Challenges and Opportunities in Delivering Oral Peptides and Proteins

Abstract

Introduction:

Rapid advances in bioengineering enable the use of complex proteins as therapeutic agents to treat diseases. Compared with conventional small molecule drugs, proteins have multiple advantages, including high bioactivity and specificity with low toxicity. Developing oral dosage forms with active proteins is a route to improve patient compliance and significantly reduce production costs. However, the gastrointestinal environment remains a challenge to this delivery path due to enzymatic degradation, low permeability, and weak absorption, leading to reduced delivery efficiency and poor clinical outcomes.

Areas covered:

This review describes the barriers to oral delivery of peptides and complex proteins, current oral delivery strategies utilized and the opportunities and challenges ahead to try and circumvent these barriers. Oral protein drugs on the market and clinical trials provide insights and approaches for advancing delivery strategies.

Expert opinion:

Although most current studies on oral protein delivery rely on in vitro and in vivo animal data, the safety and limitations of the approach in humans remain uncertain. The shortage of clinical data limits the development of new or alternative strategies. Therefore, designing appropriate oral delivery strategies remains a significant challenge and requires new ideas, innovative design strategies and novel model systems.

1. Introduction

The first protein drug, insulin, extracted from the bovine pancreas, was used for diabetes in 1921 [1]. Afterward, peptides and proteins were more routinely used as therapeutic agents. Thus far, the US Food and Drug Administration (FDA) has approved over 100 therapeutic proteins for treating diseases, including diabetes, cancer, and AIDS [2]. Among all routes of administration, oral drug administration is the most popular delivery approach because of its convenience and safety, especially in patients who require long-term medication to treat chronic diseases.

The efficacy of oral peptides or protein delivery depends on various factors, such as the physicochemical properties of the peptides or proteins and the physiological environment of the gastrointestinal tract (GIT) [3]. The exquisite spatial structure of proteins is sensitive to pH and enzymes in GIT, often resulting in hydrolysis and potential activity loss. In addition, the stomach’s acid environment, mucus layers, and intestinal epithelial cells create hurdles to the absorption of peptides or proteins into the bloodstream [4]. Therefore, improving the bioavailability of oral peptide or protein drugs requires the system design to overcome several key factors, such as safeguarding the exquisite spatial structure of peptides and proteins from corruption by GIT enzymes, navigating the physical and biological barriers of the GIT, and promoting the absorption of macromolecules into the systemic circulation. From the pharmacological perspective, the development of peptide and protein drugs has primarily focused on molecular modifications and new drug delivery systems [5].

This review summarizes the physicochemical properties of proteins and the challenges to their application in the complex GIT. Similarly, we discuss the construction of oral peptide or protein delivery systems and the application of these systems in animal models and combine this with new technologies in the medical field to provide strategies for the pursuit of improvements in oral peptide and protein delivery.

2. Structure and Properties of the GIT

2.1. Epithelial Structure of GIT (including transporters)

The degree of drug absorption varies across regions of the gastrointestinal track because of each region’s different anatomical characteristics and physiological environments. The stomach is not the primary absorption site due to its limited surface area and lower blood flow. Most absorption occurs in the intestine with the unique structure of the intestinal surface and folds in villi and microvilli, increasing the absorption area of the intestine by approximately 600 times [6]. The high absorption capacity of the intestine is attributed to the abundant blood and lymphatic capillaries in the villi. The intestine fluid contains various components, including digests, bile salts, proteases, and mineral ions. The bile salts, phospholipids, and sterols contribute to the wettability of peptide and protein drugs and facilitate the formation of micelles [6]. The premise of absorption into the systemic circulation is that the drug must cross the mucus layer and intestinal epithelial cells using mechanisms such as paracellular, transcellular, and carrier-mediated transport [7,8]. The microfold cells (M cells) in the Peyer’s patch can directly transmit nanocarriers to the lymphatic system and finally into the systemic circulation [9–11]. The paracellular pathway allows for the diffusion of small molecules (<200Da) while regulated by tight intercellular junctions [12]. The transcellular pathway refers to drug molecules running from the tip of enterocytes to the outer basement membrane, suitable for lipophilic drugs [13]. Carrier-mediated transport is the transmembrane movement of macromolecules with the help of transporters.

The proper regulation of transporters can increase protein uptake [14,15]. Transporters are membrane proteins classified as either uptake or efflux based on their function. Uptake transporters such as Peptide-transporters 1 (PEPT1) and solute carrier family 15 member 1 (SLC15A1) have transport capacity for dipeptides and tripeptides [16], and efflux transporters limit protein uptake. Both subtypes of multidrug resistance protein P glycoproteins (P-gp), adenosine triphosphate (ATP)-binding cassette subfamily B member 1 (ABCB 1) and ATP binding cassette, subfamily G member 2 (ABCG 2), exhibit efflux effects. For example, they can return linear peptides and cyclic peptides (e.g., cyclosporin) from the sites where P-gp is expressed to the lumen of the intestine, leading to multidrug resistance [17,18]. Most studies of efflux transporters have focused on the P-gp in the intestine, where efflux can be inhibited by altering the protein’s binding site and disrupting the P-gp activity [19,20]. For example, Methanone, [2-(1H-indol-3-yl)-1H-imidazol-5-yl] (3,4,5-trimethoxyphenyl) (VERU-111), a tubulin inhibitor, can enhance drug uptake in cells over-expressing P-gp [21,22].

2.2. Properties of GI Tract that Pose Challenges for Drug Delivery

2.2.1. Acid environment in GIT

The stomach is the widest part of the human digestive tract [23]. The gastric juice contains hydrochloric acid, salts, enzymes, and other substances and is acidic during fasting (pH, 1–3) [24]. The pH after feeding ranges from 3 to 7 and returns to solid acidity again after 2–3 hours [25]. As food moves through the small intestine, the pH gradually increases due to bicarbonate secretion from the pancreas and bile from the liver, which neutralizes the acidic contents transported from the stomach. The pH value of the intestines can range from 5–7.5 [26]. The spatial conformation of proteins in the oral tract can be easily disrupted due to hydrolysis, oxidation, or deamidation [27]. Proteins can aggregate and precipitate throughout different pHs in the digestive tract due to various proteins’ isoelectric points (PI) [28].

2.2.2. Enzyme

Enzymes are biochemical barriers that can hinder the uptake of peptides or proteins, leading to decreased bioavailability. This occurs due to the enzymatic degradation of the peptides and proteins into smaller peptides or amino acids [29]. The activities of proteolytic enzymes in the GIT, including pepsin, trypsin, elastase, chymotrypsin, and carboxypeptidase, require a suitable acidic environment [30]. While the trypsin, chymotrypsin, elastase, and carboxypeptidase secreted by the pancreas can lead to the rapid degradation of most peptide and protein drugs [31]. Pepsin peaks at pH 2.5 and gradually loses activity above 5 [32]. The intestine is the leading site of absorption [33], and enzymatic degradation can occur in the intestinal lumen, brush membrane border, cytoplasm, lysosomes, or other organelles of enterocytes [34].

2.2.3. Bacteria

The gut is inhabited by a diverse population of microorganisms known as the gut microbiota [35]. Commensal microbes help build a healthy immune system to resist pathogen invasion and maintain intestinal mucosa integrity [36]. Host genetics, dietary habits and drug factors all affect the homeostasis of commensal microbes [37]. The immune response and enhancing mucosal permeability are essential to secondary intestinal infections [38]. Commensal microbes inhibit the growth and reproduction of pathogens by competing for host nutrients and adhesion sites and regulating the immune system [39].

2.2.4. Mucus layer

Oral drugs interact with the mucosa in the lumen. The mucosa acts as a barrier, limiting the invasion of pathogens and toxins into the bloodstream and impeding peptide or protein absorption [40]. Dynamic hydrogels with a sieve structure in the intestinal mucosa utilize scavenging to remove proteins or hydrolysates [41,42]. In some cases, nanoparticles (NPs) can be used as peptide or protein carriers to avoid clearance by the mucus layer [43,44]. Due to mesh size, NPs below 200 nm can pass through the mucus layer [45]. The NPs with negative or neutral surface charges can minimize the binding with negatively charged mucin [46,47]. An overall discussion has been listed in Table 1.

Table 1.

Biochemical and physiological barriers to oral protein drugs in GIT.

Class	Properties	Mechanisms of hindering the absorption of oral protein drugs	Reference
pH	Fasting (pH, 1–3) feeding (pH, 3–7)	Gastric acid destabilizes protein and peptide structures and exposes peptide bonds to degradation by pepsin enzymes	[24,25,48,49]
Enzyme	Survive in suitable pH	Degrade proteins into smaller peptides or amino acids	[29,30]
Mucus layer	Comprised of water (95%) and mucin, a net negative charge	Sieve structure limits absorption; scavenging removes proteins or hydrolysates	[41,42,50]
Epithelial	Rich in villi and microvilli	Brush borders of the microvilli are filled with digestive enzymes	[6,51]

2.2.5. Surfactants

Surfactants are the primary enhancer of absorption in clinical practice by increasing solubility and intestinal permeability [52]. There are a lot of surface-active molecules, such as bile salts, digestive enzymes, phospholipids, and medium-chain fatty acids (FA) in the GIT. Bile salts are endogenous surfactants that can protect proteins by forming micelles, interacting with biological membranes and inhibiting the activity of P-gp substrates [53]. Novel glycolic acid (GCA) functionalized bilayer NPs (GCA-NPs) enhanced active transport through the apical sodium-dependent bile acid transport (ASBT) protein in the intestine [54]. In addition, sodium decanoate with medium-chain FA enhanced the penetration of oral insulin and glucagon-like peptide 1 (GLP-1) [55,56]. Endogenous surface-active molecules can also coat ingested nanocarriers, forming a protein corona [57] that enhances delivery [58]. For instance, incorporating insulin with a protein corona using cationic liposomes (PcCLs) increased oral bioavailability by up to 11.9% in type I diabetic rats, resulting in a significant hypoglycemic effect [59]. This phenomenon may be related to the coverage of protein corona on the cationic liposome surface, altering its cationic liposome surface charge (CLs). PcCLs containing bovine serum albumin (BSA) and a hexamer synthetic peptide (AT1002 with an amino acid sequence of L-Phenylalanyl-L-cysteinyl-L-isoleucylglycyl-L-arginyl-L-leucine) (Pc-AT-CLs) were used to load liraglutide (Fig 1a). The AT-1002 facilitated the opening of the tight junctions of Caco2 cells, thereby promoting the entry of liraglutide into the systemic circulation. The mucosal accumulation of Pc-AT-CLs was nearly one-fold lower than that of AT-CLs, and Pc-AT-CLs exhibited lower accumulation in the mucus layer [60] (Fig 1c–d).

Figure 1.

(a). Schematic of protein corona cationic liposomes containing BSA and AT1002 (Pc-AT-CLs) preparation. (b). TEM image of the Pc-AT-CLs. (c). Binding levels of cationic liposomes (CLs), protein corona cationic liposomes (PcCLs), cationic liposomes containing BSA and AT1002 (AT-CLs), and Pc-AT-CLs to different concentrations of mucus. (Data are presented as mean ± SD, n = 3). (d). Transmucosal transport levels of CLs, PcCLs, AT-CLs, and Pc-AT-CLs with or without pretreatment or post-treatment process to remove mucus. (Data are presented as mean ± SD, n = 3, ** p < 0.05). Reprinted (adapted) with permission from [60]. Copyright © 2023

2.2.6. Immune stimuli

Cell types in the intestine that play an essential role in the immune system include M cells, goblet cells, Paneth cells, intestinal epithelial stem cells, and other secretory cells [61]. These cells are regulated by the gut-associated lymphoid tissue (GALT) in effector and inductive sites [62], protecting the intestine and facilitating transport, and supporting immunity [63]. In particular, M cells, as the smallest mucus-protected cells, were targets of GIT vaccine preparations [64], which present antigens to potential immune cells and acquire immune responses [65,66]. Cubic and polymeric microcontainers encapsulating the model antigen ovalbumin (OVA) has demonstrated to achieve targeting or continuous delivery in the GIT. This shows promising potential directions for exploration as a viable approach for delivering oral vaccines[67]. NPs engulfed by M cells can directly pass through the lymphatic system into the blood, avoiding enzyme degradation in intestinal epithelial cells and the liver [68]. Strategies to target receptors on M cells have attracted numerous investigations [69]. Lectins are commonly used ligands capable of reversible binding to M-cell receptors. The coupling of lipid NPs loaded with insulin and lectins prolonged enterocyte retention and reduced blood glucose levels [70].

2.2.7. Disease factors

A crucial pathogenic factor for the third most lethal cancer worldwide is infection with Helicobacter pylori. (H. pylori) can reduce gastric acid secretion and allow more bacteria to survive by changing the acidic environment of the stomach [71]. Furthermore, H. pylori produce high levels of reactive oxygen species to cause cellular deoxyribonucleic acid (DNA) damage [72]. The leading cause of inflammatory bowel disease (IBD) is microbiota dysregulation, accompanied by the decreased diversity of microbiota and increased pathogens such as Escherichia coli [73]. Biopsies of patients with IBD showed significant differences in bacterial abundance in inflamed and non-inflamed regions. The two main subtypes of IBD are Crohn’s disease with transmural lesions and ulcerative colitis with continuous lesions [74], including fever, wasting diarrhea, and abdominal pain. Disorders of gut bacteria can also cause other diseases, such as lung cancer and breast cancer [75]. Disease factors including IBD, bacterial infections, or tumor may change environmental pH, microbiota metabolism and the permeability of GIT, influencing the formulation disintegration, local efficacy, absorption of proteins, and final outcome of treatment.

3. Complex Proteins

3.1. Structure

Although proteins have expanded the field of molecular pharmacology as therapeutic agents, their therapeutic effects are hampered by structural instability. Compared with peptides with a linear structure, proteins with amino acid sequences often possess more complex structures, such as the necessary secondary structures, folding, and quaternary structures to maintain their potents[76]. Structural damage at any level will result in reduced bioactivity, including subtle changes caused by exposure to conditions in the gastrointestinal microenvironment [77].

3.2. Sensitive Bonds

Peptide bonds and phosphodiester bonds in the primary structure, hydrogen bonds of the secondary structure, the disulfide bonds of tertiary structures and ionic bonds of quaternary structure are all susceptible to changes in the GIT. Disulfide bonds can be used for the cyclization of peptides or to decrease flexibility to resist protease hydrolysis by reducing intermolecular hydrogen bonds [78]. Some proteins act as drug delivery carriers by complexing with metal particles or forming complexes with other insoluble substances [79,80]. By encapsulating albendazole in a conjugate with polycaprolactone and bovine serum albumin (BSA), the drug was efficiently delivered to pancreatic tumors [81]. Albumin NPs are also used for drug delivery, and the presence of disulfide bonds may be a crucial factor for effective drug loading [82]. However, the thiol/disulfide substructures in therapeutic peptides and proteins can also serve as absorption barriers due to the form of inactive conjugates through the thiol/disulfide exchange reaction in GIT [31]. Ijaz et al. found that lanreotide was entirely degraded by thiol/disulfide exchange reaction with glutathione (GSH) within 2 hours of incubation [83].

3.3. Size

The permeability of protein macromolecules across cell membranes is characterized by the apparent permeation coefficient [84]. Absorption in intestinal epithelial cells is minimal, with a molecular weight greater than 700 Da [85]. The size of the peptides or proteins is the primary factor limiting their passive diffusion absorption. Although endocytosis allows protein macromolecules to enter cells, their therapeutic efficiency can still be impacted. The transporter-mediated efflux increases with drug molecule size [86]. Therefore, controlling the size of peptides or proteins, reducing the specific surface area by appropriate delivery methods, and decreasing the surface tension of the medium with a surfactant are valuable ways to achieve more effective absorption.

3.4. Lipophilicity

Lipophilicity is the property of molecules that allows them to dissolve in lipids, oils, or nonpolar solvents [87]. Proteins act as hydrophilic macromolecules with a log P value of less than zero, making it difficult to diffuse through the intercellular space or fuse with the cell membrane to enter systemic circulation unless carried-mediated or chemically modified [88]. A typical approach is forming hydrophobic ion pairs by competing with hydrophobic ions and mixing the protein drug into lipid-based nanocarriers. This approach prevents hydrophilic proteases from entering lipid-based carriers and protects oral protein administration [89].

3.5. Charge

The ionization of amino and carboxyl groups in proteins results in a charged state in aqueous solutions, and the resulting charge is a crucial factor in designing protein delivery systems [90]. To achieve rapid penetration of mucus, viral biomimetic NPs (P-R8-Pho NPs) composed of R8 peptide and specific anionic phosphate (Pho) were developed (Fig 2a). The morphology of P-R8-Pho NPs (Fig 2b) was imaged by TEM. It showed uniform spheres with a visible poly (lactic-co-glycolic acid) core (PLGA core) and polyethylene glycol shell structure (PEG chain). These neutral viral biomimetic NPs penetrated the mucosa almost as fast as PEGylated NPs (Fig 2c). They resulted in a significant hypoglycemic effect in diabetic rats after administration with biomimetic particles loading insulin [91] (Fig 2d–e).

Figure 2.

(a). Schematic illustration of the viral biomimetic NPs (P-R8-Pho NPs) composed of R8 peptide and specific anionic phosphate (Pho) overcoming the barriers of the mucus layer and epithelial cells. (b). TEM images of P-R8-Pho NPs. Scale bars, 50 nm. (c). Particle size and zeta potential of different NPs. (d). Fluorescent images of villi in rats after oral administration of Dio-NPs. The white symbols marked the absorption of NPs in the villi. (e). Blood glucose levels in normal rats, following subcutaneous injection of an equivalent volume (2 IU/kg) of primary insulin or insulin released from NPs. *p < 0.05 versus oral free insulin group, &p < 0.05 versus P NPs, #p < 0.05 versus P-R8 NPs. Reprinted (adapted) with permission from [91]. Copyright © 2018, American Chemical Society.

The isoelectric point of proteins is also critical, as proteins can interact with polymers through electrostatic interactions when the environmental pH is lower than their isoelectric point [92].

3.6. Polarity

The polarity of protein affects the structure and properties of protein molecules, including the solubility, activity, and encapsulation in delivery systems. The number and distribution of polar and non-polar groups on the protein surface determine its surface activity, structural arrangement in aqueous solution and intermolecular interactions [93].

4. Strategies for Oral Delivery of Peptides and Complex Proteins

4.1. Release of Peptides / Proteins Before Absorption

4.1.1. Protection of Structure and Activities

Hydrolytic enzymes in each region of the GIT degrade peptides and proteins. Therefore, regulating the pH of the microenvironment can be utilized to protect the oral proteins. The combination of citrate and salmon-derived calcitonin can inhibit the activity of enzymes by changing the local pH [94]. Circular peptides have been found to exhibit more excellent resistance to digestion because intramolecular hydrophobic bonds reduced flexibility when compared with linear peptides. This increased stability in the GIT improves the therapeutic effect of orally administered peptides or proteins [95]. The natural small-ring peptide cyclosporin also reflects this reduced enzymatic digestion [96]. More than 40 cyclic peptide drugs are on the market and used for treating cancer [97,98], diabetes, gastrointestinal dysfunction, obesity and other diseases [99]. Eudragit® is an enteric-soluble polymer sensitive to pH to target the intestine, which can resist acid degradation in the stomach and dissolve in the intestine [26]. Another way to protect peptides or proteins from the acid environment or enzymatic degradation is to encapsulate them into nanocarriers with advanced delivery strategies [100].

4.1.2. Treatment for local lumen diseases

The hyperactivation of macrophages can convert physiological inflammation into pathological intestinal damage in IBD [101]. To treat IBD, it is essential to regulate intestinal macrophages. One potential approach involves using polymer shells to enclose superoxide dismutase (SOD) and catalase (CAT) via situ polymerization on the surface of enzymes. This approach has been tested in a mouse model of dextran sodium sulfate-induced colitis. The polymer shells effectively removed intracellular reactive oxygen species and inhibited the expression of inflammatory factors [102].

4.2. Strategies for Lumen-released Preparations (gastro and intestine)

4.2.1. Physical coating

The prerequisite for protecting peptides or proteins from degradation in the GIT is whether the carrier systems resist the harsh acidic environment changes as they move from the stomach to the small intestine and withstand enzymatic degradation. The stability of the carrier systems is essential for peptides or proteins to reach the GIT. Materials are used to coat the surface of peptides or proteins to ensure integrity in the stomach, including carbohydrates to fight low pH in the gastric lumen and to promote adhesion to the GIT wall. Insulin was loaded into liposomes enclosed with chitosan, which protected the structural integrity of the insulin in the stomach [103]. Due to the adhesive properties of chitosan, there was increased uptake and permeability of insulin in intestinal epithelial cells [104]. Pepsin and trypsin in the anterior segment of the GIT where Eudragit L100 coatings protected β-casein micelles loaded with an antiretroviral to minimize release in the stomach [105,106]. Porous silicon was used to load Immunoglobulin A-2 (IgA 2), and two methods were employed on coat surfaces. The first approach involved directly covering the surface of IgA 2 NPs with Eudragit polymer, while the second method involved coating the NPs with Eudragit polymer first and then wrapping them with biodegradable capsules (Fig 3A). The sensitivity of nanocomposite formulations to pH in vitro was evaluated and the silicon NPs provided payloads and site-specific delivery for IgA 2 antibody, whereas the capsule formulation covering Eudragit delivered more significant amounts of IgA 2 [107] (Fig 3B–E).

Figure 3.

A. Schematic representation of (a). the preparation of porous silicon-loaded IgA 2 coated with Eudragit polymer and the fate of NPs in mice after oral administration. (b). the preparation of porous silicon-loaded IgA 2 NPs for capsule-based delivery. B. Total and active IgA 2 released from Eudragit L100 coated capsule packed with porous silicon loaded IgA 2 particles. C. Total IgA 2 released from Eudragit L100 coated capsule packed with IgA 2. D. Total and active IgA 2 released from Eudragit S100 coated capsule packed with porous silicon-loaded IgA 2 particles. E. Total IgA 2 released from Eudragit S100 coated capsule packed with IgA 2 particles. Reprinted (adapted) with permission from [107]. Copyright © 2022, American Chemical Society.

Although the physical coating protected the activity of therapeutic peptides or proteins through the harsh gastric environment, the absorption of peptides or proteins in the small intestine was not affected [108]. Thus, combining physical coating with permeability enhancers may facilitate peptide or protein penetration into the gut.

4.2.2. Adding anti-acid agents

A pH buffer is commonly used in clinical practice to improve the stomach’s acid environment but does not inhibit gastric acid secretion. Potassium-competitive acid blockers inhibit the H⁺/K⁺-ATP enzyme activity in the stomach by reversibly binding to K⁺ for proton pump inhibitors [109]. Unfortunately, the prolonged use of acid inhibitors predisposes the gastric mucosa to damage and can increase the risk of developing gastric cancer symptoms [110].

4.2.3. Adding Enzyme Inhibitors

The selection of enzyme inhibitors depends on the therapeutic protein structure, the target location for the enzyme, and the cellular distribution [111]. Specific information about proteases is vital to ensure the stability of proteins in the GIT when proteins and enzyme inhibitors simultaneously exist [112]. Therefore, liposomes, microcapsules, microspheres and other drug delivery carriers facilitate the encapsulation of protein and enzyme inhibitors. The presence of enzyme inhibitors can also affect the absorption of other proteins in the GIT, and improper dosage can result in negative feedback regulation, such as indigestion or hyperplasia of the pancreas. Therefore, the advantages or disadvantages should be weighed in practical applications [113]. Applying pH buffers to inactivate local digestive enzymes is another approach to inhibit enzyme activity [25].

4.2.4. 3D printing

3D printing technology, also known as additive manufacturing, has been used in the industrial, medical, dental, and pharmaceutical fields [114]. As the first batch of 3D printed drugs, the FDA approved Spritam immediate-release tablets in 2015 [115]. The advantages of 3D printing technology are generating complex structures with high accuracy and low cost, meeting personalized patient needs, and reducing side effects [116]. A 3D printing process was developed to load BSA and alginate with multi-layered particles to release BSA in the small intestine [117].

4.2.5. Microneedles

Microneedles can produce micron-sized pores in the skin, help drug penetration through the corneum barrier, provide effective percutaneous drug delivery and overcome gastrointestinal metabolism and first-pass effects of the liver in oral drug administration [118]. The length of the microneedles is between 150–1,500 nm, less than the epidermal thickness [119]. Unlike regular subcutaneous injections, microneedles provide more convenience for patients, are non-contacting with essential nerves and blood vessels, prevent the need for professional operation, are painless and are minimally invasive. [120]. Developing microneedles containing proteins for delivery is a current focus, including electrochemical microneedle technology. This approach enables the real-time transmission of human physiological status to intelligent devices, opening up new research directions for drug delivery, real-time monitoring, and related fields [121]. A double-electrode microneedle patch was developed to release glucocorticoids by electrical stimulation [122]. A closed-loop system based on microneedle platforms with wearable electronic devices was developed to regulate insulin release [123], and insulin can be added to the equipment anytime for convenience. An oral robotic pill for drug delivery was developed where the outside of the capsule was coated with an acidic enteric coating, and the inside was equipped with a microsyringe connected to a self-filled balloon (Fig 4a–b). The dissolution of the enteric coating and capsule shell in the intestine inflated the balloon, which caused the syringe to inject the drug into the intestinal wall through the soluble microneedle. The safety and feasibility of robotic pills were assessed by delivering octreotide, and the results showed that subjects tolerated robotic pills well, with bioavailability of octreotide reaching 65 ± 9% by oral robotic pill delivery [124]** (Fig 4c–d).

Figure 4.

(a). A fully assembled RP with enteric coating. (b). Schematic diagram of each component of the RP. (c). Time-course of concentrations changes of octreotide in plasma delivered by RP A and B (d). Time-course of levels changes of octreotide in plasma following drug administration, either IV (N = 6) or RP (N = 13, groups A and B combined) in subjects. Data are presented as means ± SE. Reprinted (adapted) with permission from [124]. Copyright © 2021

A luminal unfolded microneedle containing insulin was prepared for luminal delivery, where an expanded arm was enclosed in a capsule that resists degradation in the GIT. The pharmacokinetics demonstrated faster absorption than subcutaneous injection [125]*. The combined delivery of drugs and devices provides a promising approach to overcoming the challenges associated with the oral administration of macromolecules. In addition, the amount of protein delivered to GIT, either by the robotic pill or the luminal unfolded microneedle, is limited by the device size [126]. In the case of low payload, whether multiple and long-term uses of such non-sterilized surface cavity injection dosage forms will have adverse effects on patients should also need more clinical trials to confirm. Finally, this kind of precise design is expensive due to its complexity, and it should consider whether the patient can afford it.

4.2.6. Utilization of bacteria

Biological components commonly used as carriers are popular because of their biocompatibility [127]. There are 10¹⁴ colony-forming unit (CFU) bacteria per milliliter in large intestinal juice in the GIT [128]. The unique capabilities of bacteria to thrive in the harsh, hostile gastrointestinal environment make them an attractive option for oral protein delivery carriers [129]. A bacterial outer membrane vesicle (OMV) is a natural target with specific internalization by cells, which can deliver drugs without further modification [130]. Furthermore, bacteria can be chemically coupled with NPs through chemical groups on their surface [131]. The convenience of bacterial culture, surface modifications, and capability to adhere to epithelial cells provide a broad scope for developing bacteria into versatile carriers in drug delivery.

(1). Expression of proteins

Bacteria can express therapeutic proteins needed for specific diseases via recombinant DNA technology [132]. Common genetically engineered bacteria in clinical use include Lactobacillus, E. coli, Bifidobacterium, Enterococcus and others [133]. Lactobacillus lactis is considered a safe drug delivery system [134] with the effects of lowering cholesterol and regulating immunity [135]. Oral administration of Lactobacillus carrying tumor necrosis factor (TNF) single-chain fragment variable (scFv) expression vector reduced mouse colonic inflammation [136]. Bacterial-like particles (BLP) prepared from food-grade lactic acid bacteria were used to deliver insulinoma-associated protein 2 (IA-2ic) to develop an oral vaccine against autoimmune diabetes. This was achieved by combining IA-2ic with the surface of BLP, resulting in a BLP-IA-2ic oral vaccine that effectively suppresses autoimmune diabetes in animal models [137]*. A drug system for live Lactobacillus carrying the therapeutic protein P8 was developed to treat colon cancer [138].

(2). Initiate release

Building genetic circuits within bacteria to facilitate repeated and synchronous delivery of drugs to target cells while reducing damage to healthy tissue is an emerging approach [139]. Modified Salmonella as a delivery system was used as gene circuits to control protein synthesis and intercellular and intracellular release. The system resulted in 70% of the bacteria invading cancer cells and releasing protein, reducing tumor growth and metastasis [140]*.

4.2.7. Adherence

Delivery systems based on mucosal adhesive polymers can enhance the solubility of proteins and protect proteins from degradation [141]. Bioadhesive polymers are synthetic or natural polymers that adhere to mucus, including polyacrylate, polyacrylic acid, hydroxypropyl cellulose, chitosan[142,143], and various gums [23]. Unique structures with the shell of pectin and the core of folic acid-Zein (FA-Zein) were designed to target the colon to ameliorate inflammation through loading glycyrrhizic acid (GA) (Fig 5a). The NPs were spheres, and the inside core and outside shell were distinguishable by TEM (Fig 5b). The core-shell structure was adhesive and penetrated the colon of mice with ulcerative colitis [144] (Fig 5c–d).

Figure 5.

(a). Scheme of the preparation of GA@Pec-FA-Zein NPs. (b). TEM image of GA@Pec-FA-Zein NPs. (c). Fluorescence images of UC mice took free Dir, Dir@Zein NPs, Dir@FA-Zein NPs, and Dir@Pec-FA-Zein NPs at 3, 6, 12, and 24 h after oral administration. (d). Permeation of free C6 fluorescence, C6@Zein NPs, C6@FA-Zein NPs, and C6@Pec-FA-Zein NPs into the colon tissue (scale bar: 25 μm). Reprinted (adapted) with permission from [144]. Copyright © 2022, American Chemical Society.

Generally, the presence of thiol groups on polymers results in higher mucosal adhesion properties [145] due to disulfide bonds between the cysteine groups of mucus glycoproteins and thiol groups on the polymers [146]. For example, the penetration rate of thiolated chitosan was three times higher than the unmodified chitosan, which can be observed by rhodamine 123 labeling [49]. Adhesive polymers can prolong the residence time of delivery carriers in the mucus layer, facilitating the release of higher concentrations of drugs at the resident site [147]. A hydrogel polymerization grid, formed by cross-linking (polymethacrylic acid) (PMA) and PEG, protected insulin from the gastric acid environment and was released only in the neutral pH environment [148]. In contrast, the delivery system used functionalized porous silicon and mesoporous silica with mucosal adhesion and gastric retention [149,150]. Mesoporous silica NPs (MSN) with succinylated β-lactoglobulin (BL) synergistically reduced the release of gastric-sensitive drugs under acidic pH and enhanced their solubility under intestinal pH conditions [151]. However, the conjugation delivery strategy for the polymer and proteins is also affected by the turnover frequency of the mucus layer every 4–6 h [152].

4.3. Release of Peptides / Proteins after Absorption

4.3.1. Strategies for opening the paracellular barrier

Tight junctions present a series of aquaporin complexes between intestinal epithelial cells [153]. The transfer of peptides or proteins across the intestinal epithelial cell membrane is restricted when their molecular weights exceed 700 Da. Designing peptide or protein delivery systems or co-delivering adjuvant drugs to stimulate the transient opening of tight junctions between cells can promote uptake. Two mechanisms to achieve this are disrupting the tight junction protein structure or reducing the concentration of extracellular free calcium ions. Commonly used enhancers are surfactants, bile salts, and fatty acids, and nanocarriers can open the paracellular pathway. CS and zonula occlusion toxin (ZOT) derived peptides coupled with a polynucleic acid-based nanocarrier delivered insulin by regulating tight junction integrity [154]. Baicalin-AlCl₃ NPs (BA-Al NPs) were prepared using coordination bonds and hydrogen-bond induction (Fig 6a) and were sensitive to pH, and allowed for the sustained release of the loaded protein drugs. The release of BA during the disassembly of BA-Al NPs resulted in the down-regulation of the expression of tight junction proteins, improving oral insulin absorption [155] (Fig 6b–d). However, toxins and other pathogens in the gut may exploit this process to enter systemic circulation after opening tight junctions.

Figure 6.

(a). Schematic representation of the formation process of the BA-Al NPs via self-assembly. (b). Gene expression levels of Claudin-1, ZO-1 and ZO-2 mRNAs in BA- and BA-Al NPs-treated Caco2 cells. Blank control groups were Caco2 cells treated with DMEM medium. * p < 0.05, ** p < 0.01, *** p < 0.001 (c). Expression of Claudin-1, ZO-1 and ZO-2 proteins in BA- and BA-Al NPs- treated Caco2 cells was assessed by western blot. (d). Schematic illustration of mechanisms and consequences of BA-Al NPs-mediated regulation of tight junctions between intestinal epithelial cells. Reprinted (adapted) with permission from [155]. Copyright © 2022

4.3.2. Micro-and Nanocarriers

(1). Micro-carriers

Micro-carrier systems are often referred to as microcapsules, microspheres, and microemulsions. As an essential strategy for peptide or protein drug delivery, microspheres are accompanied by the efficient control of peptide or protein release. The entry of microspheres is closely related to size, with microspheres <10 μm capable of passing through the intestinal wall via Peyer’s pathway. Absorption can be achieved after the particles overcome the barrier and permeate the intestinal epithelial cell membrane, passing through the lymphatic system [156]. The self-micro emulsifying drug delivery system (SEDDS) is formed by spontaneous emulsification upon exposure to GIT, with a protective effect on drugs, mucosal penetration and simple preparation methods [157].

A successful example is cyclosporin A soft capsules, which have been marketed since 1995. Recently, a snail mucin-based oral insulin microemulsion system was developed with an encapsulation rate above 70% and blood glucose levels remained stable 8h after oral administration in diabetic rats [158]. However, excessive surfactant application can result in cytotoxicity, which limits the development of SEDDS[159]. Finding efficient and low-toxicity surfactants or optimizing the preparation process is a future direction for emphasis. To avoid emulsions precipitating at a low temperature or seeping from soft capsules, preparing solid component SEDDS (S-SEDDE) by hot-melt extrusion is a practical solution [160–162]. Other microcarriers have been developed, including biopolymer microbeads (Alg/AmCS) based on alginate and amino chitosan, which may be suitable carriers for site-specific protein delivery in the gut and colon [163].

(2). Nano-carriers

Nanocarriers of polymers, lipids and other materials can be divided into NPs [164], nano gels [165], and nano complexes. NPs offer a gradual and sustained drug release profile that promotes high permeability and long-term retention while minimizing the risks of enzymatic and immune clearance. Due to the biocompatible excipient of the matrix, solid lipid NPs are considered safe and effective drug carriers. Incorporating hydrophilic proteins into solid lipid NPs requires consideration of their affinity for lipids, and there are successful examples, such as insulin [166]. Liposomes, also act as lipid-based carriers and favor fusion with the cell membrane due to their structural properties [167]. The delivery of liposomes for peptides or proteins is usually performed after surface modification. The oral absorption of salmon calcitonin in rats was enhanced by two components (N-trimethyl CS chloride (TMC) and oligoarginine (Arg8)) [168]. Natural anionic polymers and cationic materials form nano gels through electrostatic interactions, which can be used as protein delivery carriers. As nanocarriers, pH-sensitive materials such as alginate and CS can protect proteins from degradation before absorption [169]. Hydroxyethyl methacrylate (HEMA) nano gels were prepared by emulsion polymerization and used as an alternative carrier for oral insulin delivery, as they promote intestinal absorption [170].

Nanocomplexes of sodium casein acid (NaCas) and pectin successfully avoided the hydrolysis of the encapsulated drug by pepsin, achieving controlled drug release in the intestine [171]. In situ, polymerization of zwitterions was pursued to encapsulate proteins with further encapsulation of the nano complexes in enteric capsules (Fig 7a). The polyzwitterions likely assisted nanocomplexes in permeating mucus and cellular barriers through the proton-assisted amino acid transporter 1 (PAT 1) pathway. Nanocomplexes were detected in Caco2 cells with high expression of PAT1 in vitro (Fig 7b). Insulin labeled with Cy 5.5 showed muscular retention and absorption in the small intestine of mice in vivo [172] (Fig 7c–d). Exosome-based delivery strategies are also gaining focus due to exosomes’ unique cellular messenger functions. Exosomes are dish-shaped vesicles with cellular messenger functions, which can be explored as nanocarriers to deliver macromolecules across cellular barriers [173].

Figure 7.

(a). Schematic representation of loading proteins through polymerization of zwitterion in situ. (b). Cellular uptake of M4/BSA and M8/BSA by Caco2 cells at 3 h after incubation by flow cytometry. (c). Cellular uptake images of M4/BSA and M8/BSA by Caco2 cells at 3 h after incubation, scale bar: 50 μm. (d). Absorption sites and kinetics of delivering M4/Cy5.5-insulin and M8/Cy5.5-insulin capsules in oral at different time points post-administration. Reprinted (adapted) with permission from [172]. Copyright © 2023, American Chemical Society.

4.3.3. Chemical modifications (ligands, antibodies, and others)

Modifying proteins can change the hydrophilic/hydrophobic properties and surface charge, affecting their pharmacokinetic behavior. The typical synthetic strategy is conjugation with moieties that extend half-life or improve solubility[174]. PEG is a commonly-used modification where the half-life of PEGylated proteins is longer than unmodified in vivo, possibly related to the increased protein molecular weight and the decreased glomerular filtration rate [175]. The stability of the proteins is also significantly increased due to blocking contact with digestive enzymes. Insulin tregopil (BioconLt.) is an oral hypoglycemic drug modified by PEGylation[174]. Covalent modification by PEG to adapt to the GIT environment has become the gold standard for the chemical modification of therapeutic proteins [176]. Lipidation is another way to enhance the absorption of proteins, which may be associated with increasing lymphatic transport by improving fusion with chylomicrons [177]. Long-term effects of lipidation have been shown in vivo after conjugation with fatty acids [178]. Adding functional groups that can enhance protein binding to cationic polymers is a strategy to improve protein stability and intracellular delivery.

The conjugation of functional ligands to cationic polymers reduces charge repulsion during protein complex formation [179]. Succinylation is the third of the standard chemical modification method, replacing the carboxyl group of succinic acid with the amino group of lysine [180]. After the succinylation of whey protein, solubility decreased in acidic environments while increasing in an alkaline environment, maintaining the concentration of kudzu derivatives for up to 18 hours in beagle dog plasma [181].

Conjugating specific ligands of the endogenous receptors onto the nanocarriers is conducive to the absorption and penetration of proteins in enterocytes [182]. Active targeting after functionalization is achieved due to interactions between receptor and ligand or antigen and antibody [183]. Conjugating ligands can modify CS NPs for cross-cellular uptake and specific targeted delivery [184]. The ligand-modified poly (ethylene glycol)-poly (d,l-lactic acid) (PLA-PEG) NPs [185] and micelles fabricated with Gly-Sar-conjugated PLA-PEG [186] were used to target the intestinal epithelial transport protein PepT1 to concentrate therapeutic agents on the surface of the intestinal mucosa and epithelial cells. Similarly, dipeptide-modified NPs [187] promoted cellular uptake mediated by PepT1 in a proton-dependent manner [188]. Following intestinal absorption of nutrients, vitamins (B9), biotin (B7), thiamine (B1) and glucose were used to target intestinal cells [9,189].

4.3.4. Complexation

Conventional strategies for protein delivery based on complexation processes include cyclodextrin (CD) or hydrogel systems. The CD is a promising pharmaceutical excipient that can change guest compounds’ physicochemical properties with good biocompatibility, improved organ targeting, low degradability and enhanced adhesion [190]. The CD consists of 6–8 glucose molecules linked by 1,4-glycosidic bonds, while β CD is more commonly used due to the moderate size of the central cavity [191]. The hydrophobic nature of this cavity can accommodate hydrophobic molecules or hydrophobic portions of macromolecules [192]. Oral protein delivery systems based on CS NPs and antigen-modified CD inclusion complexes were prepared to deliver Ovalbumin (OVA). These systems showed enhanced stability and improved controlled release of OVA. After oral administration with β-CD/CS NPs, the OVA-specific sIgA levels in Balb/c mice were 3.6 and 1.9 times higher than OVA solution and OVA-loaded CS NPs, respectively [193]. Hydrogels can also be used to deliver proteins by stereo-complexation [194]. A composite gel [methacrylic acid grafted PEG] [P (MAA-g-EG)] was prepared for the oral delivery of peptides with enhanced effects on transporting insulin using a Caco2 cell monolayer [195]. Another advantage of the hydrogel system is that it can be tailored to different pH by adjusting the number of carboxylic acid groups or other chemical substituents. pH-responsive hydrogels are widely used to prevent proteins from being degraded [196]. To alleviate the side effects caused by chemotherapeutic drugs and address the shortage of human lactoferrin, silk sericin hydrogel (SSH) delivery systems with uniform porous structures were prepared for delivering recombinant human lactoferrin (rhLF). The effects of different temperatures, pHs and enzymes on the SSH were evaluated. The SSH protected rhLF from degradation, and oral SSH-rhLF at a low dose had a significant therapeutic effect on immune organs in cyclophosphamide (CTX)-treated mice [197]. Protein polysaccharide-based nanogels are promising delivery carriers for bioactive components owing to their high loading capacity, controlled release properties, improved bio-accessibility, good chemical stability, and intelligent response to environmental stimuli [198]. For example, the temperature can regulate gel size and control internalization efficiency [199]. Significantly, these delivery systems have been designed and studied for light-responsive, electromagnetic-responsive, biomolecular responsive and other stimuli [200].

(1). Metals

Metal-organic frameworks (MOFs) are porous grid structures with uniform and adjustable sizes formed by coordinating metal ions and organic ligands [201]. The grid structure can provide fixed sites for biological macromolecules (Da＜7 kD), such as proteins and nucleic acids, to protect them from the adverse effects of acids and enzymes [202]. MOFs can be modified with pH-responsive materials or linked with the targeted ligands, which allows the protein to be released after absorption to achieve controlled release [203]. The epithelial barrier was overcome by encapsulating insulin into acid-resistant MOFs NPs (UiO-68-NH2) and decorating externally with a targeting protein transferrin. The insulin delivery by the MOFs lasted for 10 hours, and the relative bioavailability increased to 29.6% compared with free insulin by oral administration [204]. Based on the theory that clustered oligonucleotides can combine with the scavenger receptor to promote cell transfection, nucleic acid-MOFs NPs were synthesized and loaded with insulin, resulting in significantly improved intracellular uptake of insulin [205]. Biodegradable nanocomposite microspheres containing MOFs were developed, and these iron-based MOFs were loaded with insulin and then modified with sodium dodecyl sulfate (Ins@MIL100/SDS) (Fig 8a). Ins @ MIL100/SDS @ MS was spherical and uniform, and the insulin labeled with rhodamine was equally evenly distributed in the microspheres (Fig 8b–c). The strongest fluorescence signal of RhoB-Ins in mice treated with Ins@MIL100/SDS@MS, particularly in the kidneys, was detected (Fig 8d). Due to the antacid protection of biodegradable microspheres, blood glucose levels in diabetic rats orally administrated with Ins@MIL100/SDS@MS significantly decreased than those treated with free insulin or Ins@MIL100/SDS. [206].

Figure 8.

(a). Schematic representation of the preparation of Ins@MIL100/SDS@MS. (b). SEM image of Ins@MIL100/SDS@MS. (c). Distribution of the Rhodamine B-labeled insulin in Ins@MIL100/SDS@MS. (d). Images of kidneys at different time points after the oral administration of insulin, Ins@MIL100/SDS, or Ins@MIL100/SDS@MS. Rhodamine B-Ins were used as the model protein for all tests. Reprinted (adapted) with permission from [206]. Copyright © 2020, American Chemical Society.

(2). Polyelectrolytes

Polyelectrolytes are molecules with many functional groups which are charged or can obtain charge under certain conditions [207]. Proteins are a class of natural polyelectrolytes that interact with oppositely charged polyelectrolytes to form self-assembled micelles to exert control of drug release effects. Chitosan-coated and alginate-coated NPs formed pH-sensitive polyelectrolyte complexes by self-assembly. When loaded with insulin, the complexes extended the release time and continuously reduced blood glucose levels [208].

4.3.5. Other systems

Toxins can be used as carriers to deliver therapeutic proteins to achieve targeted therapeutic effects. A single-chain bacterial toxin delivered the target protein Cas9 to human neuron cells without transfection and infection [209]. The structure and transport property of non-toxic derivatives of botulinum neurotoxin was used to deliver blocking nanoantibodies into the neuronal cytoplasm, conjugated with botulinum and alleviated toxicity in mice [210]*. The strategies discussed in this review are listed in Table 2.

Table 2.

Oral protein delivery strategies are discussed in this review.

Strategy	Representative cases	Properties or mechanism of action	Reference
Adding antacid Agents	pH buffer (e.g., Potassium-competitive acid blockers)	Inhibit H+/K+-ATP enzyme activity, but long-term application harmful to the gastric mucosa	[109,110]
Adding Enzyme Inhibitors	Sodium glycocholate, trypsin inhibitor, bacitracin soybean	Resistance to enzymes in GIT but the improper dosage can result in negative feedback regulation	[88,113]
Adhesion	Functionalized porous silicon, mesoporous silica or other bioadhesive polymers	Enhance targeted delivery and permeability	[149,150,211]
Advanced technologies	3D printing, M\microneedles	Release of proteins at specific sites	[117,123]
Formulation vehicles	SEDDS, S-SEDDS, Liposomes, NPs, physical coating, nanocomplexes	Sensitive to pH and protect macromolecules from degradation; enhance penetration of mucosa.	[107,157,160,167,171]
Chemical modifications	PEGylation, succinylation, coupling specific ligands or antibodies	Enhance targeting and bioavailability of oral proteins	[175,180,183]
Other systems	Utilization of endogenous carriers (bacteria, toxin)	Enhance intercellular delivery to target cells, suitable for small molecules	[138,209,212]

5. Status of Oral Peptides / Proteins in Clinical Trials

5.1. Salmon Calcitonin

The initial oral dosage form of salmon calcitonin coating with citrate and polymer increased local acidity to protect molecules from enzymatic degradation, while the polymer coating was regulated by pH [213]. The formulation was changed into structurally stable complexes by reversible ligation of non-covalent bonds with the enhancer (8-[(5-chloro-2-hydroxybenzoyl) amino] octanoic acid (5-CNAC). Unlike semaglutide and salmon calcitonin was not approved by the FDA because the results of phase III clinical trials (NCT00525798)) still showed low bioavailability [214].

5.2. Dolcanatide

Dolcanatide is considered adequate as an agonist of guanylyl cyclase for targeting the early stages of colon cancer. Dolcanatide consists of natural L-amino acids, while D-asparagine and D-leucine were used to replace nature amino and carboxyl groups to enhance stability and persistence in the intestinal lumen and improve delivery to the colorectum [215]. The results of phase I (NCT03300570) double-blind trial indicated that oral GUCY2C agonist replacement of the hormone could prevent the development and progression of colorectal cancer [216].

5.3. Tregopil

Tregopil (IN-105) is a PEGylated recombinant insulin with 100% sequence identity with endogenous insulin. It contains a single methoxy-trimethylene-ethylene glycol-propionyl unit linked to the lysine-β 29-amino group of human insulin via an amide bond [217]. Tregopil is being developed for oral administration for type II diabetes, with ongoing phase II clinical trials.

5.4. OPRX-106

The most crucial pathogenesis of ulcerative colitis is immune dysfunction. OPRX-106 comprises lyophilized tobacco cells expressing the TNFR 2-Fc fusion protein, with resistance in the gastric acid environment and a prolonged blood half-life. Oral OPRX-106 has completed a phase Ⅱa (NCT02768974) clinical trial with acceptable safety and immunomodulatory effects [218].

5.5. PTG-100

Targeting integrins is one option for inflammatory bowel disease. The antagonist peptide of integrins can be exposed to intestinal lymphoid tissue after oral administration. The oral preparation of PTG-100 has completed clinical phases II (NCT02895100). After oral administration in healthy volunteers, exposure in plasma binds to targets dose-dependently. Patients who suffered ulcerative enteritis after 12 weeks of oral PTG-100 intervention acquired effective clinical remission with safety and tolerability [219]. Table 3 includes the representee oral protein drugs in clinical trials.

Table 3.

Oral therapeutic peptides/proteins tested in clinical trials.

Name	Target disease	Phase	Technology	References
Salmon Calcitonin	Osteoporosis	III	Citrate increases local acidity, PH-sensitive polymer protects molecules from enzymatic degradation, and covalent ligation of enhancer (5-CNAC) stabilizes the formulation	[213,214]
Dolcanatide	Early stages of colon cancer	I	D-asparagine and D-leucine replace natural amino acids and carboxyl groups	[215,216,220]
Tregopil	Type II diabetes	II	Recombinant technology and chemical modification	[217]
OPRX-106	Ulcerative colitis	IIa	Recombinant technology	[218]
PTG-100	IBD	II	Monoclonal antibody	[219]

6. Oral Peptides in the Market

6.1. Semaglutide

Semaglutide, an agonist of glucagon peptide for the treatment of type II diabetes as well as weight management, stimulates insulin secretion, slows gastric emptying time, and is designed to be injected weekly. To reduce the pain of the injection, N-(8-[2-benzoyl] amino) octoate (SNAC) forms a noncovalent bond with semaglutide to increase lipophilic properties and provide a local buffer against pH [221]. Oral tablets of Semaglutide have completed clinical trials and were approved for treatment in the United States in September 2019 [222].

6.2. Octreotide

Abnormal secretion levels of growth hormone and insulin-like growth factors trigger acral hypertrophy. The conventional treatment choice is the injection of somatostatin analogs (SSA) or growth hormone-receptor antagonists (GHRA); however, 55.7% of patients were satisfied with these injections, while others expressed anxiety and frustration with the deep tissue injections [223]. Octreotide, combined with a transient penetration enhancer, was transformed into oily suspensions coated by capsules, which have completed phase II (NCT01412424) and III (NCT03252353) clinical trials [224]. Octreotide was approved as an oral drug for treating acromethotrophy in 2020 [225].

6.3. Linaclotide Acetate

Guanylate cyclase C (GUCY2C) is a tumor suppressor receptor. Oral GUCY2C agonists may target chemotherapy agents. The FDA approved Linaclotide acetate, a cyclic peptide composed of 14 amino acids, as the only guanylate cyclase agonist for treating irritable bowel syndrome and chronic idiopathic constipation [226].

6.4. Desmopressin Acetate

Desmopressin acetate, the first choice for treating central insipidus, was marketed in 1995 with solid antidiuretic effects and safety. Desmopressin can be used as an injection and a nasal preparation [227]. The artificial synthetic analog of desmopressin acetate as a diuretic hormone was prepared by deamination of the first amino acid and substituting D arginine with L arginine [28]. Table 4 includes the representee oral protein drugs in the market.

Table 4.

Oral peptides in the market.

Name	Target disease	Approval year	Technology	References
Semaglutide	Type II diabetes	2019	Formation of noncovalent bonds by semaglutide and SNAC to increase lipophilic properties and provide a local buffer against pH	[221,222]
Octreotide	Acral hypertrophy	2020	Octreotide combined with a transient penetration enhancer transformed into an oily suspension coated by capsules	[223–225]
Linaclotide Acetate	Irritable bowel syndrome	2012	A cyclic peptide is composed of 14 amino acids	[226,228]
Desmopressin Acetate	Central insipidus	1995	Deamination of the first amino acid and substituting D arginine with L arginine	[28,227]

7. Conclusions

Due to the high specificity and fewer side effects, proteins are designed and applied to cancer, diabetes, inflammatory diseases, and many other indications. Protein-based therapies can be administered orally in the case of some peptides. Protein delivery strategies with higher efficiency, flexibility, low cost, and good patient compliance are in pursuit. However, simultaneously overcoming gastrointestinal acid environments, enzyme digestion, mucus, and epithelial barriers makes developing oral protein delivery strategies difficult. Most peptides and protein drugs used in clinical practice are inseparable from penetration enhancers [229]. Conventionally, structural modification can improve protein stability and immunogenicity. Replacing desmopressin with native vasopressin achieved an extended half-life, and PEGylation allows the protein to escape from the stomach and reduces renal clearance. A focus on probiotics without harm to the human body as carriers for protein delivery is an emerging direction. Engineered bacteria may be able to integrate multiple target genes to achieve precise local intestinal delivery and regulate metabolic or other immune-related diseases. Patients should benefit from safe-living biological drugs. In addition, the combined application of advanced technologies supports the oral administration of proteins.

8. Expert opinion

Proteins and peptides are active and promising categories in biologics, but clinical translation for oral delivery remains problematic. The bioavailability of oral proteins by any preparation technology is still far lower than that of injections and is accompanied by significant challenges. The metabolism of nanocarrier materials in the body, the burden of surfactants to the GIT, intestinal perforation, the infection caused by new delivery devices, and the preparation and large-scale production of these systems are all problems to be solved. Although most current studies on oral protein delivery rely on in vitro and in vivo animal data, the safety and limitations of the approach in humans remain uncertain. The shortage of clinical data limits the development of new or alternative strategies. Therefore, designing appropriate oral delivery strategies remains a significant challenge and requires new ideas, innovative design strategies and novel model systems. Regardless of the chemical modifications, delivery carriers or combinations of drugs and devices, the practical significance or impact of the treatment comes first. Thus, control of organic solvents and compatible materials to ensure safety is critical. The protein delivery system should be designed and integrated to avoid chemical attacks (acid or alkali erosion), enzymatic degradation, mucosal confinement, endothelial limitation, and immune provocation. Microenvironmental factors containing gastric acid, GIT enzyme, mucus diffusion, and foci-specific delivery should be considered for proteins that function directly at the intestinal lumen. Besides the microenvironment above, interfering factors in the blood should also be considered for functions post-absorption. Any existing single strategy may not solve all the challenges of oral delivery of peptides and proteins. Aside from the valuable starting points of those drugs that are now approved and work well in the oral delivery mode, advanced technologies, including new in vitro tissue models to rapidly screen designs, the application of artificial intelligence to identify strategies, 3D printing technology, and nano- or micro-scale microelectromechanical systems (MEMS) should further help to continue to propel the field forward.

Scheme 1.

Schematic of the structure of GIT and transport mechanisms of oral peptides and proteins: (a) paracellular pathway; (b) transcellular pathway; (c) carrier-mediated transport; (d) M cells-mediated transport.

Abbreviations

FDA

US Food and Drug Administration

GIT

Gastrointestinal tract

M cells

Microfold cells

PEPT 1

Peptide-transporters 1

SLC15A1

Solute carrier family 15 members 1

P-gp

P glycoproteins

ATP

Adenosine triphosphate

ABCB 1

(ATP)-binding cassette subfamily B member 1

ABCG 2

ATP binding cassette, subfamily G member 2

VERU-111

Methanone, [2-(1H-indol-3-yl)-1H-imidazol-5-yl] (3,4,5-trimethoxyphenyl)

PI

Isoelectric point

NPs

Nanoparticles

FA

Fatty acids

GCA

Glycolic acid

ASBT

Sodium-dependent bile acid transport protein

GLP-1

Glucagon-like peptide 1

PcCLs

Protein corona using cationic liposomes

BSA

Bovine serum albumin

GALT

Gut-associated lymphoid tissue

OVA

Ovalbumin

H. pylori

Helicobacter pylori

DNA

Deoxyribonucleic acid

IBD

Inflammatory bowel disease

PLGA core

Poly (lactic-co-glycolic acid) core

SOD

Superoxide dismutase

CAT

Catalase

IgA 2

Immunoglobulin A-2

CFU

Colony-forming unit

OMV

Outer membrane vesicle

TNF

Tumor necrosis factor

BLP

Bacterial-like particles

scFv

Single-chain fragment variable

IA-2ic

Insulinoma-associated protein 2

FA-Zein

Folic acid-Zein

MA

Methacrylic acid

MSN

Mesoporous silica NPs

BL

β-lactoglobulin

ZOT

Zonula occlusion toxin

BA-Al NPs

Baicalin-AlCl3 NPs

SMEDDS

Self-micro emulsifying drug delivery system

S-SMEEDS

Solid component SMEDDS

HEMA

Hydroxyethyl methacrylate

NaCas

Nanocomplexes of sodium casein acid

PAT 1

Proton-assisted amino acid transporter 1

PLA-PEG

Poly (ethylene glycol)-poly (d,l-lactic acid)

P (MAA-g-EG

Methacrylic acid grafted PEG

SSH

Silk sericin hydrogel

rhLF

Recombinant human lactoferrin

CTX

Cyclophosphamide

MOFs

Metal-organic frameworks

5-CNAC

(8-[(5-chloro-2-hydroxy benzoyl) amino] octanoic acid

SNAC

N-(8-[2-benzoyl] amino) octoate

SSA

Somatostatin analogs

GHRA

Growth hormone-receptor antagonist

GUCY2C

Guanylate cyclase C

MEMS

Microelectromechanical systems