Two major types of biochemical barriers exist for orally administered peptides: variable pH and gastrointestinal proteases (Figure 1). Orally administered bioactive peptides travel through the oral cavity to the stomach, then to the duodenum, jejunum, ileum, and finally to the colon and rectum (17). Although digestion begins in the oral cavity, due to the extremely short oral action time, the oral cavity not typically cited as a major factor hindering the absorption and utilization of orally administered bioactive peptides. The main factors affecting the absorption and utilization of oral bioactive peptides mainly come from the stomach and small intestine. The first thing to overcome when taking bioactive peptides orally is the variable pH of the gastrointestinal tract. The pH value of gastric juice is 1.5–3.5, that of the duodenum is about 5–6, and that of the jejunum and terminal ileum rises to 7–8 (18). Variable pH gradients have a great impact on the physiological efficacy of some bioactive peptides. The antioxidant activity of the pentapeptide ATSHH from whitefish protein will show a significant decrease trend under acidic conditions (pH = 2) (19).

In addition, after the bioactive peptides reach the stomach, they will stimulate the gastric mucosa to secrete pepsin from the gastric lining cells. Pepsin can hydrolyze the polypeptide with aromatic residues such as phenylalanine, tryptophan, and tyrosine. Bioactive peptides hydrolyzed by pepsin will lose their inherent biological activity. After the bioactive peptide enters the small intestine through the stomach, the trypsin and chymotrypsin present in the small intestine will also specifically hydrolyze the peptide chain (20). The hydrolysis of the above enzymes will change the structure and activity of the bioactive peptide. Li et al. (21) performed in vitro simulated digestion experiments on rice protein hydrolyzate and found that the anti-hypertensive IC₅₀ (half maximal inhibitory concentration) value of rice protein increased from 140 to 180 μg/mL in the presence of digestive enzymes (pepsin and pancreatic enzymes), indicating that the anti-hypertensive activity of rice protein hydrolyzate was significantly reduced. In addition, after the bioactive peptides reach the stomach, they will stimulate the gastric mucosa to secrete pepsin from the gastric lining cells. Pepsin can hydrolyze the polypeptide with aromatic residues such as phenylalanine, tryptophan, and tyrosine. Bioactive peptides hydrolyzed by pepsin will lose their inherent biological activity. After the bioactive peptide enters the small intestine through the stomach, the trypsin and chymotrypsin present in the small intestine will also specifically hydrolyze the peptide chain (20). The hydrolysis of the above enzymes will change the structure and activity of the bioactive peptide. Li et al. (21) performed in vitro simulated digestion experiments on rice protein hydrolyzate and found that the anti-hypertensive IC₅₀ (half maximal inhibitory concentration) value of rice protein increased from 140 to 180 μg/mL in the presence of digestive enzymes (pepsin and pancreatic enzymes), indicating that the anti-hypertensive activity of rice protein hydrolyzate was significantly reduced.

After bioactive peptides are digested in the stomach and successfully reach the small intestine, the intestinal mucus layer covering the intestinal surface is one of the main factors limiting the bioavailability of oral bioactive peptides. The intestinal mucus layer is a kind of intelligent hydrogel with high viscoelasticity and adhesiveness, which contains highly branched polysaccharides and negatively charged mucin (22). The intestinal mucus layer plays a protective role by forming a sieve-like structure on itself. This structure can effectively prevent 10–200 nm particles from passing through the mesh, and has the function of selectively transmitting nutrients (23). Mucin, glycolipids, and glycoproteins in the mucus layer act as both barriers and transmit signals (24). When bioactive peptides reach the intestinal mucus layer, their further diffusion may be affected by mucin adhesion.

After bioactive peptides pass through the mucus layer and reach the surface of epithelial cells, the epithelial cells located under the mucus are another major factor limiting the bioavailability of oral bioactive peptides. The small intestine epithelial cells are a continuous monolayer that separates the intestinal lumen from the underlying lamina propria. There is a tight junction (TJ) between adjacent epithelial cells, which only allows small molecules such as water and ions to pass through. In addition, the small intestine cell membrane acts as a barrier to prevent extracellular substances from freely entering and exiting the cells by selectively absorbing nutrients (25). Based on the above reasons, the small intestinal epithelium is impermeable. Bioactive peptides need to pass through the TJ or intestinal epithelial cell membrane to reach the bloodstream and ultimately bind to the target to exert physiological activity. However, most bioactive peptides cannot effectively penetrate intestinal epithelial cells due to the lack of targeted carrier proteins on the intestinal epithelial cell membrane, which seriously affects the bioavailability of bioactive peptides.

The carrier-mediated transport pathway primarily relies on oligopeptide transporters (34). The important feature of transporters is that they can select peptides. Transporters have been found to recognize and transport over 8,000 different peptides (35). There are two main types of transporters: PepT1 and PepT2. Both PepT1 and PepT2 can be used for the transport of dipeptides and tripeptides (36). Currently, there are more studies on PepT1 than PepT2 on the transport of polypeptides. PepT1 is mainly expressed in intestinal epithelial cells and is responsible for the transport and absorption of bioactive peptides. As mentioned in the section on the physicochemical properties of peptides, the charge of peptides affects the mode of transport, and PepT1 preferentially recognizes peptides with neutral charge and high hydrophobicity, and preferentially binds residues rich in non-polar amino acids. Fan et al. (37) studied the transport modes of IW, IWH, and IWHHT peptides in Caco-2 cells, which further verified that PepT1 preferred to select small peptides with high hydrophobicity. Table 1 summarizes the transport pathways of different bioactive peptides through the Caco-2 cell model, aiming to provide a solid experimental basis for subsequent research and product development.

The paracellular transport pathway is currently the most reported passive absorption pathway for bioactive peptides with more than tripeptides (38). The driving force for oligopeptide transport comes from the electrochemical gradient formed by protons as high-energy electrons are transferred along the respiratory chain, and the diffusion process does not require a carrier or energy consumption (39). The paracellular transport pathway is mediated through the TJ between epithelial cells, a tight biological barrier with selective permeability (40). It has been shown that TJ tends to transport negatively charged peptides and is selective for positively charged peptides (41), and bioactive peptides with small hydrophilic molecular weights are more inclined to this transport mode (42). In general, when the molecular diameter of a bioactive peptide exceeds 15 Å, the peptide cannot undergo paracellular transported. However, it is still possible for bioactive peptides with larger molecular sizes to diffuse through TJ if their structures have high conformational flexibility (43). Chiasma has successfully developed an oral formulation of octreotide, named Mycapssa®, utilizing its innovative Transient Permeation Enhancer (TPE™) technology. In this approach, sodium caprate serves as an osmotic enhancer, inducing the reversible opening of tight junctions between intestinal epithelial cells to facilitate the paracellular transport of peptides. The successful development of Mycapssa® not only strongly confirms the feasibility of the paracellular transport strategy for the oral delivery of peptide drugs but also paves the way for further research into the oral delivery of bioactive peptides (44).

Endocytic transport is an energy-dependent transcellular transport pathway and is the main transport pathway for long-chain peptides. In this pathway, bioactive peptides are transported into cells through the formation of vesicles formed by invagination of the cell membrane (45). Bioactive peptides with smaller molecules can enter the blood circulation through carrier transport and paracellular pathways, while most large molecule peptides need to be transported through endocytosis. The study by Xu et al. (46) showed that 17-peptide (casein 193–209) can be completely absorbed by the Caco-2 cell monolayer model, and its absorption process is mainly carried out through endocytosis transport. The first step in endocytic transport is the interaction of polypeptides with the cell membranes. Since the cell membrane is composed of a lipid bilayer, endocytic transport is considered an ideal pathway for the transport of lipophilic peptides. The anti-oxidant peptide YWDHNNPQIR is transported across the Caco-2 cell monolayer via endocytosis, primarily because it is composed of hydrophobic amino acids (47). Xiao et al. have innovatively designed and prepared a hybrid liposome system named mExos@DSPE-Hyd-PMPC. This system significantly improves drug encapsulation efficiency and enhances endocytic transport efficacy by effectively integrating functional liposomes with milk-derived exosomes (mExos). Notably, this hybrid liposome exhibits adaptive surface characteristics, enabling it to intelligently adjust its physicochemical properties based on the pH microenvironment of the intestinal mucosal surface. This adaptability facilitates a more efficient endocytic transport process (48).

Notably, research has demonstrated that the hydrophilicity and charge state of bioactive peptides play a significant role in their transport within the body (49). The charge can influence the interactions of bioactive peptides with cell membranes, transport carriers, and other molecules in the gastrointestinal environment. Table 2 summarizes the relationship between various transport mechanisms and the properties of peptides. However, it is important to emphasize that hydrophilicity and charge state are not the only factors determining the transport pathways of bioactive peptides. The transport pathways are also influenced by several other factors, including molecular weight, peptide structure, hydrophobicity, the gastrointestinal environment, and the selection of transport carriers (133).

Liposomes are a kind of spherical closed vesicle formed by concentric phospholipid molecules linked end to end through hydrophobic interactions, which can protect the loaded materials from being broken down by enzymes and improve their bioavailability in the body (Figure 3a) (67). Gong et al. (68) the bioavailability of peanut peptides was effectively improved after being encapsulated in nanoliposomes. The main reason is that the nanoliposomes prepared in this study exhibited good stability under different pH conditions and different morphologies, which allows the peanut peptides encapsulated in the nanoliposomes to retain a relatively complete structure and high ACE inhibitory activity. Compared with other delivery systems, liposomes have the advantages of easy encapsulation, large encapsulation capacity, and minimal residual organic solvents. Liposomes can encapsulate both hydrophobic and hydrophilic bioactive peptides. Hydrophobic peptides can be embedded within the phospholipid bilayer, while hydrophilic peptides can be encapsulated in the aqueous core (69).

However, liposomes also have some limitations. Firstly, the phospholipid membrane of liposomes is sensitive to adverse factors such as high temperature, enzymes, and ionic strength. These adverse factors may cause the liposomes to decompose during storage or before reaching the small intestine, causing the bioactive peptides wrapped inside to leak out in advance (70). To overcome this limitation, researchers have found that surface modification of liposomes with polymers such as chitosan, pectin, and polyethylene glycol can effectively improve the stability and sustained release ability of liposomes (71). Ramezanzade et al. (72) developed a novel composite nano-carrier of triphosphorus sodium cross-linked chitosan coated liposomes, and differential scanning calorimetry showed that this composite nano-carrier had better thermal stability than ordinary liposomes. Wu et al. (73) used sodium alginate (SA) to coated liposomes containing DPP-IV inhibitory collagen peptides and found that compared with uncoated liposomes, SA-coated collagen peptide liposomes exhibited higher storage stability, gastrointestinal stability and transcellular permeability. Secondly, due to the large size structure of liposomes, they may not be absorbed by intestinal epithelial cells, and the penetration mechanism of liposomes is not yet clear. Therefor the best approach is to choose vesicles as small as possible for the delivery of active substances, with particle diameters below 100–200 nm (Figure 4) (74). Additionally, cationic charged liposomes are often chosen to deliver bioactive substances because they are more easily attracted to the negatively charged mucus layer. Cuomo et al. (75) employed liposomes for the oral delivery of all-trans-retinoic acid and observed that cationic liposomes could interact with saliva in the oral cavity, which carries a net negative charge. Importantly, when cationic liposomes were coated with mucoproteins from oral saliva, the charge on the cationic surface interaction changed from positive to negative. This prevented the liposomes from being attracted to the negatively charged mucus layer during other stages of digestion, providing further protection for the loaded molecules.

An emulsion is a thermodynamically unstable colloidal dispersion formed by two immiscible liquids (usually oil and water), in which one liquid is dispersed as small droplets in the other liquid (76). According to their structural characteristics, emulsions can be divided into single-layer emulsions (water-in-oil, oil-in-water) (Figure 3b) and multi-layer emulsions (water-in-oil-in-water, oil-in-water-in-oil) (Figure 3c) (77). As a complex multi-phase system, multi-layer emulsion has various system types, among which W1/O/W2 is the most commonly used in food. The main structural state of W1/O/W2 type emulsions is that small water droplets (internal water phase, W1 phase) are trapped in larger oil droplets, and are subsequently dispersed in the external water phase (W2 phase). Multi-layer emulsions are complex multiphase systems. W1/O/W2 type is more common in food, where small water droplets (inner aqueous phase, W1 phase) are trapped in larger oil droplets, which are then dispersed in the outer aqueous phase (W2 phase) (78). Like singlelayer emulsions, the formation of multilayer emulsions also requires the addition of emulsifiers. Previous studies have found that the type of emulsifier can affect the stability of multilayer emulsions. Jo et al. (79) found that the hydrophilic and lipophilic balance value of the emulsifier can significantly affect the stability of W1/O/W2 emulsion loaded with collagen peptides, and emulsifiers with significant amphiphilicity can make W1/O/W2 emulsion more stable. Ying et al. (80) used polyglycerol ricinoleate and modified starch as emulsifiers to successfully prepare an emulsion system with a soybean peptide encapsulation rate of more than 80%. The results of in vitro simulated gastrointestinal digestion showed that the emulsion system showed strong resistance to the decomposition of pepsin, and the retention rate of soybean peptide was higher than 70% after simulated gastric digestion. In some cases, even with the addition of emulsifiers, the properties of multilayer emulsions are still not stable enough. This is because the system has two interfaces with a large interfacial area, making the multiphase structure prone to destruction during storage (81). Currently, there are various methods to stabilize the structure of multiple emulsions. One effective method to improve the stability of multiple emulsions is to add proteins or polysaccharides to limit the movement of components. For example, the addition of gelatin to multiple emulsions could significantly improves their stability (82). Furthermore, studies have shown that emulsion delivery systems not only improve the gastrointestinal stability of peptides, but also have the characteristics of masking the bitter taste of bioactive peptides (79). Gao et al. (83) used water-in-oil high internal phase emulsions (W/O HIPE) to encapsulate bitter peptides and found that W/O HIPE had a significant masking effect on the bitter taste of peptides.

Although both single-layer emulsions and multi-layer emulsions need to be stabilized by adding emulsifiers, some synthetic low molecular weight surfactants still need to be considered for their potential harm to the human body (84). Specifically, surfactants with a high HLB (Hydrophilic–Lipophilic Balance) value may disrupt the skin barrier due to their strong interfacial activity, which can increase the skin’s permeability to harmful substances, leading to skin irritation and even triggering allergic reactions and skin inflammation. Secondly, during the preparation of emulsions, although surfactants are renowned for their emulsifying properties, there is also a risk of causing emulsion instability, such as phase separation, coalescence, or creaming. These instability phenomena not only affect the appearance and texture of the product but may also compromise its actual efficacy. Moreover, the interactions between surfactants and bioactive ingredients may lead to structural changes in the bioactive components, resulting in the loss of their original functions, which is crucial for maintaining the integrity of bioactive ingredients. Surfactants may interfere with the permeability and retention time of bioactive components, thereby affecting their distribution and metabolism within the organism, ultimately reducing their bioavailability and therapeutic effects (84). Therefore, researchers have been on the way to seek other safer methods to stabilize the emulsion structure. At this time, a special emulsion, Pickering emulsion, came into the attention of researchers. Cai et al. (85) found that the natural Pickering emulsion system formed by composite nanoparticles that interacted/conjugated antimicrobial peptide Parasin I with chitosan significantly improved the stability and antibacterial activity of Parasin I. The solid particles in Picorling emulsions are irreversibly adsorbed on the surface of the emulsion droplets and play a role in stabilizing the emulsion system. This characteristic of Picorling emulsion avoids the use of surfactants, so its advantage is that there is no need to consider the safety of surfactants in food systems (86). In view of the characteristics and high safety of Pickering emulsions, it has a large application space in the field of bioactive substance delivery, but its specific mechanism of action and application characteristics still require further extensive research.

Polymer nanoparticles are solid colloidal particles with an average particle size ranging from 10 to 1,000 nm (Figure 3d). Polymer nanoparticle delivery system is a kind of system that uses natural, semi-synthetic or synthetic polymer nanoparticles as delivery carriers to load bioactive substances through non-covalent methods such as electrostatic adsorption, hydrophobic interaction, hydrogen bonding and so on (87). Compared to lipid-based carriers and emulsions, polymer nanoparticles have a simple preparation process, smaller system size, better stability which can protect bioactive peptides from being decomposed in harsh gastrointestinal environments (88), thereby improving the oral bioavailability of bioactive peptides. Additionally, high lipid intake may in due obesity and cardiovascular diseases (89), while the commonly used materials of polymer nanoparticles are proteins, polysaccharides and their composite derivatives, such as gelatin, sodium alginate, chitosan, and their derivatives, etc. Thus, polymer nanoparticles are more healthier and easilier to be accepted by consumers. Currently, various polymer nanoparticle delivery systems have been designed and applied to bioactive peptides delivery. Zhu et al. (90) used lysozyme-xanthan gum nanoparticles as carriers of selenium-containing peptides and prepared lysozyme-xanthan gum-selenopeptide composite nanoparticles. In vitro release test results showed that the composite nanoparticles successfully delayed the release of selenium-containing peptides and improved their in vitro antioxidant activity. Uhl et al. (91) developed a surface-modified PLA nanoparticles that can be loaded with liraglutide, which increased the oral bioavailability of liraglutide by 4.5-fold.

Some polymers can reversibly open TJs between intestinal epithelial cells, help bioactive peptides to be transported through the paracellular pathway, and promote the penetration and absorption of bioactive peptides, such as chitosan and its derivatives (92). In addition, chitosan also has good degradability and is one of the commonly used materials for constructing polymer nanoparticle delivery systems (93). Auwal et al. (94) used sodium tripolyphosphate cross-linked chitosan nanoparticles as the carrier to encapsulate ACE-inhibitory peptides, and found that not only the physical and chemical stability of the peptides was significantly improved in vitro, but also the ACE inhibitory effect of the peptides was significantly improved after simulated gastrointestinal digestion. Han et al. (95) prepared a pH-sensitive complex through the electrostatic self-assembly of chitosan derivative N-trimethyl chitosan, peanut peptide, and sodium alginate. This complex exhibited a regular spherical shape with good stability, and the highest entrapment efficiency for peanut peptide reached 91%.

Hydrogel is a highly cross-linked hydrophilic polymer with a three-dimensional network structure and abundant pores that can absorb and retain a large amount of water (96) (Figure 3e). A hydrogel system is a very effective delivery system for bioactive peptides, which can be prepared by mixing bioactive peptides with a solution containing biopolymer molecules before gel formation, or also by loading bioactive peptides into a microgel after microgel formation (97). Ma et al. (98) developed a novel type of fish skin gelatin-based hydrogel that successfully loaded codfish peptides after gel formation and exhibited good mechanical properties and biocompatibility. Because different types of materials have greatly different molecular and physicochemical properties, the physical and chemical differences of materials have a greater impact on the encapsulation effect of the system. Therefore, when preparing hydrogels, materials need to be selected according to specific purposes and applications. Protein and polysaccharide are commonly used materials for the preparation of ingestible food-grade microgels. Huang et al. (99) used the emulsion template method to successfully loaded ACE inhibitory peptides into biopolymer microgels composed of chitosan and alginate, which effectively reduced the in vitro release rate of ACE-inhibitory peptides. Ma et al. (100) used hydrogel made of alginate and chitosan to contain sericin with anti-inflammatory activity, and animal experiments showed that sericin loaded by hydrogel could more effectively alleviate ulcerative colitis in mice. These experimental results indicate that hydrogels have great potential in oral delivery systems.

In addition, pH, temperature and other stimuli will lead to the morphological changes of some polymer hydrogels, which will eventually lead to the phase transition of hydrogels (101). The hydrogels with this phenomenon are called smart hydrogels, which can respond to environmental stimuli, also known as environmentally responsive hydrogels. Environmentally responsive hydrogels can make corresponding shrinkage and swelling changes when single or multiple changes occur in external temperature, pH, light, electric field, salinity and other conditions, ultimately achieving targeted release of bioactive peptides (102). The environmental responsiveness of smart hydrogels shows important application potential and value in the field of substance delivery. Specifically, some temperature responsive smart hydrogels can exhibit different morphologies through corresponding phase transitions at elevated or low temperatures depending on the ambient temperature. This temperature responsiveness allows the hydrogel to adjust the position and rate of drug release in response to fluctuations in body temperature or environmental temperature, resulting in precise delivery of internal embedding. For example, Chuang et al. (103) cleverly designed a thermosensitive hydrogel based on the fact that tumor tissue is slightly hotter than normal tissue. This hydrogel will precisely undergo phase transition and release the embedded drug in the high temperature environment of the tumor site, allowing effective tumor treatment with minimal drug damage to normal tissues. In addition, there are some pH-responsive smart hydrogels that can adjust their morphology or properties according to changes in environmental pH, a property that enables the embedded material to respond to release in a specific pH environment, such as the slightly acidic environment of tumor tissue or the acidic environment of the stomach. Xie et al. (104) designed a pH-sensitive hydrogel that expands and releases drugs in the acidic environment of the stomach, which could facilitate precision treatment of gastric ulcer sites. In addition to the temperature and pH response, some smart hydrogels can undergo morphological changes upon the induction of light, which are called photoresponsive hydrogels. In the treatment of skin diseases, Huiwen et al. (105) use photosensitive hydrogels to deliver drugs precisely to lesions, which can significantly reduce the damage of drugs to surrounding normal tissues and improve the accuracy and safety of treatment.

Due to their unique environmental responsiveness, smart hydrogels have the ability to precisely regulate the drug release process, which makes them show broad application prospects in the field of drug delivery. Similarly, with appropriate design and preparation strategies, smart hydrogels are also suitable for quantitative, timed, and site-directed delivery of bioactive peptides. Ye et al. (106) found that the pH-responsive carboxymethyl cellulose/polyvinyl alcohol hydrogel effectively prevented the release of soy peptides in the stomach and could basically achieve the directional release of soy peptides in the intestine. This precise delivery strategy not only enhances the retention rate of bioactive peptides but also significantly improves their bioavailability, thereby optimizing therapeutic effects. In addition, it needs to be acknowledged that although smart hydrogels can effectively control the directional release of bioactive peptides, because the human body environment is complex and changeable, the changes and safety of smart hydrogels in the body need to be further studied.

Another, it needs to be acknowledged that hydrogels also have some disadvantages that are difficult to avoid. Typically, hydrogels are very porous and have weak structural strength, which allows bioactive peptides (especially small peptides) to easily diffuse out of them. At present, some studies have shown that improving the capture rate of bioactive peptides by hydrogels by ensuring that the pores are small enough or enhancing the interaction between bioactive peptides and the biopolymer network within the microgel (107). Two polymers with complementary properties can form a double crosslinked hydrogel to increase the stability of the hydrogel (108). Chen et al. (109) successfully prepared strong gelatin hydrogels by dual-crosslinking gelatin with transglutaminase and carrageenan, which improved the mechanical properties and thermal stability of gelatin hydrogel. In addition, since hydrogels are mostly hydrophilic substances, they have certain limitations when embedding hydrophobic substances. Studies have found that polymerizing hydrogels with nanoparticles, micelles and cyclodextrins can significantly improve the encapsulation rate of hydrophobic substances in hydrogels. Shabkhiz et al. (110) successfully encapsulated a β-cyclodextrin inclusion complex containing glycyrrhizic acid and thyme essential oil into alginate hydrogel beads, increasing the peptide encapsulation rate to 89%. However, there are few reports on the use of this technology in bioactive peptide entrapment, and further investigation is required. In summary, with the further development of smart hydrogel delivery systems, more innovative breakthroughs will be achieved in the application of smart hydrogels in the delivery of bioactive peptides.