Nanoparticles with Cell-Penetrating Peptides for Oral Delivery: A Case for Oral Delivery of Insulin

Introduction

The protein-based drug market is growing at an average annual rate of 20%, which is significantly higher than the overall pharmaceutical market’s annual growth of 9%and reached $369.11 billion by 2024.¹ And it is projected to reach a value of $1326.16 billion by 2032. Protein-based drugs are primarily delivered through intravenous (IV), intraperitoneal (IP), intramuscular (IM), and subcutaneous (SC) routes. These delivery systems cause physical discomfort, pose a risk of infection, and are costly due to needle-based administration, which often leads to disapproval by patients, particularly those suffering from chronic conditions like diabetes who require regular dosing.^1,2 The oral insulin delivery system is a groundbreaking treatment for patients with diabetes and is preferred by those struggling with hyperinsulinemia, discomfort, infections, and adherence issues.³ However, the FDA has approved only a few protein-based drugs for oral delivery because the gastrointestinal tract presents significant physiological barriers. The bioavailability of oral insulin is reduced to less than 2% due to the action of stomach acid and proteases, as well as the impermeable mucus layer of the intestinal epithelium.^4,5

The researchers have addressed the above-mentioned obstacles by developing various nanoparticle (NP) platforms for oral insulin delivery. Recently, many nanocarriers, including lipid-based nanocarriers (liposomes, micelles), carbon nanotubes (CNTs), inorganic nanocarriers (quantum dots), gold nanoparticles and polymeric nanoparticles (PNPs, including polysaccharides, proteins, peptides, nucleic acids, metal-organic frameworks (MOFs), porous organic polymers (POPs)) have been reported.⁶ Although NPs protect encapsulated protein-based drugs from acidic denaturation and enzymatic degradation, several factors still limit their application in oral insulin delivery systems. Firstly, the viscous, semi-permeable mucus layer acts as an initial barrier to the diffusion of NPs toward the surface of the intestinal epithelium.⁷ Secondly, the tight junctions linking intestinal epithelial cells significantly hinder the uptake of macromolecules via the paracellular route.^8,9 Thirdly, macromolecules’ large size and hydrophilic nature also prevent their transcellular passage.⁸

Biomolecules such as vitamin B12 and transferrin have been observed to cross the mucus layer and intestinal epithelial cells through various carriers and receptor-mediated transport systems.^10,11 However, these systems face multiple challenges, including low expression of target receptors and insufficient uptake capacity.¹²

Recently, cell-penetrating peptides (CPPs), composed of 4–30 amino acids and capable of penetrating cell membranes,^13,14 have been employed to facilitate the systemic delivery of biological molecules across the intestinal mucosa and epithelial cells.¹³ CPPs are encapsulated in NPs or specialized devices to protect them from harsh environments, such as low-pH gastric conditions and intestinal proteases.^15,16 Additionally, CPPs can be integrated with various drug carriers to combine their benefits, forming innovative multifunctional drug delivery systems that enhance stability during blood circulation.^17–19 These advancements have also greatly facilitated the development of novel cargos, enabling targeted delivery and controlled release of biomolecules. The widespread application of CPPs in developing drug delivery cargos will help overcome the physiological barriers encountered by biomacromolecules, such as protein-based drugs, during oral delivery.^20–22

This review summarizes the latest developments in CPPs-NPs as nanocarriers and their use for oral insulin delivery. Specifically, it covers (i) the design, synthesis, and classification of CPPs; (ii) the challenges and mechanisms involved in the cellular uptake of CPPs-cargos; and (iii) a detailed analysis of the advancements in utilizing CPPs-NPs as oral delivery systems for insulin. The aim is to assist researchers in designing and developing CPPs-NPs for drug delivery applications.

Cell-Penetrating Peptides

Definition of CPPs

Cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs) or membrane transduction peptides (MTPs), are typically composed of 4–30 amino acids²³ and can penetrate cell membranes without significant toxicity.^17,18 CPPs enter the cell through various mechanisms; therefore, they can enable the intracellular delivery of bioactive substances, which are either covalently or non-covalently bound, such as nucleic acids and low molecular weight drugs.²⁴ In 1988, the first CPPs, known as Tat, were reported.²⁵ In the following years, CPPs have been extensively employed for the treatment of various ailments, including cancer,^26–28 muscular dystrophy,²⁹ anti-prion diseases,³⁰ both viral and bacterial infections,^31–33 and diabetes.^34–39 CPPs can be derived from DNA- and RNA-binding proteins,⁴⁰ homeoproteins,⁴¹ heparin-binding proteins,³ bacterial membrane proteins,^42,43 and signal peptides.⁴⁴ CPPs database (CPPsite 2.0: http://crdd.osdd.net/raghava/CPPsite/) was updated in 2015 and contains 1855 entries, including peptide sequences, the nature of the peptides, chemical modifications, experimental validation techniques, peptide structures, and types of cargo delivered.⁴⁵

Classification of CPPs

CPPs are generally categorized based on their physicochemical properties, origin, structural conformation and cell targeting (Figure 1).⁴⁶

Figure 1 Schematic diagram illustrating the types of CPPs.

Physicochemical Properties of CPPs

CPPs can be categorized into cationic, amphipathic, and hydrophobic CPPs based on their physicochemical properties (Table 1). Cationic CPPs comprise positively charged amino acid residues (Arg & Lys).^47,48 Amphiphilic CPPs are made up of both hydrophilic- (Arg & Lys) and hydrophobic amino acid residues (Val, Leu, Ile, and Ala).⁴⁹ Amphiphilic CPPs are classified into primary amphipathic-, secondary amphipathic- and Pro-rich CPPs.^50,51 Primary-amphipathic CPPs inherently possess hydrophilic and hydrophobic amino acid residues distributed across their molecular surfaces. This dual nature allows them to interact effectively with aqueous environments and lipid-rich cell membranes.⁴⁹ Secondary-amphipathic CPPs are conformationally dynamic and exhibit amphipathic properties only upon adopting a specific secondary structure, such as an α-helix or β-sheet.⁵² Another interesting class of amphiphilic peptides are Pro-rich CPPs, for which various families with different sequences and structures have been reported, but all contain a proline-pyrrolidine template. Pro has unusual properties among the 20 genetically encoded amino acids due to the rigidity imparted by the pyrrolidine ring. Additionally, since the α-amino group lacks hydrogen, it cannot provide hydrogen bonds (in the peptide structure) to stabilize α-helices or β-sheets. Unlike other amino acids that are found almost exclusively in the trans structure of a polypeptide, proline can be found in the cis structure of a polypeptide.⁴⁹ Hydrophobic CPPs contain hydrophilic amino acid residues (Leu, Ile, Val, Phe, and Ala) and few hydrophilic amino acid residues.⁵³

Table 1 The Name, Sequences, Physicrochemical Properties, Origin, Confirmation and Cell Targeting of CPPs

Origin of CPPs

CPPs are also categorized into three types based on their sources: protein-derived, chimeric, and synthetic CPPs. Protein-derived CPPs originate from naturally occurring proteins, which are inherently equipped to penetrate cell membranes.⁹⁴ Tat CPPs (transcription activator of the human immunodeficiency virus) is one of the earliest and well-studied examples of protein-derived CPPs.⁴⁵ Tat CPPs interacts with proteoglycans on the cell surface through its positively charged amino acid residues. It efficiently delivers drugs into the cell without requiring or saturating the specific ligand-receptor. Tat CPPs lacks selectivity, which limits its use in synthesizing systems needed for targeted oral delivery.^55,56 Chimeric CPPs are synthesized by combining two distinct peptides of different origins. For instance, the transportan (TPGWTLNSAGYLLGKINLKAKISIL) is produced by fusing a segment of the hormone galanin and wasp venom mastoparan.⁹⁵ Synthetic CPPs are designed and synthesized artificially; therefore, their features, such as stability in blood circulation, resistance to endolysosomal breakdown, enhanced cellular uptake, and pH sensitivity, can be tuned.¹⁹ MAP-amphipathic model peptide (KLALKLALKALKAALKLA), a synthetic CPPs, is a highly amphiphilic α-helical structure that has been several times used to deliver multiple bioactive molecules into the cell through endocytosis.⁶⁶ Glutamate (Glu) and aspartate (Asp) function as pH sensors due to their inherent trait of losing organic protons depending on the environmental pH.⁹⁶ His, with its imidazole group’s pKa of 6.5, is a buffering agent in physiological conditions and demonstrates a proton-sponge effect, which facilitates the molecule’s escape from endolysosomes. Additionally, novel synthetic CPPs can be engineered with customized features using the appropriate proportion of Glu, Asp, and His residues.⁴⁸

Structural Conformations of CPPs

CPPs are classified into two groups based on their structural conformation: linear and cyclic CPPs (Figure 2). Linear CPPs have disadvantages such as poor stability, endosomal entrapment, toxicity, and suboptimal cell penetration, whereas cyclic CPPs can avoid these limitations. Therefore, cyclic CPPs demonstrate increased cell permeability, higher resistance to proteolysis, effective endosomal escape, a higher affinity for target receptors, and nuclear-targeting properties. The amphipathic cyclic CPPs are composed of positively charged amino acid residues (His, Arg, and Lys) and hydrophobic amino acid residues (Trp, Phe, and Val), which enable them to interact efficiently with and penetrate cell membranes. Positive and negatively charged amino acid residues on the ring of cyclic CPPs are important factors affecting cellular uptake and their use for drug delivery.⁸⁹

Figure 2 The chemical structures of (a) linear and (b) cyclic CPPs.55,58,86–89

Cell Targeting by CPPs

CPPs are categorized into two types based on their targeting specificity: tissue-specific CPPs, which selectively target particular cells, and non-tissue-specific CPPs, which target cells indiscriminately to deliver the drugs.⁹⁷ Non-tissue-specific CPPs are categorized into three types based on their structural characteristics: cationic peptides, hydrophobic peptides, and amphipathic peptides.⁹⁸ Cationic peptides comprise 6–12 residues of positively charged amino acids such as TAT-PTD,^35,54 Tat-HA2⁵⁷ and penetratin,⁵⁸ etc. The positive charge of these peptides facilitates cellular uptake by promoting interactions with the negatively charged cellular membranes.⁹⁹ Hydrophobic peptides, rich in hydrophobic amino acids, are derived from the leader sequences of secreted growth factors or cytokines. Due to their hydrophobic nature, these peptides easily traverse lipid membranes.⁸⁵ Amphipathic peptides are formed by fusing hydrophobic sequences with nuclear localization signals (NLSs), creating a structure that allows them to effectively penetrate cell membranes and deliver their cargo to the nucleus.¹⁰⁰ However, non-tissue-specific CPPs will appear off-target in vivo. Tissue-specific CPPs could address this issue. Tissue-specific CPPs are identified through extensive screening of large peptide phage display libraries. This approach enables researchers to isolate the peptides that exhibit high affinity for specific tissue types.¹⁰¹ These peptides have widespread applications in both diagnostic and therapeutic fields. For instance, they facilitate the delivery of various compounds, including fluorescent or radioactive molecules, for imaging purpose. Moreover, these peptides can transport therapeutic peptides and proteins. Additionally, they significantly enhance the uptake of nucleic acids such as DNA, RNA, and siRNA and enable the delivery of viral particles. The broad range of applications underscores the importance of CPPs in advancing therapeutic strategies and enhancing the effectiveness of treatment modalities.¹⁰²

Designing of CPPs

The following parameters must be considered when designing CPPs: guanidinium groups, hydrophobic residues, structure, and targeted performance.

Guanidinium Group

Due to its unique chemical properties, the guanidinium group is essential for designing CPPs because it significantly enhances membrane permeability and cellular uptake. The guanidinium group of the Arg side chain demonstrates a stronger affinity for hydrophobic anions than Lys, which is why arginine-rich peptides form strong bonds with the lipid bilayer.¹⁰³ This interaction helps to reinforce transient pores formation and allows the peptides to embed themselves within the bilayer. The distance between the guanidine group and the peptide backbone influences its cellular uptake capability. As the flexibility of the backbone or side chain increases, the guanidine group interacts more closely with the cell surface, thereby neutralizing the negatively charged regions of the cell membrane more effectively.^104,105

Cell-permeable polydisulfides (CPDs) are derived from a guanidine-rich polyarginine (pArg) peptide backbone that is substituted with disulfide polymers.¹⁰⁶ These CPDs demonstrate improved cell penetration capability by employing dynamic covalent disulfide exchange reactions that occur on the cell surface with thiol compounds.^107,108

Hydrophobic Residues

Hydrophobic amino acid residues play a crucial role in the insertion and penetration of CPPs into the lipid bilayer of the cell membrane.¹⁰⁹ A proposed mechanism for CPPs penetration suggests the formation of a hydrophobic counter-ion around the guanidinium-rich backbone of CPPs.¹¹⁰ This counter-ion formation is initiated through hydrogen-bonding interactions between arginine residues in the CPPs and components of the cell membrane. The resulting complex then facilitates the crossing of the CPPs across the cell membrane. However, the binding strength of counter-ions in Lys-rich CPPs is lower than that of the guanidinium moieties of arginine, highlighting the critical role of the guanidinium group in this process.¹¹¹ The self-activating characteristics of these peptides are associated with the formation of counter-ion complexes by the hydrophobic segments of CPPs. Consequently, while hydrophobicity warrants further investigation, exploring the functional contributions of individual aliphatic and aromatic groups is imperative.¹⁰⁵ Hydrophobicity can be achieved through the incorporation of either aliphatic or aromatic moieties.¹¹² The process of lipidation entails attaching hydrocarbon chains of varying lengths to the N-termini of well-characterized CPPs.¹¹³ This alteration is crucial for improving the capability of these peptides to penetrate cellular membranes. In addition, alkylation is recognized as a conventional technique to bolster the internalization of peptides by reinforcing their hydrophobic interactions with cellular membranes. This approach utilizes the fundamental characteristics of alkyl groups to enhance the interaction between the peptide and the lipid elements of the membrane, consequently increasing the efficiency of cellular uptake. Through these strategies, the bioavailability of cell-penetrating peptides can be significantly improved, which is essential for their effectiveness in therapeutic applications.¹¹⁴ The inclusion of aromatic amino acid residues, like Trp, Phe, and Tyr, can also improve hydrophobicity. In addition to boosting hydrophobicity, aromatic groups can engage in π-π stacking with membrane proteins that feature aromatic residues. This interaction can help promote and stabilize the binding of CPPs to the membrane, thus assisting in their translocation and drug delivery.⁵¹

Secondary Structure

The secondary structure of CPPs (eg, α-helices and β-sheets) also significantly influences their ability to penetrate the cells. The influence of secondary structures on the translocation capability of CPPs is determined through two key factors: the strength of the interaction between CPPs and cell membranes, and their capacity to fold in the presence of these membranes.⁵¹ Weak non-covalent interactions, such as electrostatic interactions, hydrogen bonds, hydrophobic effects, and van der Waals forces, primarily influence the internalization of CPPs. Therefore, understanding the role of these non-covalent forces is essential to unveil the driving factors that support the ability of CPPs to penetrate cellular membranes. Furthermore, this knowledge will facilitate the design of novel CPPs with improved cell-penetrating capabilities, as well as more effective drug delivery cargos.¹¹¹ Yamashita et al¹¹⁵ investigated the effect of the secondary structure of CPPs on cell penetration activity. They developed a cationic dAA (α,α-disubstituted amino acid), ApiC2Gu, as an Arg mimic and replaced the hydrophobic Aib residues in peptide A (FAM-β-Ala-(L-Arg-L-Arg-Aib)₃-NH₂) with cationic ApiC2Gu residues. The cationic peptide B (FAM-β-Ala-(LArg-L-Arg-Aib)₃^C2Gu-NH₂) also formed a stable helical structure and exhibited greater cell permeability than nona-arginine (R9).²⁰ The results showed that peptides capable of altering their secondary structure in response to environmental conditions may demonstrate superior cell penetration capability compared to CPPs with a fixed helical secondary structure.

Rigidity is gaining recognition as an important concept in the design of CPPs, as it can influence the interaction of CPPs with cell membranes and their overall efficiency. In the case of linear CPPs, the efficiency of internalization is affected by multiple factors: sequence length, quantity of arginine residues, and positioning of arginine residues.¹¹⁶ The comparison of free Arg residues in linear CPPs with Arg residues in cyclic CPPs revealed that cyclization enhances the membrane interaction by facilitating improved charge distribution and greater penetration efficacy. Previously, the insertion of a spacer, such as Gly, into pArg CPPs was shown to enhance the cell permeability. This improvement is attributed to the increased distance between guanidinium groups, which resultantly reduces the steric hindrance and allows more effective interactions with the negatively charged components of cell membrane.¹⁰⁴ Traditional CPPs derived from natural _L-amino acids are sensitive to proteolytic enzymes, which presents a significant obstacle to their use in vivo for drug delivery. Consequently, the chirality of the amino acids plays a fundamental role in the development and optimization of these CPPs.

Hence, the stability of the primary structure can be improved through the incorporation of non-natural amino acids and their chiral isomers, especially _D-stereoisomers, which enable these CPPs to withstand proteolytic degradation. This characteristic allows them to remain stable and functional in the biological environment. However, the exact mechanism of how chirality of amino acid residues plays a role in enhancing the stability and cellular penetration of CPPs remains elusive, but it is evident that the strategic use of _D-stereoisomers significantly enhances the performance of CPPs in cellular applications.⁵¹

Targeted Performance

The researcher design targeting CPPs through employing three different strategies: (i) the development of novel CPPs that function as targeting ligands; (ii) the attachment of CPPs to specific targets, including small molecules, peptides, and proteins; and (iii) the modulation of CPPs absorption through stimuli-responsive signals, such as UV light, ultrasound, temperature variations, enzymatic activity, and pH levels.¹⁰⁵ Pep-1 was designed from native CPPs by incorporating fusion sequences, which include a hydrophobic segment composed of a patch of five tryptophan residues, a positively charged segment of Lys residues, and a Pro-rich spacer region. The hydrophobic segment enhances cellular penetration, the positively charged segment improves nuclear traversal capability, and the Pro-rich region increases the flexibility of the Pep-1 CPPs.¹¹⁷ Fusing RGD (arginine-glycine-aspartic acid; target integrin receptors) or homing peptides (specific for tissue markers, cells, or pathological conditions, eg, tumors or inflamed tissues) improves the specificity and affinity of CPPs.^118,119 The pH-responsive CPPs are engineered by substituting Lys with His residues. These CPPs target selectins that are overexpressed due to the acidic microenvironment produced in inflamed tissues or tumor sites.¹²⁰ Additionally, thermoresponsive polymers are coupled with CPPs to enable them to detect variations in the temperature of the cellular microenvironment. As a consequence, this enhances their capability for cell penetration and drug delivery.¹²¹

Synthesis of CPPs

Generally, CPPs are synthesized through two methods: microbial synthesis and chemical synthesis.¹²² The synthesis of CPPs by microorganisms mainly involves enzymatic hydrolysis, genetic engineering, and fermentation (Figure 3a). Enzymatic hydrolysis uses enzymes to degrade plant or animal proteins into small peptides. However, this process fails to meet industrial production levels due to its low yield, significant investment, long cycle time, and the severe pollution associated with it.¹²³ The production of CPPs through genetic engineering offers several advantages, including solid expression orientation, safety and health benefits, a comprehensive source of raw materials, and low cost. However, the production of CPPs through genetic engineering also encounters setbacks, including difficulties in achieving industrial-scale production levels due to issues with efficient expression, separation challenges, and low yield. The fermentation method involves the use of simple nutrients by microbial organisms to produce peptides, such as ɛ-polylysine (ɛ-PL), γ-polyglutamate (γ-PGA), and cyanobacterial peptide.¹²⁴ Microbial synthesis is only helpful for synthesizing peptides with natural amino acid residues¹²⁵ and is typically employed for the production of longer peptides (> 40 amino acids). However, due to their low yield and the long synthesis time required, CPPs are rarely synthesized on an industrial scale using microorganisms.¹²⁶

Figure 3 CPPs synthesis methods. (a) microbial synthesis; (b) chemical synthesis.

Nowadays, chemical synthesis is the standard technique for peptide production because it is fast, allows the incorporation of non-standard amino acid residues, and poses minimal risk of endotoxin contamination.¹²⁶ Three approaches are commonly used for the chemical synthesis of polypeptides: solid-phase peptide synthesis (SPPS), solution-phase coupling, and ring-opening polymerization (ROP) of α-amino acid N-carboxy anhydrides (NCAs) (Figure 3b).¹²⁷ The SPPS is recognized as the benchmark method for synthesizing peptides, and its efficiency has significantly improved since Merrifield’s pioneering synthesis. The linker, which bears reactive functional groups (either inherent to the resin or added at the outset of synthesis), secures the first amino acid to the resin. This linker is designed to facilitate the release of the complete peptide from the resin once the synthesis is completed. In SPPS, peptides are constructed on the resin from the C-terminus to the N-terminus using side chain-protected amino acids, which prevent side reactions during the coupling process.¹²⁸ SPPS is commonly employed to synthesize short peptides consisting of fewer than 50 residues.¹²⁵

The solution-phase coupling technique utilizes the short peptide sequence as the starter to produce larger quantities of CPPs. Therefore, this method is always used to synthesize CPPs containing repetitive short sequences (ca. 3 to 10 residues in length).¹²⁹ Nevertheless, this method also faces challenges in synthesizing initial oligopeptides, the cyclization processes, and the low molecular weight of the resulting products.¹²⁷ The ROP of NCAs represents a fundamental and widely embraced methodology for synthesizing cyclic- and linear polypeptides.¹³⁰ The NCAs are synthesized through two methods: (i) the Leuchs method, which involves the reaction between N-alkyloxycarbonylamino acids and halogenating agents like thionyl chloride, and (ii) the Fuchs-Farthing method, where α-amino acids react with phosgene (COCl₂).¹³⁰ These methods are essential for creating a robust framework for synthesizing NCAs, which subsequently undergo polymerization initiated by a diverse array of nucleophiles and bases, including amines and metal alkoxides. There are two predominant mechanisms: the activated monomer (AMM) pathway and the normal amine (NAM) pathway through which NCAs are aggregated.¹³¹ The AMM mechanism uses bases such as tertiary amines and alkoxides to initiate the polymerization reaction. These bases abstract a proton from the nitrogen of the NCA (designated as 3-N) and form an NCA anion. This deprotonation process allows the NCA anion to initiate the polymerization by attacking the carbonyl group of another NCA; as a consequence, carbon dioxide (CO₂) is released with the formation of a new anion that continues the polymerization reaction.¹²⁵ However, it is essential to note that polymerizations based on the AMM are usually uncontrolled and less desirable for synthesizing well-defined cyclic peptides due to their inherent unpredictability. In contrast, the NAM mechanism is typically used for NCA polymerizations initiated by nonionic initiators with at least one mobile hydrogen atom, such as primary and secondary amines, alcohols, and water. The NAM mechanism offers advantages by providing control over the molecular weight of the resulting polymer and ensuring the fidelity of end groups, which is crucial for synthesizing high-quality cyclic peptides.¹³² Traditional NCA polymerization is typically a slow process, often lasts 2–3 days or more, and is sensitive to moisture, frequently necessitating using anhydrous solvents and handling within a glovebox. Recently, Wu et al¹³⁰ introduced a superfast NCA ring-opening polymerization method initiated by lithium hexamethyldisilazane (LiHMDS), conducted in an open vessel outside glovebox environments.

Clinical Challenges of CPPs-Cargo

CPPs maintain the integrity of cell membranes and are therefore regarded as both highly effective and safe in contrast to the traditional methods of sonoporation, microinjection, electroporation, and bead loading.^133–135 Furthermore, other key aspects that must be evaluated for clinical use of CPPs include therapeutic effectiveness, the practicality of large-scale production, physical and chemical characteristics, formulation, administration routes, stability, toxicity, and pharmacokinetics (PK).¹³⁶ CPPs are rapidly degraded by proteases in systemic circulation and subjected to intracellular digestion by lysosomes and proteasomes, significantly limiting their plasma half-life and stability. The rapid barrier penetration and cellular uptake of CPPs result in the clearance of CPP conjugates from the bloodstream within minutes, thereby reducing protease-mediated degradation in circulation. However, this characteristic complicates pharmacokinetic analysis through plasma concentration measurements of these conjugates. The short half-life is a primary factor diminishing the in vivo efficacy of CPP-cargo complexes.¹⁷ To address these limitations, peptide stabilization strategies have been developed, including: (i) D-amino acid and unnatural amino acid substitutions,¹³⁷ (ii) N-terminal or C-terminal modifications,¹³⁸ (iii) fatty acid conjugation,¹³⁹ (iv)formulation optimization¹⁴⁰ and (v) cyclizing.¹⁴¹ Additionally, the coupling of CPPs with therapeutic cargo peptides to generate fusion peptides is often not cost-effective for large-scale synthesis. Given the limited development of peptide-based therapeutics for human diseases, drug design strategies must be rigorously evaluated by integrating scientific and economic considerations. CPP conjugates may accumulate in non-target cells/tissues, particularly the liver and kidneys, posing potential toxicity risks.¹⁴² Consequently, comprehensive assessment of the pharmacokinetics, biodistribution, and hepatotoxicity/nephrotoxicity of CPP conjugates is essential to mitigate toxicity. Localized administration rather than systemic delivery can minimize systemic toxicity. Encouragingly, CPP-based therapies have demonstrated preliminary safety in Phase I clinical trials. However, immunogenicity concerns persist for non-human-derived or synthetic CPPs, underscoring the preference for human-derived CPPs in clinical applications.¹⁴³ While novel CPP-based candidates continue to enter clinical trials with recent advancements. Ja-Hyun Koo et al discussed this in detail.¹³⁶ However, no FDA-approved drugs incorporating CPPs are currently available due to limitations in human trial validation. Importantly, in the clinical application of CPPs, it is imperative to rigorously uphold patient autonomy through informed consent, ensure transparency in trial design, prioritize safety-first pharmacovigilance, and enforce stringent screening of raw materials to mitigate environmental contamination risks.

Cellular Uptake Mechanisms of CPPs

A defining characteristic of CPPs lies in their capacity to perturb cellular membranes, thereby enabling subsequent intracellular transduction. The efficiency of cellular transduction and active targeting (extending beyond peptide-specific interactions) is constrained and modulated by molecular size and polarity. Small, nonpolar molecules traverse cell membranes via simple diffusion, a process characterized by rapid, concentration-dependent, and energy-independent kinetics. Recent studies highlight that loosened packing of lipid bilayers has been recognized as a pivotal factor governing CPP-mediated membrane translocation. Strategic augmentation of hydrophobic residues within the peptide backbone facilitates penetration through the lipophilic core of the membrane. A proposed mechanism suggests that CPPs may mediate transduction by inducing membrane curvature. Furthermore, enzymatic degradation in extracellular environments (eg, proteases in the gastrointestinal tract) compromises CPPs stability prior to cellular uptake. Beyond intrinsic CPPs properties, membrane composition critically influences translocation efficacy. Notably, specific lipids such as bis (monoacylglycero) phosphate (BMP), enriched in late endosomes, are identified as key mediators of endosomal escape-a rate-limiting step for intracellular delivery.^144–146 The precise molecular mechanism governing the entry of CPPs into cells has yet to be fully explained, although many investigators have proposed mechanisms through which CPPs penetrate the plasma membrane.¹⁰⁵ The mechanistic models have unveiled several factors which impact the cellular penetration of CPPs: i) the type of cell, ii) the mass and structure of the cargo, iii) the binding method, iv) the concentration of CPPs, and v) the duration and temperature of incubation. The primary mechanisms proposed for CPPs internalization include endocytic routes and direct penetration. (Figure 4).¹⁴⁷

Figure 4 Cellular internalization/uptake mechanisms of CPPs. Endocytosis mechanisms mainly include macropinocytosis, clathrin-/cavedin-mediated endocytosis, and clathrin-/cavedin-independent endocytosis. Direct penetration mainly includes the inverted micelles, carpet-like model, membrane thinning, transient pore formation, and induction of multilamellarity alongside the stalk-pore hypothesis.

Endocytic Routes

Most CPPs enter cells via endocytic pathways, which comprise two key steps: endocytosis and subsequent endosomal escape.¹⁴⁸ Endocytosis is the process by which intracellular and extracellular substances are internalized through the invagination of the plasma membrane, forming vesicles that enclose the ingested material.¹⁴⁹ Endocytosis is an energy-dependent process that occurs in all cells. The CPPs are translocated into cells primarily through endocytosis rather than direct translocation due to the rapid turnover of the plasma membrane.¹⁵⁰ Endocytosis consists of various pathways, including macropinocytosis, clathrin-/cavedin-mediated endocytosis, and clathrin-/cavedin-independent endocytosis (Figure 4).¹⁵¹ All of these endocytic pathways are inhibited under energy-depleting conditions, such as low temperature (4°C) or with sodium azide (NaN₃) treatment.¹⁵² Macropinocytosis is a unique cellular process that does not initiate through the typical mechanism of membrane invagination; instead, it arises from increased membrane ruffling and cellular activation.¹²⁵ This form of membrane dynamics leads to protrusions that do not encapsulate a ligand-coated particle but fold back to integrate into the plasma membrane, forming structures referred to as macropinosomes.¹⁵³ A large body of experimental evidence has indicated the crucial role of macropinocytosis in the cellular penetration of different CPPs.^154,155 The mechanism by which the M918 peptide translocates across the cell membrane appears to involve both macropinocytosis and clathrin-mediated endocytosis (CME). Similarly, the TP10 peptide penetrates the cell membrane primarily via CME.¹⁵⁶ This suggests that cellular uptake mechanisms are complex and multifaceted. In addition to macropinocytosis, various endocytic processes, such as CME, also play a vital role in the uptake of CPPs by neural cells. The Tat peptide and related transporters rely on caveolin-mediated endocytosis.^157,158 Furthermore, the cellular uptake of Tat was partially inhibited by a blocking agent, indicating that caveolin-mediated endocytosis is not solely responsible for its uptake, as other pathways also work in conjunction. A comprehensive study revealed that Tat and pArg utilize three endocytic pathways for cellular translocation: clathrin-mediated endocytosis, caveolin-mediated endocytosis, and micropinocytosis. However, the involvement of these pathways in uptake varies across different CPPs, highlighting the complexity and specificity of their cellular entry mechanisms.¹⁵⁹ J.P. Richard proposed that the clathrin-dependent pathway might be involved in the cellular uptake of CPPs and demonstrated that TAT uptake in HeLa cells was reduced by 50% following chlorpromazine-mediated inhibition.¹⁶⁰ It is widely accepted that CPPs bind to the plasma membrane before being internalized via any of the aforementioned endocytic pathways.¹⁶¹ The electrostatic interaction between CPPs and glycosaminoglycans is not merely a passive cellular internalization process. In fact, it prompts endocytosis by inducing the aggregation of membrane proteins. The heparan sulfate proteoglycans have been implicated in playing a crucial role in the cellular internalization of CPPs. However, the precise role of proteoglycans in the context is still ambiguous.^162,163 Recently, it has been reported that scavenger receptors play a crucial role in the endocytic-mediated internalization of PepFect CPPs, indicating that internalization also depends on these receptors.¹⁶⁴ After endocytic internalization, CPPs and CPPs-cargo complexes must escape from endosomes to avoid lysosomal degradation, reach their target site, and exert their biological activity. Endosomal escape is a major limiting factor for the efficient intracellular delivery of functional macromolecules.⁴⁹ Several reports have shown that the formation of a pH gradient, increased vesicle concentration, and the attraction of CPPs can cause membrane stiffening and rupture, contributing to the escape of CPPs from endosomes.^165,166 The endocytic pathway is ideal for delivering drugs to the area of interest. However, the number of NPs that can enter cells is limited by factors such as endocytosis, endosomal maturation, and exocytosis. Studies have shown that NPs must be smaller than 100 nm to efficiently penetrate the cells through vesicle-based transport systems. This transport mechanism is particularly effective for the transcytosis of macromolecules across endothelial cells.¹⁶⁷

Direct Penetration

Although nerve-dependent endocytosis is considered the main pathway for the cellular internalization of various CPPs, increasing evidence indicates that some CPPs are internalized through endocytosis-independent pathways. Certain CPPs, for example, are internalized at low temperatures, which may explain why they avoid energy-dependent endocytosis.^168–172 Direct penetration is a straightforward, single-step process that occurs independently of energy. In this mechanism, positively charged CPPs interact with negatively charged components of the cell membrane, such as the phospholipid bilayer and heparan sulfate.¹⁷³ It has been shown that Arg-CPPs translocate through the plasma membrane by the direct penetration mechanism.^167–171 The direct translocation of the Arg-rich CPPs is thought to be facilitated by their strong interactions with the lipid head groups of the plasma membrane. Specifically, the positively charged Arg residues bind to negatively charged lipids (eg, phosphatidylserine (PS) and phosphatidylglycerol (PG)). Additionally, the guanidine group forms hydrogen bonds with phosphate, carboxylic acid, or sulfate groups, leading to structural changes in the membrane.^174,175 Direct penetration of CPPs can even occur in the presence of cryogenic and endocytic inhibitors. CPPs can penetrate cells via five distinct direct penetration mechanisms: (i) the formation of inverted micelles, (ii) the carpet-like model, (iii) the model of membrane thinning, (iv) transient pore formation, and (v) the induction of multilamellarity alongside the stalk-pore hypothesis (Figure 4).^176,177 The first pathway involves the formation of inverted micelles, where CPPs aggregate and insert themselves into the lipid bilayer of the membrane. The carpet-like model demonstrates that CPPs adhere to the membrane surface and disrupt the lipid organizational arrangement, consequently allowing them to penetrate the membrane. Additionally, the membrane thinning model proposes that CPPs rely on reducing the membrane thickness, which subsequently creates favourable conditions for their cellular internalization. The transient pore formation model relies on CPPs forming temporary openings in the cell membrane by interacting with lipid molecules. The induction of multilamellar and the stalk-pore hypothesis highlight how CPPs can promote the formation of multiple lipid layers and transient structures that facilitate their uptake into the cell. These diverse internalization pathways highlight the versatile applications of CPPs in cellular drug delivery, functioning without the need for energy input.

Strategies for CPPs Conjugation to Cargo

CPPs and cargos bind through covalent binding or physical mixtures Recently, the ConjuPepDB database was launched, and it contains information on over 1600 drug molecules conjugated with CPPs, their biomedical applications, and the specific chemical conjugation procedures employed.¹⁷⁸

Covalent Binding

Numerous types of covalent bonds exist, including peptide bonds,¹⁷⁹ disulfide bonds,^180,181 sulfanyl bonds,¹⁸² maleimide bonds,¹⁸³ imine bonds,¹⁸⁴ and triazole bonds.¹⁸⁴ The attachment of CPPs to cargo through covalent binding enhances the stability, improves cellular uptake, endosomal escape and allows precise control over positional selectivity.¹⁸⁵ Morishita et al found that the _D-R9-insulin conjugate promoted intestinal insulin absorption and demonstrated an improved hypoglycaemic effect after pulmonary administration in diabetic rats.¹⁸⁶ Noriyasu et al noticed an improvement in the intestinal absorption of insulin due to the covalent attachment of _D-R8 with insulin. They also observed that the binding ratio between insulin and _D-R8 plays a significant role in enhancing intestinal absorption.²⁰ However, covalent conjugation can alter the CPPs or cargo molecule’s natural conformation and functional groups, which may affect their biological activity. And this method faces the challenge of synthesis complexity and inflexibility for dynamic adaptation.^187–189 The active fragment of parathyroid hormone (PTH1-34), conjugated with the N-terminal, demonstrated superior biological activity compared to the C-terminal conjugated (VP22-PTH (1–34) of ((CD40 + CD86) / (MHC II + CD11C) and relative SEAP activity increased by 5% and 0.3 compared to PTH (1–34) -VP22, respectively). The coadministration approach proved to be more effective for delivering PTH 1–34 across the Caco-2 monolayer.¹⁸⁸ In such cases, non-covalent conjugation strategies are more appropriate.

Physical Mixture

The CPPs-cargo complexes are formed through intermolecular interactions, including hydrophobic interactions, electrostatic forces, hydrogen bonds, and others.⁷⁷ This method can preserve cargo bioactivity, simplify preparation, and enable dynamic ratio adjustment, as demonstrated by R8-insulin mixtures enhancing intestinal absorption. Kamei et al discovered that the simultaneous administration of insulin and R8, including VEC and RRL helices, following physical mixing, notably enhanced insulin absorption in the ileum of rats. Among various CPPs, the _L-CPPs demonstrated the highest efficacy for intestinal absorption of insulin.¹⁹⁰ Non-covalent complexes also shield cargo from enzymatic degradation, prolonging serum half-life. Despite limitations in stability under physiological conditions and lower loading efficiency, non-covalent strategies hold significant clinical potential for delivering sensitive therapeutics (eg, nucleic acids, proteins) due to their adaptability, stimulus-responsive release, and reduced immunogenicity. Future advancements may integrate nanocarriers or targeted ligands to address stability and toxicity challenges, positioning non-covalent systems as promising tools for oral biologics, combination therapies, and precision medicine. For instance, negatively charged oligonucleotides interact with positively charged CPPs through electrostatic and hydrophobic forces. In this strategy, CPPs shield the bioactive conjugate, thereby protecting it from degradation by proteases or nucleases, which, in turn, prolong the serum half-life of the cargo.⁴⁹ Table 2 summarizes the results of more CPPs combined with cargo.

Table 2 List of the Reported Conjugates of CPPs and Cargos

CPPs and PNPs for Oral Delivery of Insulin

Recently, many nanocarriers, including lipid-based nanocarriers (liposomes, micelles), carbon nanotubes (CNTs), inorganic nanocarriers (quantum dots), gold nanoparticles and polymeric nanoparticles (PNPs) have been reported.⁶ However, lipid-based nanocarriers (liposomes, micelles) exhibit limitations including immunogenicity, stability challenges, and scalability issues. For instance, polyethylene glycol (PEG) coatings on liposomes may induce anti-PEG antibodies, leading to accelerated blood clearance (ABC phenomenon) or hypersensitivity reactions, compromising long-term efficacy. Repeated dosing of lipid formulations can also trigger complement activation, resulting in drug leakage and toxicity. Furthermore, phospholipid bilayers in solid lipid nanoparticles (SLNs) are prone to drug leakage during storage, while large-scale production via microfluidic techniques remains inefficient. Lipid carriers may also interfere with lipid metabolism by binding to lipoproteins (eg, LDL), potentially exacerbating atherosclerosis.^196,197 CNTs are generally limited by poor biodegradability and challenges in systemic clearance.¹⁹⁸ Inorganic nanocarriers (quantum dots) and gold nanoparticles, face biocompatibility and toxicity concerns. Quantum dots containing heavy metals (eg, cadmium) pose long-term toxicity risks, while gold nanoparticles often require complex surface modifications to achieve targeting, increasing costs.^199,200 The PNPs are oral drug delivery systems which are typically range between 100 to 500 nm. They are classified into two types: natural polymers and synthetic polymers.²⁰¹ PNPs can improve insulin loading capacity, gastrointestinal stability, higher bioavailability, low toxicity and preparation controllability.^202–205 It is especially suitable for the treatment of diseases that require precise delivery and long-term efficacy. However, these systems face multiple challenges, including low expression of target receptors and insufficient uptake capacity.¹² CPPs have been widely used in the delivery of protein drugs due to their cell membrane, penetrating and designed targeting function, and good biocompatibility.^17–20 The intestinal permeability of insulin is influenced by factors such as the chain length, amphipathic nature, hydrophobic characteristics, and basic properties of CPPs. The attachment of CPPs with macromolecules enhances the permeability of membranes for the associated cargoes.¹⁸⁹ Tat-CPPs not only efficiently penetrate the cell membrane but also enhances the translocation of 100 times larger macromolecular drugs. Tat (YGRKKRRQRRR) interacts non-covalently with proteins to mediate their delivery into cells.^206,207 In 2005, the first CPPs employed as a peptide vector for insulin transport through the intestinal epithelium was TAT-PTD.³⁴ Since then, additional CPPs have been utilized as potential carriers for intestinal transepithelial and transmucosal delivery of biopharmaceuticals in both in vitro and in vivo studies. Mariko et al found that oligoarginine is likely to be a powerful tool for overcoming insulin’s low permeability through the epithelial cell membrane.¹⁸⁶ According to Kristensen et al. Arg residues within the peptide are essential for facilitating insulin transport across the epithelium.¹⁸⁸ This is because Arg is the most polar of all the proteinogenic amino acids. The cationic Arg residue is translocated through a nonpolar lipid bilayer, and more acid residues form stronger hydrogen bonds. This forms a salt bridge between the cationic amino acids and the acidic residues.²⁰⁸ As stated by Patel et al²⁰⁹ the covalent linkage of _D-R9 to insulin plays a crucial role in its translocation mechanism across the alveolar epithelium in rats. According to Patel et al, the covalent attachment of _D-R9 to insulin is essential for the translocation process across the alveolar epithelium in rats. The _D-R9-insulin conjugate demonstrated an enhanced hypoglycaemic effect after pulmonary administration in diabetic rats and also promoted the intestinal absorption of insulin.¹⁸⁶ Kamei et al demonstrated that the binding rate of insulin to _D-R8 is a critical factor in _D-R8’s ability to enhance intestinal insulin absorption.²⁰ The initial group demonstrated that CPPs facilitated the oral absorption of a peptide through a physical mixing formulation without the use of PNPs or devices. Nielsen et al observed that, in diluted gastrointestinal fluid, _D-penetration exhibited greater stability than _L-penetration. By interacting non-covalently with insulin, _D-penetration extends its half-life from 25 minutes to 91 minutes. Nonetheless, the effectiveness of this approach was only moderate (PA = 1.2%) compared to the subcutaneous delivery of insulin.²¹⁰ Bioavailability, stability, selectivity, and in vivo efficacy are enhanced by incorporating CPPs into or coupling with nanomaterials.^6,211

Natural Polymers

Natural polymers, such as polysaccharides, proteins, peptides, and nucleic acids, offer numerous advantages for encapsulating and delivering active drug components into the cell. These benefits include biodegradability, biocompatibility, nontoxicity, and non-immunogenic properties.^212,213 Biocompatible and biodegradable, natural polymer nanoparticle carriers are the most promising materials for developing innovative oral insulin delivery systems.²¹⁴

Natural Polysaccharide

Natural polysaccharides primarily include dextran, chitosan (CS), pectin, alginate (ALG), hyaluronic acid (HA), starch, and others.

CS is a cationic polysaccharide copolymer composed of glucosamine and N-acetylglucosamine linked by β (1→4) bonds.²¹⁵ The structure of CS features numerous active amine and hydroxyl groups. Consequently, chemical modification of CS, along with further modification of its derivatives, can yield new compounds with desired characteristics.²¹⁶ CS improves the intestinal permeability of biological macromolecules by enhancing adhesiveness and temporarily opening tight junctions, ensuring good biocompatibility and safety for drug delivery applications.²¹⁷ Guo et al²¹⁸ developed a delivery system (Tat-CS-NPs) consisting of NPs incorporated with Tat and amphiphilic chitosan derivatives (aCS). The resulting delivery system demonstrated a significant increase in the colonic absorption of the medication. The study on a diabetic rat model revealed that Tat-CS-NPs produced an insulin-lowering effect that was 6.89 times greater than PVA-NPs and 1.79 times greater than CS-NPs, indicating that Tat enhanced penetration efficiency. The nanocarriers, developed by coupling chitosan (CS) with a novel cell-penetrating peptide (SAR6EW), demonstrated embedding and drug loading rates for insulin of 75.36% and 7.58%, respectively. The oral administration of SAR6EW/CS/insulin NPs produced a greater hypoglycaemic effect in diabetic rats compared to CS/insulin NPs without inducing NP-mediated toxicity. The findings indicated that chitosan nanocarriers mediated by SAR6EW effectively deliver insulin orally. Furthermore, this delivery method shows promise for the oral administration of other protein-based medications.²¹⁹ Liu et al²²⁰ employed CRT-modified trimethylchitosan (TMC) NPs for the oral delivery of insulin. Their results showed that CRT-NPs exhibited improved cellular uptake, superior transport across the Caco-2 monolayer, and enhanced absorption via villi, particularly when compared to transferrin receptor-targeted modified NPs (HAI NPs). Furthermore, in vivo investigations confirmed that the hypoglycemic effect and absorption of insulin (INS) were significantly greater in diabetic rats treated with CRT peptide-modified NPs compared to those receiving HAI NPs targeting the transferrin receptor (TfR). These findings suggest that CRT peptides represent a promising alternative for the oral delivery of peptides and proteins.

Modified starch derivatives, such as carboxymethyl short-chain amylose and cyclodextrin (CD), are extensively utilized in drug delivery due to their low toxicity, excellent biocompatibility, and unique biodegradability. CD are cyclic oligosaccharides widely regarded as valuable pharmaceutical excipients.²²¹ β-cyclodextrin (β-CD) is a ring-shaped oligosaccharide featuring a hydrophobic cavity that interacts with specific surface residues of insulin through hydrophobic interactions.^222,223 The R8-CM-β-CD conjugate enhanced the intestinal absorption of insulin through various endocytosis mechanisms, inhibited the function of P-glycoprotein (P-gp) efflux pumps, and demonstrated an extraordinary hypoglycemic effect. These outcomes highlight R8-CM-β-CD as a promising candidate for an effective drug delivery system.²²⁴ Nanocomplexes loaded with insulin were prepared through self-assembly using CPPs or their derivatives modified with bis-β-cyclodextrin (P-bis-CD), enhancing stability and delivery efficiency. In diabetic rats, the intestinal administration of P-bis-CD nanocomplexes demonstrated a significant hypoglycemic effect that persisted for 6 h. The bioavailability of EN NC and P-bis-CDNC was measured at 3.5% and 10.6%, respectively. These findings indicate that P-bis-CD effectively enhances the epithelial penetration of insulin.¹²

Alginate, a polyanionic polysaccharide, is widely used in various fields, including medicine and food. Its reliability and biocompatibility make it highly suitable for numerous applications in medical materials.²²⁵ In a study, Li et al¹⁹⁴ developed R8-modified insulin-SA NPs (INS-SA/R8 NPs) to enhance the oral delivery of insulin. These NPs significantly improved insulin absorption, increased intestinal permeability and enhanced the uptake of villi. Notably, INS-SA/R8 NPs also stimulated the production of endogenous nitric oxide (NO), a naturally occurring signalling molecule that enhances insulin absorption at specific concentrations. When administered orally, INS-SA/R8 NPs exhibited superior hypoglycemic effects and biocompatibility compared to the oral administration of INS-SA NPs in diabetic rats.

Natural Proteins

Proteins are essential natural biomaterials for developing innovative nano-vehicles due to their exceptional properties, including biodiversity, biodegradability, low immunogenicity, non-toxicity, and biocompatibility.^226,227 Protein-based nano-vehicles, with their well-defined primary structure, can be customized through pre- or post-functionalization. This unique characteristic allows various drugs, components, and carriers to be combined with the hydrophobic or hydrophilic regions of proteins using a range of reagents. In addition to serving as insulin delivery systems, protein NPs can also provide energy to individuals with diabetes.^228,229 Liu et al²³⁰ utilized conventional transferrin receptor-targeting ligand (HAIYPRH)-modified NPs (HAI-NPs) for the oral delivery of insulin. The HAI-NPs demonstrated good hypoglycaemic efficacy and insulin absorption. However, while their hypoglycemic efficacy and insulin absorption were significantly improved, they remained lower than those of CRT peptide-modified NPs.

Synthetic Polymers

Synthetic polymers (SPs) offer several advantages over natural polymers and other materials, particularly due to their ability to be tailor-made for specific pathological needs and patient requirements.²²⁹ Examples of synthetic polymers (SPs) include poly(lactic-co-glycolic acid) (PLGA), hydrogels, silica, porous coordination polymers such as metal-organic frameworks (MOFs), and porous organic polymers (POPs), among others.²³⁰ Synthetic polymers have been shown to enhance bioavailability and slow the release of insulin in oral insulin delivery systems.

PLGA

PLGA is a widely recognized biomaterial used in drug delivery and approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA).^231–233 It is metabolized into lactide and glycolic acid in the body, which are further converted into carbon dioxide and water via the Krebs cycle. This indicates that PLGA possesses a low toxicity profile for living organisms. Importantly, as a copolymer composed of two monomers (lactide and glycolate), PLGA’s degradation rates and drug release profiles can be regulated by modifying the ratio of its monomers and the molecular weights of lactide and glycolate.³ Consequently, PLGA is regarded as one of the most essential carriers in modern biomedical research, enabling targeted and extended drug delivery to address various health issues, including diabetes and cancer.^234–237

In 2013, Yan et al²³⁸ developed Tat-mediated PLGA NPs for delivering insulin to the brain. Their findings indicated that CPPs have the potential to facilitate the transport of macromolecules to cerebral tissue. In 2013, Liu et al²³⁹ introduced a method using CPPs-functionalized PLGA-NPs to enhance the oral delivery of insulin. Their results showed that the use of _L-R8-modified NPs increased the relative bioavailability of insulin by 3.2 times, while _D-R8-modified NPs increased it by 4.4 times. Corresponding improvements in hypoglycemic effects were observed at 2.5 times and 3.7 times, respectively. In 2016, J. Sheng and associates⁵⁹ developed an insulin-LMWP conjugate, which was subsequently loaded onto mucoadhesive PLGA NPs coated with N-trimethyl chitosan chloride, resulting in the formation of insulin-LMWP NPs. These findings indicated that the pharmacological availability of oral insulin was 17.98 ± 5.61% compared to subcutaneously administered insulin, representing an improvement twice as significant as that observed with MNPs containing native insulin. In 2022, Yang et al²⁴⁰ developed PLGA-NPs (Pep/Gal NPs) modified with pH-responsive stretchable CPPs and a liver-targeting fraction (Gal). They demonstrated effective intestinal absorption and significant intrahepatic deposition of insulin. The highest pharmacological availability (PA) of Pep/Gal-PNPs was 10.1%. Notable, Pep/Gal PNPs supported glucose homeostasis for effective diabetes management by increasing hepatic glycogen production by 7.2-folds.

Hydrogel

Hydrogel systems consist of two or more components, including a stable three-dimensional polymer network and water molecules that fill the spaces between macromolecules. These hydrogels can absorb and retain substantial amounts of water (typically between 70% and 99%); therefore, they are ideal for formulating hydrophilic and sensitive drugs in semi-solid delivery systems.²⁴¹ The water content influences drug diffusion through the polymer network in the hydrogel. The diffusion coefficient is determined by the number and size of the pores, while water absorption depends on the type of bonds.²⁴² The hydration of hydrophilic groups in the polymer chain triggers the swelling phenomenon. As these polar groups hydrate, the network expands, exposing hydrophobic groups that can interact with water molecules. This process allows the polymer network to absorb additional water, driven by osmotic forces that push it toward a state of infinite dilution. This additional expansion is limited by covalent cross-links, which establish equilibrium. Free water refers to excess water absorbed after the saturation of ionic, polar, and hydrophobic groups. The “free water” occupies the voids within the polymeric network.²⁴³ Fukuoka et al³⁸ investigated the effectiveness of combining P(MAA-g-EG) hydrogel with oligoarginine R6 to enhance intestinal insulin absorption. Their findings suggest that P(MAA-g-EG) hydrogel carriers provide protection and enable the controlled release of drugs, presenting a promising approach for oral insulin delivery.

Silica

Silicon NPs (SNs) are widely used as inorganic drug carriers. Mesoporous silica NPs (MSNs) (Mobil Composition of Matter (MCM), Santa Barbara Amorphous (SBA), and Mesocellular Foam (MCF)) have been extensively studied in medical applications such as diagnosis, engineering, and treatment. Among various drug delivery systems, SN-based oral delivery systems have been extensively studied and demonstrate significant potential for the delivery of oral therapeutic proteins and peptides (TPPs) delivery, particularly insulin.^244,245

In 2021, Rao et al synthesized porous SNs and modified them with poly (pyridyl disulfide ethylene phosphate/sulfobetaine) polymers, resulting in the formation of P(PyEP-g-SBm)n-AmPSiNPs NPs. The insulin-loaded P(PyEP-g-SB)-AmPSiNPs demonstrated enhanced stability and biocompatibility in-vitro. However, their hypoglycemic effect and bioavailability were suboptimal in vivo, possibly due to the mucus layer acting as a barrier to the nanoparticle’s passing through the intestinal epithelium.²⁴⁴ The same year, Zhang et al²⁴⁶ engineered mesoporous SNs with modification groups (MSN-NH₂@COOH/CPPs5) that effectively traversed the mucus layer and passed through the intestinal epithelium by mimicking the surface of viruses. These MSN-NH₂@COOH/CPPs5-NPs significantly improved the apical-to-basal transcytosis, primarily through caveolae-mediated endocytosis. Interestingly, the transepithelial transport rate of MSN-NH₂@COOH/CPPs5 across the Caco-2 cell monolayer was 2.4-fold higher than that of MSN@NH₂ and 2.0-fold higher compared to MSN-NH₂@COOH. The bioavailability of insulin encapsulated in the MSN-NH₂@COOH/CPPs5 NPs was 2.1 times higher than insulin administered directly into the jejunum. Additionally, these modified NPs demonstrated no significant toxicity in preliminary in vitro and in vivo experiments. This work demonstrates the efficient delivery of peptide or protein drugs by overcoming dual barriers, such as the intestinal mucus layer and epithelium.

Others

Many synthetic polymers, such as carrier peptides and silica-alginate NPs, can orally deliver insulin but have been less extensively studied. Diedrichsen et al²⁴⁷ studied the effect of the physical mixing of carrier peptides with insulin on intestinal absorption. Carrier peptides are novel and promising excipients for oral delivery of therapeutic peptides. The study demonstrated that insulin’s transepithelial permeability was influenced by the extent of complex formation with the insulin-carrier peptide and depended on the stereochemistry of petromax. However, it was not affected by penetration or shuffle. He et al²⁴⁸ suggested that encapsulating CPPs-insulin conjugates into silica-alginate-NPs enhances the intestinal shelf life of insulin. The presence of alginate within the NPs highlights its adhesive role in facilitating transport to the intestinal mucosa. Upon accumulation at the mucosal surface, the strong cell-penetrating capabilities of CPPs enable the rapid passage of released CPPs-insulin conjugates across the epithelial cell layer, directly delivering insulin into the blood circulation. Additionally, the swift internalization of CPPs-mediated NPs significantly reduces the time insulin spends in the luminal cavity, thereby minimizing its degradation by endogenous proteases. In 2020, Abdelhamid et al²⁴⁹ introduced a method for loading simple oligonucleotides (ONs) and achieving efficient release through a synergistic approach. This method combines the adaptable features of ZIF-8 NPs as chemically tunable nanocarriers with those of CPPs, specifically PepFects (PF), functioning as a capping system. Their findings revealed that the use of ZIF-8 and its composites enhances cell transfection and improves the cellular uptake of ONs while maintaining high biocompatibility. Thus, combining CPPs with POPs may significantly enhance drug loading and enable efficient delivery. Surprisingly, the sublingual insulin drops developed by the University of British Columbia (UBC) team, incorporating fish-derived cell-penetrating peptides, enable rapid insulin absorption via sublingual capillaries, demonstrating hypoglycemic efficacy comparable to injections. This technology has been validated in murine models and has entered the commercial licensing phase.²⁵⁰

In summary, CPPs play a vital role as transporters for nanodrugs and demonstrate promising outcomes in the treatment of diabetes. Further, significant research findings are outlined in Table 3.

Table 3 Application of CPPs in the Oral Insulin Delivery

Conclusion and Perspective

In recent years, numerous nanoparticle systems intended for the oral administration of insulin have been developed. However, the efficacy of novel drug delivery systems (NDDS) is often constrained by the low expression of receptors on enterocytes and their limited absorption capacity. Incorporating or coupling CPPs with nanoparticles (NPs) can enhance bioavailability, stability, selectivity, and in vivo efficacy. Despite being the safe and effective carrier for macromolecular delivery, CPPs have not received significant attention for the oral delivery of insulin. Currently, porous organic polymers (POPs), including hyper-cross-linked polymers (HCPs), metal-organic frameworks (MOFs), and covalent organic frameworks (COFs), are widely utilized in oral insulin delivery systems. Their unique pore architecture, adjustable pore dimensions, customizable structures, and modifiable surface properties make them particularly suitable for this application. POPs can enhance bioavailability and enable controlled insulin release, making them an excellent candidate for oral insulin delivery systems. In the future, the development of multimodal delivery systems (POPs-CPPs nanocomposite carriers) and stimuli-responsive CPPs engineering (eg, pH/enzyme-sensitive peptide motifs) provides a promising strategy to overcome mucosal barriers, achieve enhanced cellular internalization, and optimize future oral insulin delivery platforms. The implementation of CPPs-mediated intranasal and pulmonary delivery systems could establish a complementary therapeutic regimen to oral administration, effectively mitigating dose-dependent complications associated with single-route delivery. Furthermore, CPPs-enabled co-delivery of insulin with metabolic regulators (eg, GLP-1 analogues) facilitates synergistic glycemic control through insulin-GLP-1 combination therapy. To accelerate clinical translation, it is imperative to forge interdisciplinary collaborations between academic institutions and pharmaceutical enterprises, ultimately enabling pain-free and therapeutically efficient treatment modalities for diabetic patients.