalcetide C - terminal d -amino acid backbone Low immunogenicity 323 Degarelix Multiple d - amino acids + hydrophobic substitutions Enhanced GnRH receptor binding; increased plasma T 50 324 Examples of peptide drug modifications by chirality changes and their benefits 321 , 322 , 323 , 324 . Drug delivery via various administration routes could be developed by tailoring the particle size and physicochemical properties of carrier systems. One of the most effective strategies for enhancing the stability and therapeutic efficacy of peptides and proteins is their incorporation into nanoparticulate drug delivery systems, which have been extensively investigated to address the inherent stability challenges of peptide-based therapeutics 325 , 326 . Nanoparticles, defined as multiphase carrier vesicles with a particle size below 1000 nm, can encapsulate a broad range of bioactive compounds, thereby improving their stability, bioavailability, and therapeutic performance 326 , 327 . Compared to conventional drug delivery systems, such as microspheres and microparticles, nanoparticulate formulations offer several advantages: (1) high stability and drug-loading capacity, enabling the encapsulation of both hydrophilic and hydrophobic compounds while being compatible with multiple administration routes, including oral, injectable, and inhalation delivery 328 ; (2) the capacity to deliver drugs in a modified and controlled release pattern, thereby prolonging therapeutic effects; and (3) enhanced resistance to enzymatic peptidolysis and proteolysis in the GIT and bloodstream, improving cellular uptake via endocytosis, phagocytosis, and micropinocytosis 329 . These properties collectively enhance drug bioavailability while reducing dosing frequency 328 . Common nanoparticulate delivery systems encompass liposomes, microemulsions, nanoparticles, nanogels, and niosomes, as depicted in Fig. 5 A 330 . As presented in Table 9 333 , 334 , 335 , 336 , 337 , 338 , 339 , 340 , 341 , 342 , 343 , 344 , this overview summarizes advanced delivery systems developed for peptide and protein therapeutics, highlighting representative examples of both marketed drugs and clinical-stage candidates. These delivery platforms are designed to enhance stability, bioavailability, and controlled release, while also offering protection against enzymatic degradation and physiological barriers, thereby improving the overall therapeutic performance of biologics 331 . The cellular uptake of nanocarriers is mediated by multiple endocytic pathways, including clathrin-dependent and caveolae-mediated endocytosis, facilitating efficient intracellular drug delivery, as depicted in Fig. 5 B 332 . The release kinetics of encapsulated peptides and proteins are influenced by factors such as lipid matrix composition, particle size, and preparation techniques, allowing for sustained and controlled drug release. Optimization of surfactant concentration and lipid structure can minimize burst release while enhancing drug retention. These characteristics position nanoparticulate formulations as a promising approach for improving therapeutic outcomes across diverse administration routes, including oral, topical, dermal, and transdermal delivery, as demonstrated in Fig. 5 C and D. Figure 5 (A) Schematic representations of various nanoparticulate drug delivery carriers for peptide therapeutics, including polymeric nanoparticles, solid lipid nanoparticles (SLN), nanogels, microemulsions, liposomes, and niosomes. (B) Illustration of the different endocytic pathways involved in nanocarrier uptake. (C) Mechanisms by which nanocarriers traverse intestinal barriers for oral drug delivery. (D) Mechanisms by which nanocarriers penetrate skin barriers for topical and transdermal drug delivery. Figure 5 Table 9 Overview of key delivery systems for peptide and protein therapeutics, highlighting examples of marketed drugs and clinical-stage candidates 333 , 334 , 335 , 336 , 337 , 338 , 339 , 340 , 341 , 342 , 343 , 344 . Table 9 Delivery system Marketed drugs Clinical-stage candidates Ref. Microemulsion Cyclosporine A, Saquinavir, Neoral® OPBP-1, ORMD-0801 333 , 334 Liposome Doxil®, Marqibo®, Oral-Lyn™ Semaglutide hybrid, oral EPO, Biphasix™, BLP25/Stimuvax® 335 , 336 Niosome None (cosmetics only, e . g ., Lancome) TP5 niosomes, catechin niosomes, Diclofenac niosomal gel 337 , 341 Nanogel Eligard® pH-responsive nanogels, HA nanogels, cCHP Nanogel 338 , 342 Polymeric NPs Sandostatin LAR®, Bydureon®, Afrezza®, Zoladex®, Lupron Depot® Oral insulin (spore-based), RL-QN15 NPs 339 , 343 PEGylation Pegasys®, Neulasta®, Doxil®, Cimzia® (certolizumab pegol), Yorvipath (palopegteriparatide), Palynziq® (pegvaliase) PT302 (exenatide), PEGylated brain targets, NLY01 (PEGylated exenatide) 340 , 344 (A) Schematic representations of various nanoparticulate drug delivery carriers for peptide therapeutics, including polymeric nanoparticles, solid lipid nanoparticles (SLN), nanogels, microemulsions, liposomes, and niosomes. (B) Illustration of the different endocytic pathways involved in nanocarrier uptake. (C) Mechanisms by which nanocarriers traverse intestinal barriers for oral drug delivery. (D) Mechanisms by which nanocarriers penetrate skin barriers for topical and transdermal drug delivery. Overview of key delivery systems for peptide and protein therapeutics, highlighting examples of marketed drugs and clinical-stage candidates 333 , 334 , 335 , 336 , 337 , 338 , 339 , 340 , 341 , 342 , 343 , 344 . Microemulsions are clear, thermodynamically stable, and isotropic mixtures consisting of oil, water, surfactants, and co-surfactants, typically featuring droplet sizes below 100 μm 345 , 346 . They can be categorized into three primary classifications: oil-in-water (O/W), water-in-oil (W/O), and bicontinuous microemulsions. The choice of surfactants influences the phase properties of the microemulsion, which can significantly enhance drug solubilization and protect peptide and protein drugs from enzymatic degradation 345 . For example, the bioavailability of the water-soluble peptide SK&F-106760 (arginylglycylaspartic acid) increased more than tenfold when incorporated into a W/O microemulsion with a mean droplet size of 15 nm, compared to the bioavailability of the pure drug solution 347 . However, the application of microemulsions is constrained by their relatively low drug loading capacity and the inclusion of unnecessary excipients, which can exhibit toxic effects when administered at high doses. A promising advancement in microemulsion-based peptide delivery is the development of a fish oil-based system for oral administration of the PD-1/PD-L1 blocking peptide OPBP-1, as shown in Fig. 6 348 . This formulation, prepared without the need for co-surfactants, exhibited favorable physicochemical properties, including a transparent, light-yellow appearance, good flowability, and a droplet size of 152 ± 0.73 nm. It demonstrated sustained drug release (56.45 ± 0.36% over 24 h) and remained stable under harsh intestinal conditions. In vitro studies confirmed enhanced peptide uptake and transport across Caco-2 cells, resulting in a 4.1-fold increase in oral bioavailability compared to the peptide solution. Mechanistic analysis identified clathrin- and caveolae-mediated endocytosis as the primary absorption pathways. In vivo , this system facilitated CD8 + T cell infiltration in tumors, increased IFN- γ secretion, and significantly suppressed murine colonic carcinoma (CT26) growth. Notably, fish oil-induced ferroptosis in tumor cells, demonstrating a synergistic effect with OPBP-1 in cancer immunotherapy. These findings highlight fish oil-based microemulsions as a promising, naturally derived platform for oral peptide delivery, leveraging their ability to enhance bioavailability while promoting reactive oxygen species (ROS)-mediated ferroptosis in tumors. Figure 6 Schematic of a fish oil-based microemulsion for oral delivery of a PD-1/PD-L1 blocking peptide for cancer immunotherapy. Reprinted with the permission from Ref. 348 . Copyright © 2023 Elsevier B.V. Figure 6 Schematic of a fish oil-based microemulsion for oral delivery of a PD-1/PD-L1 blocking peptide for cancer immunotherapy. Reprinted with the permission from Ref. 348 . Copyright © 2023 Elsevier B.V. Another promising application of microemulsion-based peptide delivery is its use in skin treatments, particularly for facial oil control and skin hydration. A hydrophilic tripeptide-3 nanoemulsion was developed to enhance skin penetration and reduce sebum production, as illustrated in 333 Fig. 7 . The formulation process was optimized using water titration and pseudoternary phase diagrams, identifying key factors such as surfactant selection, hydrophilic-lipophilic balance (HLB), and co-solvent composition. A combination of Cremophor® RH40 and polyglycerol-3-diisostearate at an HLB of 13, along with a water-to-co-solvent (propylene glycol) ratio of 1:1, significantly reduced interfacial tension, facilitating stable nanoemulsion formation. Employing a low-energy emulsification method, the optimized formulation achieved a translucent oil-in-water nanoemulsion with a droplet size of 25.7 ± 1.20 nm, a narrow polydispersity index 0.237 ± 0.129, and high transmittance 70.6 ± 0.58%. In vitro skin permeation studies demonstrated superior penetration and retention of Tripeptide-3 compared to high-surfactant microemulsions and conventional emulsions. A 28-day clinical evaluation in healthy volunteers confirmed reduced sebum secretion and improved skin hydration without irritation. Together with the oral fish oil-based microemulsion for PD-1/PD-L1 blockade in cancer therapy, this study underscores the versatility of microemulsion systems in peptide delivery. These formulations improve both bioavailability and therapeutic effectiveness while also enabling targeted delivery for systemic and dermatological treatments. Figure 7 Schematic of microemulsions for topical delivery of tripeptide-3 for anti-sebum efficacy on facial skin. Reprinted with the permission from Ref. 333 . Copyright © 2024 MDPI. Figure 7 Schematic of microemulsions for topical delivery of tripeptide-3 for anti-sebum efficacy on facial skin. Reprinted with the permission from Ref. 333 . Copyright © 2024 MDPI. Liposomes are spherical vesicles, ranging in particle size from 1 to 2000 nm, consisting of one or more layers of phospholipids 349 . They offer several advantages for dermal and transdermal applications, including: 1) biocompatibility and biodegradability, 2) controlled drug release, 3) enhanced skin or topical deposition, 4) improved permeability, and 5) increased stability 350 , 351 , 352 . Numerous studies have shown that liposomes can improve the skin penetration of various actives for transdermal and topical applications, including tetracaine, corticosteroids, and ciclosporin 352 , 353 . Studies have also been explored to increase the oral bioavailability of bioactives like insulin using liposomal formulations, with some studies reporting a decrease in plasma sugar levels following oral administration 354 , 355 , 356 . Similarly, other protein therapeutics, such as erythropoietin, calcitonin, and parathyroid hormone, have shown improved pharmacological effects when incorporated into liposomes for oral delivery 357 , 358 , 359 . However, liposomal formulations are not without their drawbacks. Issues such as hydrolysis, aggregation, fusion, and oxidation can lead to reduced stability and a reduced shelf life of liposomal products 360 . Additionally, the high cost of phospholipids contributes to the increased expense of liposomal formulations, limiting their widespread use 346 . A novel advancement in liposomal drug delivery is the development of hybrid vesicles that combine liposomes with exosomes, enhancing their stability and bioavailability for oral applications. One successful approach involves the fusion of functionalized liposomes with milk-derived exosomes to create a self-adaptive delivery system, as illustrated in 335 Fig. 8 . This hybrid vesicle leverages a pH-sensitive hydrazone bond between zwitterionic polymers and phospholipids, enabling surface property transformation in response to the pH microenvironment of the jejunum. In the intestinal lumen, the hydrophilic and neutrally charged surface of the hybrid vesicle facilitates mucus penetration while preserving membrane proteins. Upon reaching jejunal epithelial cells, the vesicle undergoes a charge shift, improving cellular uptake and intracellular transport. This adaptive mechanism enhances oral bioavailability to 8.7% and significantly improves therapeutic efficacy. This advancement builds on prior research demonstrating the potential of liposomes for oral protein and peptide drug delivery. Liposomal formulations have been explored to improve the bioavailability of insulin, erythropoietin, calcitonin, and parathyroid hormone, showing enhanced pharmacological effects. However, conventional liposomes face limitations such as hydrolysis, aggregation, and high production costs. The integration of exosomes addresses these challenges by improving liposomal stability and transport efficiency, representing a promising strategy for next-generation oral delivery systems. Figure 8 Schematic representative of exosome−liposome hybrid drug carrier for oral delivery of Semaglutide. (A) The preparation process and structural composition of the hybrid vesicle, and (B) the exosome−liposome vesicle effectively overcomes the GI tract and tissue barriers in the oral route. Reprinted with the permission from Ref. 335 . Copyright © 2024 American Chemical Society. Figure 8 Schematic representative of exosome−liposome hybrid drug carrier for oral delivery of Semaglutide. (A) The preparation process and structural composition of the hybrid vesicle, and (B) the exosome−liposome vesicle effectively overcomes the GI tract and tissue barriers in the oral route. Reprinted with the permission from Ref. 335 . Copyright © 2024 American Chemical Society. Another promising skin application of liposomal technology is its role in enhancing collagen delivery for anti-aging effects, as illustrated in Fig. 9 361 . Collagen-encapsulated liposomes, prepared via high-pressure homogenization, demonstrated superior skin penetration and retention compared to native collagen. Significantly, these vehicles prolonged collagen retention in the artificial membranes by a factor of two, even after several wash cycles. Additionally, real-time PCR analysis demonstrated that 3D skin models in the presence of collagen-encapsulated liposomes showed increased expression of keratin, involucrin, and collagen, even at the post-exposure to ethanol. This suggests that liposomal vehicles not only enhance collagen stability and absorption but also promote skin regeneration and barrier function, making them effective vehicles for anti-aging formulations. This application aligns with advancements in liposomal delivery systems, including their integration with exosomes to improve oral bioavailability and stability. While conventional liposomes have been explored for oral protein delivery, challenges such as low stability and high production costs remain. The ability of liposomes to facilitate transdermal and oral delivery underscores their versatility in biomedical applications, from enhancing skin hydration and elasticity to improving systemic bioavailability of therapeutic peptides and proteins. Figure 9 Schematic of liposome-assisted penetration and antiaging effects of collagen. Reprinted with the permission from Ref. 361 . Copyright © 2023 Wiley Periodicals LLC. Figure 9 Schematic of liposome-assisted penetration and antiaging effects of collagen. Reprinted with the permission from Ref. 361 . Copyright © 2023 Wiley Periodicals LLC. Niosomes are non-ionic surfactant-based vesicular systems, structurally analogous to liposomes, but composed primarily of non-ionic surfactants and cholesterol. They form bilayered vesicles capable of encapsulating both hydrophilic and lipophilic drugs, offering improved stability, biocompatibility, and drug delivery potential. This unique niosomal structure offers several advantages: 1) niosomes are easier and more cost-effective to produce, 2) they enhance drug loading capacity, provide higher solubilization and encapsulation efficacy, particularly for the transdermal and topical delivery of peptide and protein drugs 362 . For instance, Manosroi et al. 363 showed improved delivery of human tyrosinase plasmid using cationic niosomes in an abdominal rat skin model, observing a six-fold enhancement in drug permeability compared to a pure drug solution. Nevertheless, the incorporation of non-ionic surfactants in niosomes can reduce their physicochemical stability 364 . These surfactants may cause particle aggregation, therefore limiting the overall niosomal stability 362 . Additionally, charged nanocarriers may exhibit toxicity to human cells, emphasizing the need for careful consideration of safety when developing niosomal products 365 . A promising application of niosomes in oral drug delivery involves thymopentin (TP5), an immunomodulatory peptide prone to rapid degradation in the digestive system. Niosomal nanocarriers were fabricated using the thin film hydration method to enhance TP5 stability and prevent intestinal degradation. As shown in Fig. 10 , TP5-niosomes exhibited superior protection against enzymatic degradation in ex vivo intestinal luminal contents and mucosal homogenates for up to 6 h, compared to the pure drug solution 366 . These findings suggest that lipid-based niosomes can effectively reduce peptide degradation, potentially improving the oral bioavailability of TP5. Figure 10 Schematic of lipid-based niosomes of TP5 for ex vivo studies towards oral application. Reprinted with the permission from Ref. 366 . Copyright © 2022 Taylor & Francis Group. Figure 10 Schematic of lipid-based niosomes of TP5 for ex vivo studies towards oral application. Reprinted with the permission from Ref. 366 . Copyright © 2022 Taylor & Francis Group. In dermatological applications, niosomes have been explored for the topical delivery of (+)-catechin, a potent antioxidant. Catechin-loaded niosomes, developed via film hydration, exhibited spherical nanoscale size (204 nm), high drug entrapment efficiency (49%), and a sustained drug release profile up to 24 h, as shown in Fig. 11 A and B 367 . Ex vivo studies demonstrated significantly improved drug deposition and penetration in the viable layers of human skin ( P < 0.05), and indicated enhanced cellular protection through an endocytosis-mediated uptake process, as shown in Fig. 11 C 367 . These findings underscore the potential of niosomes as effective topical carriers for antioxidant and peptide delivery in skincare. Figure 11 Non-ionic niosomes of catechin on skin application. (A) The morphology of catechin-loaded niosomes under SEM; (B) Drug release profiles of niosomes of catechin and its solution in 24 h; and (C) Fluorescence microscopic images of a cross-section of human skin treated with niosomes and ethanol solution control (15% ethanol in water). Reprinted with the permission from Ref. 367 . Copyright © 2020 Taylor & Francis Group. Figure 11 Non-ionic niosomes of catechin on skin application. (A) The morphology of catechin-loaded niosomes under SEM; (B) Drug release profiles of niosomes of catechin and its solution in 24 h; and (C) Fluorescence microscopic images of a cross-section of human skin treated with niosomes and ethanol solution control (15% ethanol in water). Reprinted with the permission from Ref. 367 . Copyright © 2020 Taylor & Francis Group. Nanogels, or polymeric hydrogels, are networks at the nanometer scale (typically under 1000 nm) made up of swollen structures formed from amphiphilic or hydrophilic polyionic polymers, which may be derived from artificial synthesis or natural extraction 368 , 369 . Nanogels have emerged as an advancing and promising drug carrier because of their distinct characteristics and versatile applications. The promising properties of nanogels involve customizable physicochemical structures, the ability to deform and adapt within the nano-range, a high surface area for multivalent conjugations, and a significant water content. Furthermore, nanogels are made from biocompatible materials, offering high drug loading and enhanced stability. They enhance the efficacy of targeted delivery to specific cells and compartments, exhibit desirable immunomodulatory effects, and demonstrate sensitivity to environmental changes 370 , 371 , 372 . These characteristics make nanogels a compelling option for advanced drug delivery systems. As a nanocarrier, it can be directed to specific sites following injection into bodily fluids, with the versatility to adjust its physicochemical properties to address key biological barriers, such as evading the reticuloendothelial system, reducing renal clearance via the glomeruli, and minimizing nonspecific accumulation in various organs in vivo . Nanogels have been mainly designed to improve drug stability through several approaches, including: physical self-assembly of interactive polymers, polymerization of monomers in homogeneous or micro/nanoscale heterogeneous systems, cross-linking of pre-synthesized polymers, and template-assisted nanofabrication techniques 373 . A variety of natural biodegradable polymers are commonly used in nanogel development, such as heparin, hyaluronic acid, dextran, dextrin, poly- l -lysine, poly( γ -glutamic acid) ( γ -PGA), pullulan, mannan, chitosan, and alginate. Additionally, synthetic biocompatible and biodegradable polymers like poly( ε -caprolactone) (PCL), poly(methyl methacrylate) (PMMA), poly(glycolic acid) (PGA), poly( d , l -lactic acid) (PLA), and poly( d , l -lactic- co -glycolic acid) (PLGA) are also frequently used. Importantly, many of these polymers have been approved by the US Food and Drug Administration (FDA) for human use, making them suitable candidates for the development of nanoparticulate delivery systems 374 . A promising example of nanogels in oral application is the development of pH-responsive, reactive oxygen species (ROS) scavenging, and highly swellable nanogels as shown in Fig. 12 342 . The nanogels are synthesized by acrylamide monomers with itaconic acid at a molar ratio of 4:1 through a free radical polymerization approach. The resulting spherical nanogels, measuring 180 ± 20 nm in size, are verified by field emission scanning electron microscopy. Dynamic light scattering analysis indicates a hydrodynamic diameter of 270 ± 25 nm and a surface charge of −6.9 ± 2.3 mV. The presence of various functional groups on the nanogels is confirmed through Fourier-transform infrared spectroscopy, X-ray photoelectron spectroscopy, and nuclear magnetic resonance analysis. The nanogels demonstrate a significant swelling (10-fold) at colonic pH (pH 7.4) compared to gastric pH (pH 1.2), highlighting their pH-responsive behavior. Furthermore, the nanogels exhibit a fivefold increase in ROS scavenging activity compared to the control. Figure 12 Schematic of pH-responsive swellable nanogel for colon-targeted oral drug delivery. Reprinted with the permission from Ref. 339 . Copyright © 2024 American Chemical Society. Figure 12 Schematic of pH-responsive swellable nanogel for colon-targeted oral drug delivery. Reprinted with the permission from Ref. 339 . Copyright © 2024 American Chemical Society. Hyaluronic acid-based nanogels have gained considerable attention in topical and transdermal delivery applications due to their self-assembling nature, biodegradability, and ease of preparation. A recent study explored the relationship between peptide structure and encapsulation efficiency within these nanogels, as illustrated in Fig. 13 375 . This study explored the effects of key peptide properties, such as charge and hydrophobicity, on their encapsulation within octenyl succinic anhydride-modified hyaluronic acid nanogels. The size and surface characteristics of the peptide-loaded nanogels, assembled using microfluidic techniques, were assessed through small-angle neutron scattering, laser Doppler electrophoresis, and dynamic light scattering. Moreover, this study explored changes in the protein's secondary structure following encapsulation, its release profile, and in vitro antimicrobial efficacy. The findings revealed that peptides with higher hydrophobicity demonstrated stronger interactions with the nanogel, preferentially localizing internally rather than at the surface. This interaction resulted in nanogels with smooth surfaces, a more spherical morphology, and prolonged release profiles. Conversely, cationic and hydrophilic peptides were primarily located at the surface of the nanogel, leading to looser structures and a faster, more complete release in biorelevant media. These resulting data underscore that the performance of nanogel delivery systems is influenced by the specific properties of the therapeutic peptides, highlighting the importance of peptide characteristics in tailoring nanogel formulations for targeted applications. Figure 13 Schematic nanogel delivery systems for cationic peptides. Reprinted with the permission from Ref. 375 . Copyright © 2024 Elsevier. Figure 13 Schematic nanogel delivery systems for cationic peptides. Reprinted with the permission from Ref. 375 . Copyright © 2024 Elsevier. Nanoparticles are defined as colloidal particles with sizes in the range below 1000 nm. As for transdermal delivery, the optimal particle size typically ranges below 400 nm. Nanoparticles can adopt a core‒shell structure, referred to as nanocapsules, or a continuous monolithic structure, known as nanospheres 376 , 377 . These nanoparticles offer significant advantages in improving the bioavailability of encapsulated therapeutic peptides and proteins. Research has demonstrated that encapsulating drugs in polymeric nanoparticles made from materials such as poly(lactic acid) (PLA) 378 , poly lactic- co -glycolic acid (PLGA) 379 , gelatin 380 or chitosan 381 enhances their stability and bioavailability. Particle size is a crucial factor influencing drug absorption, with smaller nanoparticles generally demonstrating superior absorption. Studies have shown that particles larger than 500 nm face significant challenges in being absorbed through the human skin 382 . Due to their small size, nanoparticles can also exhibit bioadhesion to the gastrointestinal tract wall, lodging in the intervillar spaces, which increases their residence time and enhances bioavailability 383 , 384 . Several studies have reported promising results from incorporating therapeutic peptides and proteins into solid lipid nanoparticles, such as thymopentin, streptacidin, and insulin 383 , 385 . However, polymeric nanoparticles may exhibit toxic side effects, making it essential to focus on developing nanoparticles with non-toxic characteristics and high stability for safer and more effective formulations. One novel application for oral peptide and protein administration is motivated by the innate physicochemical characteristics of spores. This approach involves the use of deoxycholic acid-modified Heyndrickxia coagulans spores, which are loaded with insulin (DA-Spore/Ins). This system aims to overcome absorption barriers and enhance the oral delivery of insulin. As illustrated in Fig. 14 , the DA-Spore/Ins formulation offers superior stability and facilitates effective mucus permeation via spore germination in the digestive microenvironment 386 . The system self-assembles into nanoparticles (NPs) by disintegrating the DA-covalently amphipathic protein coat and the hydrophilic protein/peptide drug, enabling efficient transport across epithelial cells via the bile acid pathway. In vivo studies showed that the DA-Spore/Ins system achieved a 15.1% oral relative bioavailability and superior hypoglycemic effects in type I diabetic rats, with good biocompatibility. These findings suggest that the biological properties of Heyndrickxia coagulans spore-based nanogenerators hold promise for oral insulin and other protein drug therapies. This approach is linked to the broader application of nanoparticles in oral delivery systems. Nanoparticles, ranging from 1 to 1000 nm in size, offer improved bioavailability for encapsulated peptides and proteins. Nanocapsules (with core‒shell structures) and nanospheres (with continuous monolithic structures) have been optimized to improve drug absorption. Particle size plays a crucial role in drug absorption, with smaller particles (200–400 nm) being optimal for transdermal administration. Nanoparticles can improve bioavailability by enhancing stability and facilitating absorption through bioadhesion to the GI tract wall, increasing residence time. However, attention must again be given to ensuring non-toxicity and stability in the formulation. Figure 14 Schematic representation of a Heyndrickxia coagulans spore-based nanoparticle system designed for enhanced oral insulin delivery and hypoglycemic treatment. Reprinted with the permission from Ref. 386 . Copyright © 2025 Elsevier. Figure 14 Schematic representation of a Heyndrickxia coagulans spore-based nanoparticle system designed for enhanced oral insulin delivery and hypoglycemic treatment. Reprinted with the permission from Ref. 386 . Copyright © 2025 Elsevier. Hollow polydopamine nanoparticles (HPDA) have shown promise for delivering the pro-healing peptide RL-QN15 to skin wounds. As shown in Fig. 15 , HPDA nanoparticles were synthesized and loaded with RL-QN15 (HPDAlR), an amphibian-derived peptide with pro-healing properties 343 . The characterization, biodistribution, and clearance of both HPDA nanoparticles and HPDAlR were systematically examined. Additionally, the loading efficiency of RL-QN15 and its sustained-release profile from HPDAlR were also assessed. Both HPDA nanoparticles and HPDAlR exhibited non-toxicity towards keratinocytes, macrophages, and mice. Although HPDA nanoparticles did not show significant pro-healing effects, HPDAlR significantly improved the therapeutic efficacy of RL-QN15, accelerating wound healing and modulating cytokine release from macrophages. In animal models, HPDAlR showed a 50-fold increase in regenerative potency for mouse skin wounds and a 10-fold increase in oral ulcers in rats compared to RL-QN15 alone. Additionally, HPDAlR accelerated healing in skin scalds in mice and full-thickness skin wounds in swine. These findings suggest that HPDAlR holds significant potential for skin wound healing therapies, highlighting the growing role of nanoparticles in enhancing drug stability, controlled release, and tissue regeneration. Figure 15 Schematic of hollow polydopamine nanoparticles loading with peptide RL-QN15 for skin wounds. Reprinted with the permission from Ref. 341 . Copyright © 2021 Springer Nature. Figure 15 Schematic of hollow polydopamine nanoparticles loading with peptide RL-QN15 for skin wounds. Reprinted with the permission from Ref. 341 . Copyright © 2021 Springer Nature. PEGylation is chosen as the preferred additive as it is one of the most effective and common modifications among the nanoparticulate drug delivery systems 387 . PEGylation refers to the chemical modification of small molecules and therapeutic bioactives by attaching polyethylene glycol (PEG) molecules of varying lengths to improve their pharmaceutical properties 388 . PEGs are synthetic, highly water-soluble, and inert polymers that come in a wide array of molecular weights. These polymers are commonly found in consumer healthcare products, including laxatives, toothpaste, and shampoos, and are particularly prominent in biopharmaceutical applications 389 . The process of PEGylation offers several advantages, including prolonged circulation time of proteins or nucleic acids, enhanced water solubility of therapeutics, protection against biological inactivation ( e.g. , peptidolysis and proteolysis), and a reduction in immunogenicity 390 . The primary benefit of PEGylation is its ability to extend the half-life of therapeutic agents, thus improving drug stability in vivo and reducing renal clearance of the drug 391 . PEGylating a drug delivery system improves systemic circulation time and decreases immunogenicity to obtain higher stability. The impact of PEG coatings on the surface of systemically administered nanoparticulate drug delivery formulations has, and continues to be, widely studied 392 . Some examples can be PEGylated SLNs, PEGylated Niosomes, and PEGylated nanogels, whose structures are shown in Fig. 16 393 . PEGylation of a nanoparticulate drug delivery system not only enhances the bioactives' in vivo stability but also improves the biocompatibility and therapeutic effect 394 . Figure 16 The proposed structures of PEGylated SLNs, niosomes, and nanogels. Figure 16 The proposed structures of PEGylated SLNs, niosomes, and nanogels. One of the PEGylated nanocarrier applications is PEGylated liposomes. In the study from Du et al. 344 , the influence of PEG chain lengths (2, 3.4, 5, and 10 kDa) on BBB penetration and brain targeting using Angiopep-2 peptide-decorated liposomes was explored, as shown in Fig. 17 . The results revealed that shorter PEG chains facilitated more efficient in vitro cell uptake through endocytosis, while longer PEG chains enhanced BBB penetration via transcytosis. In vivo , liposomes with longer PEG chains showed superior brain accumulation in both normal and glioblastoma (GBM)-bearing mice due to prolonged circulation and improved BBB penetration. These findings highlight the critical role of PEG chain length in designing nanocarriers for targeted delivery, particularly for brain diseases. This study illustrates the potential of PEGylated nanocarriers for peptide delivery, offering key insights into the design of nanocarriers for enhanced bioavailability and targeted treatment. PEGylation, which involves attaching polyethylene glycol (PEG) molecules to the surface of nanoparticles, is widely used to improve the stability, solubility, and circulation time of therapeutic peptides. This modification not only enhances the biocompatibility and stability of the peptides but also facilitates their controlled release and efficient delivery to specific tissues, such as the brain. The varying lengths of PEG chains can significantly affect the properties of the nanocarriers, such as the release rate and bioadhesion, which is crucial for optimizing peptide delivery systems for clinical applications. Figure 17 Schematic representation of PEGylated liposomes. (A) Composition of liposomes with varying PEG chain lengths; (B) Cellular uptake of U87MG-Luc cells incubated with PEGylated liposomes for 6 h; (C) Median DiO intensity of cells incubated with different PEGylated liposomes, as measured by flow cytometry. Data are presented as mean ± SD, n = 3; (D) Confocal laser scanning microscopy images of U87MG-Luc cells following 6 h of incubation with various PEGylated liposomes; (E) DiO fluorescence intensity in U87MG-Luc cells measured by Zen, mean ± SD, n = 6; Reprinted with the permission from Ref. 343 . Copyright © 2024 Elsevier. Figure 17 Schematic representation of PEGylated liposomes. (A) Composition of liposomes with varying PEG chain lengths; (B) Cellular uptake of U87MG-Luc cells incubated with PEGylated liposomes for 6 h; (C) Median DiO intensity of cells incubated with different PEGylated liposomes, as measured by flow cytometry. Data are presented as mean ± SD, n = 3; (D) Confocal laser scanning microscopy images of U87MG-Luc cells following 6 h of incubation with various PEGylated liposomes; (E) DiO fluorescence intensity in U87MG-Luc cells measured by Zen, mean ± SD, n = 6; Reprinted with the permission from Ref. 343 . Copyright © 2024 Elsevier. In addition to chemical, physical, and nanotechnological strategies, the choice of administration route and anatomical site exerts a critical influence on the pharmacokinetic behavior, therapeutic efficacy, and patient acceptability of protein and peptide therapeutics 395 . Due to their large molecular size, structural fragility, and susceptibility to enzymatic degradation, these macromolecules often exhibit limited membrane permeability and poor oral bioavailability 396 . Consequently, delivery routes that circumvent first-pass hepatic metabolism and proteolytic barriers are pivotal for clinical success. The pulmonary route offers several pharmacological advantages owing to the lung's extensive absorptive surface area (∼100 m 2 ), dense capillary network, and ultrathin alveolar epithelium (∼0.1–0.2 μm), which collectively facilitate rapid systemic absorption and avoidance of hepatic metabolism 397 . Approved products such as inhaled insulin ( e.g. , Afrezza®) exemplify the feasibility of this approach 398 . Nevertheless, barriers such as mucociliary clearance, alveolar macrophage uptake, and enzymatic degradation in the epithelial lining fluid pose significant challenges. Moreover, efficient alveolar deposition necessitates particles with aerodynamic diameters between 0.5 and 3 μm, as larger particles are retained in the upper airways while smaller ones are exhaled 399 . Inhaled protein formulations also require stabilization against shear-induced aggregation during aerosolization and pH-sensitive degradation. Similarly, the nasal route has garnered attention due to its high vascularity and approximately 150 cm 2 of absorptive surface, enabling rapid systemic uptake and, uniquely, direct nose-to-brain transport via the olfactory and trigeminal neural pathways 400 . This allows for the non-invasive delivery of neuropeptides and protein-based CNS therapies. However, short residence time (∼15–30 min), mucociliary clearance, and tight junctions limiting paracellular diffusion, in addition to enzymatic barriers such as aminopeptidases, compromise bioavailability 401 . Strategies to enhance mucosal permeation—such as mucoadhesive polymers, enzyme inhibitors, and surfactants like bile salts or chitosan—must be carefully optimized to balance absorption enhancement against risks of epithelial irritation and long-term toxicity. The buccal and sublingual mucosae provide direct access to systemic circulation via the jugular and facial veins, bypassing hepatic first-pass metabolism and minimizing enzymatic degradation 402 . These tissues are more enzymatically stable than the gastrointestinal tract, offering a relatively favorable milieu for peptide absorption. However, the stratified squamous epithelium (∼500–800 μm) presents a significant diffusion barrier, and the lack of active transport mechanisms restricts uptake primarily to low-molecular-weight, lipophilic peptides 403 . Advanced delivery strategies under development include mucoadhesive films, permeation enhancers, and electro-driven systems ( e.g. , iontophoresis), although few have progressed beyond preclinical evaluation due to limited permeability and difficulty sustaining therapeutic levels. SC injection remains the most widely adopted route for peptide and protein drugs ( e.g. , insulin analogues, GLP-1 receptor agonists) due to its relatively convenient administration and consistent systemic exposure 404 . However, SC absorption is influenced by local blood flow, enzymatic activity, and lymphatic drainage, resulting in inter- and intra-patient variability. By contrast, intradermal delivery, particularly via microneedle arrays, provides access to the richly vascularized dermis, enabling rapid onset, dose sparing, and improved patient adherence. Microneedle-mediated delivery also allows for targeted immune activation, making it a compelling platform for peptide vaccines and long-acting depot formulations 405 . Finally, site-specific routes, such as intra-articular, intravitreal, or intrathecal administration, are employed for localized therapeutic effects, especially in inflammatory, ophthalmic, or central nervous system disorders 406 . These specialized approaches offer high local concentrations while reducing systemic exposure, but necessitate highly controlled formulations with stringent sterility and stability requirements to mitigate risks of immunogenicity and toxicity. In overview, tailoring the delivery route and anatomical target site, in conjunction with formulation design, represents a fundamental strategy to overcome the pharmacological and biopharmaceutical limitations of peptide and protein therapeutics. A route-specific approach that accounts for anatomical barriers, enzymatic environments, and patient-centric considerations is indispensable for advancing these therapies from bench to bedside.

This article has offered an in-depth analysis of the key challenges associated with peptide drug delivery, including physical, enzymatic, and membrane-related barriers that limit their clinical effectiveness. By examining the complexities of peptide metabolism and the challenges inherent to various administration routes, such as oral, parenteral, topical, and transdermal, this paper highlights the necessity for innovative approaches to improve peptide/protein bioavailability and therapeutic efficacy. Understanding the interplay between therapeutic peptides/proteins and their barriers to delivery to their site of action is essential for the development of advanced drug delivery systems that offer high entrapment efficiency, multifunctionality, and cost-effectiveness. The integration of chemical modifications, tailored formulation strategies, and nanoparticulate delivery systems holds significant potential for improving peptide stability, bioavailability, and patient adherence. Recent advancements in nanotechnology-based carriers, such as liposomes, polymeric nanoparticles, and nanoemulsions, have demonstrated promising outcomes in protecting peptides from enzymatic degradation and facilitating targeted delivery. Furthermore, systematic approaches, including quality-by-design and formulation-by-design, provide rational frameworks for optimizing peptide and protein formulations and streamlining their clinical translation. This review serves as a valuable resource for researchers in peptide and protein therapeutics, offering critical insights into formulation strategies, emerging trends, and translational pathways for overcoming delivery challenges. With continued research and innovation, there is considerable potential to bridge existing gaps in peptide- and protein-based drug delivery, ultimately realizing their full clinical potential and improving therapeutic outcomes.

Peptides are short chains of amino acid residues, typically consisting of fewer than 50 amino acids, linked together by peptide bonds 1 . Proteins, on the other hand, are larger biomolecules composed of one or more polypeptide chains, generally exceeding 50 amino acids in length 2 . Compared to small-molecule drugs, peptides and proteins exhibit high specificity in their biological activity, resulting in fewer off-target effects and reduced toxicity. This has led to their increasing application in pharmaceuticals, functional foods, and cosmetics over the past decade. As of 2025, the global peptide and protein therapeutics market continues to expand, with over 80 peptide-based drugs currently approved for clinical use, contributing around $50 billion USD in pharmaceutical revenues 3 . Additionally, more than 150 peptide candidates are in clinical trials, with another 600–700 in preclinical development 4 . The market for peptide and protein-based drugs is expected to expand at a compound annual growth rate (CAGR) of 9% to 10%, exceeding $100 billion USD by 2030 5 . This surge is driven by advancements in drug delivery technologies, bioengineering, and recombinant protein production, making peptides and proteins key players in the future of biopharmaceuticals. In recent years, major pharmaceutical companies have filed more patent applications for biopharmaceuticals than for small-molecule drugs, with this trend continuing to grow. This shift is reflected in global sales data; in recent years, peptide- and protein-based therapeutics, especially monoclonal antibodies, have continued to dominate the pharmaceutical market. For instance, by 2024, drugs such as adalimumab (Humira®, AbbVie) and infliximab (Remicade®, Johnson & Johnson and Merck & Co.) remain among the top-selling biologics worldwide 6 . The global market for monoclonal antibodies alone was valued at over US$200 billion in 2024, and projections reach over US$400 billion by 2029, reflecting sustained growth driven by increasing demand for targeted therapies 6 . These data highlight the expanding clinical and commercial importance of peptide- and protein-based drugs. In fact, seven out of the ten best-selling pharmaceuticals are based on amino acid sequences 6 . The growing preference for larger biopharmaceuticals over traditional small-molecule drugs introduces unique challenges for formulation scientists. Therapeutic peptides and proteins are increasingly being integrated into clinical treatment strategies, owing to their high specificity and ability to target disease pathways with precision 7 . The main challenge in commercializing peptide and protein-based drugs stems from their limited stability, due to both physical and biochemical barriers encountered during oral or transdermal administration 8 . As a result, invasive parenteral routes remain the most viable means of delivery. However, there is no systemic analysis or overview of the different routes of administration for peptide/protein therapeutics. Therefore, this article will highlight recent advancements in the development and application of peptide and protein therapeutics. In particular, the comprehensive challenges in delivering peptides and proteins through different administration routes and the advanced strategies to overcome these challenges are presented and discussed in this review.

Peptide- and protein-based therapeutics hold significant clinical promise due to their high specificity, potent bioactivity, and favorable safety profiles. The recently approved protein and peptide drugs are selected and listed in Table 10 418 , 419 , 420 , 421 , 422 , 423 , 424 , 425 , 426 , 427 , 428 , 429 , 430 , 431 , 432 , 433 , 434 . Based on this, a growing number of peptide and protein therapeutics have gained FDA approval in recent years, showcasing diverse innovations in formulation and delivery, ranging from oral absorption enhancers ( e.g. , Rybelsus® and Mycapssa®) to long-acting injectables ( e.g. , Cabenuva® and Mounjaro®) and advanced biologics such as cell and gene therapies ( e.g. , Skysona® and Lantidra™). These advances highlight the evolving landscape of biologic drug delivery, where physicochemical challenges such as enzymatic degradation, poor membrane permeability, and short half-lives are being addressed through tailored delivery technologies. Table 10 Examples of recently FDA-approved peptide and protein drugs 418 , 434 . Table 10 Drug Indication Route Approval year Type Formulation/Innovation Ref. Rybelsus® (Semaglutide) Type 2 diabetes Oral 2019 Peptide SNAC-mediated absorption enhancer (Novo Nordisk) 418 Vyleesi® (Bremelanotide) Hypoactive sexual desire disorder SC 2019 Peptide Melanocortin receptor agonist 419 Evenity® (Romosozumab) Osteoporosis SC 2019 Protein Anti-sclerostin monoclonal antibody 420 Lyumjev® (insulin lispro-aabc) Diabetes SC 2020 Protein Ultra-rapid formulation with citrate/treprostinil 421 Imcivree® (Setmelanotide) Genetic obesity syndromes SC 2020 Peptide MC4R agonist 422 Cabenuva® (Cabotegravir + Rilpivirine) HIV-1 IM 2021 Protein Monthly long-acting injectable 423 Nulibry® (Fosdenopterin) MoCD type A IV 2021 Peptide Cyclic pyranopterin monophosphate analogue 424 Mounjaro® (Tirzepatide) Type 2 diabetes SC 2022 Peptide Dual GIP/GLP-1 receptor agonist 425 Terlivaz® (Terlipressin) Hepatorenal syndrome IV 2022 Peptide Vasopressin analogue 426 Skysona® (Elivaldogene autotemcel) Cerebral adrenoleukodystrophy IV 2022 Protein Gene-modified autologous cell therapy 427 Zegalogue® (Dasiglucagon) Severe hypoglycemia SC 2021 Peptide Glucagon analogue with enhanced stability 428 Bkemv® (Pegcetacoplan) Paroxysmal nocturnal hemoglobinuria SC 2023 Protein C3 complement inhibitor 429 Lantidra™ (Donislecel) Type 1 diabetes IV 2023 Protein Allogeneic pancreatic islets 430 Ozempic® Type 2 diabetes SC 2022 Peptide Extended dosing of GLP-1 analogue 431 Zepbound™ (Tirzepatide) Obesity SC 2023 Peptide Dual GIP/GLP-1 agonist for chronic weight control 432 Octreotide (Mycapssa®) Acromegaly Oral 2020 Peptide Enteric-coated capsule with permeability enhancers 433 Oramed ORMD-0801 (insulin) Type 2 diabetes Oral 2025– Protein Protease inhibitors with enteric coating (in trials) 434 Examples of recently FDA-approved peptide and protein drugs 418 , 434 . Despite this positive progress, the full therapeutic potential of peptide and protein drugs remains partially unrealized, largely due to the inherent limitations and practical complexities of current delivery enhancement strategies. For example, nanoparticle- or carrier-based systems often involve multistep and resource-intensive manufacturing processes, which are difficult to scale up while maintaining product consistency and reproducibility 407 . Moreover, formulation complexity introduces challenges in quality control, stability assurance, and cost–effectiveness, limiting their accessibility and commercial viability. The use of excipients such as surfactants, co-surfactants, and stabilizers, although necessary for enhancing drug stability and bioavailability, can generate intermediate degradation products or excipient–drug interactions that compromise therapeutic efficacy or introduce safety concerns, especially when administered chronically 408 . These potential risks require comprehensive safety and toxicity testing, which can delay development timelines and increase regulatory scrutiny 409 . Furthermore, interpatient variability in pharmacokinetics, coupled with the possibility of immune responses to delivery vehicles or protein aggregates, adds an additional layer of complexity to clinical translation. Immunogenicity risks are especially concerning for repeated administration of modified proteins or formulations involving novel materials. In some cases, enhancement strategies such as PEGylation, while extending half-life, may inadvertently alter receptor binding or lead to accelerated blood clearance phenomena after repeated dosing, undermining long-term efficacy 410 . Thus, while promising strategies have emerged, bridging the gap between laboratory research and clinical implementation continues to demand multidisciplinary innovation, robust translational frameworks, and regulatory harmonization. A systematic approach during formulation development could mitigate these challenges. Strategies such as ‘quality-by-design’ and ‘formulation-by-design’ provide a rational and scientific framework for optimizing formulations, thereby improving their clinical translation potential 411 , 412 . Regulatory considerations are also crucial for the clinical success of peptide/protein-loaded nanoparticulate drug delivery systems. Safety and toxicity assessments are essential when seeking regulatory approval, as these formulations often involve complex systems containing multiple excipients, such as surfactants, co-surfactants, and stabilizers. These excipients must be selected from the Generally Recognized as Safe (GRAS) list maintained by global regulatory agencies, ensuring their established safety and toxicity profiles 413 , 414 . However, specific regulatory guidelines for nanocarrier-based therapeutics remain lacking, as agencies such as the FDA and the European Medicines Agency (EMA) have yet to establish comprehensive frameworks for their approval 415 . To address this gap, the USFDA has formed a Nano-Technology Interest Group to facilitate communication and educate regulatory staff on advancements in nanomedicine 416 , 417 . Looking forward, emerging technological trends are expected to further reshape the field. Intelligent delivery systems incorporating stimuli-responsive materials, AI-driven formulation design, and bioinspired delivery platforms ( e.g. , exosome-mimetic carriers) offer transformative potential for overcoming current barriers. Additionally, convergence with digital health tools, such as smart injectors and wearable biosensors, may enable real-time dosing feedback and personalized therapeutic regimens. The integration of synthetic biology with delivery science is also anticipated to produce next-generation biotherapeutics with programmable release and enhanced tissue targeting. By aligning regulatory frameworks and industrial scale-up with such innovations, the field is poised to move toward more precise, effective, and patient-centric delivery of peptide and protein therapeutics. Overall, while peptide/protein-based therapeutics offer significant clinical potential, overcoming challenges related to delivery, regulatory approval, and large-scale production remains essential. Through systematic formulation development, regulatory alignment, and innovative strategies such as chemical modifications and advanced nanoparticulate systems, the next generation of peptide and protein therapeutics can achieve improved efficacy, stability, and patient compliance.

The authors have no conflicts of interest to declare.