Abstract
Subcutaneous and inhaled insulins are associated with needle phobia, lipohypertrophy, lipodystrophy, and cough in diabetes treatment. Oral nanoinsulin has been developed, reaping the physiologic benefits of peroral administration. This review profiles intestinal receptors exploitable in targeted delivery of oral nanoinsulin. Intestinal receptor targeting improves oral insulin bioavailability and sustains blood glucose–lowering response. Nonetheless, these studies are conducted in small animal models with no optimization of insulin dose, targeting ligand type and content, and physicochemical and molecular biologic characteristics of nanoparticles against the in vivo/clinical diabetes responses as a function of the intestinal receptor population characteristics with diabetes progression. The interactive effects between nanoinsulin and antidiabetic drugs on intestinal receptors, including their up-/downregulation, are uncertain. Sweet taste receptors upregulate SGLT-1, and both have an undefined role as new intestinal targets of nanoinsulin. Receptor targeting of oral nanoinsulin represents a viable approach that is relatively green, requiring an in-depth development of the relationship between receptors and their pathophysiological profiles with physicochemical attributes of the oral nanoinsulin.
Significance Statement Intestinal receptor targeting of oral nanoinsulin improves its bioavailability with sustained blood glucose–lowering response. Exploring new intestinal receptor and tailoring the design of oral nanoinsulin to the pathophysiological state of diabetic patients is imperative to raise the insulin performance to a comparable level as the injection products.
I. Diabetes Mellitus and Therapy
Diabetes mellitus in humans is characterized by an inability to metabolize blood glucose for energy (Xiao et al., 2020). In 2019, approximately 463 million adults worldwide were found to be living with diabetes (Pinchevsky et al., 2020). The International Federation of Diabetes predicted that by 2045, 700 million adults worldwide would be diabetic. Insulin is employed to manage specifically type 1 diabetes and late-stage type 2 diabetes where oral therapy alone is inadequate to attain euglycemia (Xiao et al., 2020). It is most commonly available as subcutaneous formulations. Despite the good bioavailability it confers, the subcutaneous route is associated with needle phobia, trauma, and inconvenient administration as well as lipodystrophy, local tissue necrosis, and infection (Gedawy et al., 2018; Xiao et al., 2020). These drawbacks lead to poor patient compliance, with around 60% of patients failing to attain long-term blood glucose control (Wong et al., 2016; Gedawy et al., 2018), and risks developing diabetic complications such as macrovascular and microvascular complications, retinopathy, nephropathy, peripheral neuropathy, and periodontal disease (Wong et al., 2016; Gedawy et al., 2018; Polak et al., 2000; Stöhr et al., 2021). These in turn provide the impetus for significant research into developing insulin formulations for administration by less invasive routes, such as the buccal, ocular, rectal, oral, pulmonary, and transdermal routes (Mutalik, 2011; Gedawy et al., 2018).
In 2006, Pfizer launched Exubera, an inhaled insulin (Wong et al., 2016). This product was withdrawn from the market a year later owing to the undesirable respiratory burden of inhaled insulin, along with a lack of cost effectiveness (Hollander et al., 2004; Wong et al., 2016). Undeterred by the failure of Exubera, MannKind Corp obtained Food and Drug Administration approval in June 2014 for Afrezza, another inhaled insulin. Afrezza managed to capture some market share, possibly due to improvements in design and formulation over Exubera. However, Afrezza is not routinely recommended by physicians due to side effects and cost concerns (Al-Tabakha, 2015). The failure of inhaled insulins diverted focus to other routes of administration, especially the oral and buccal routes, for insulin formulation research. Despite drawbacks of buccal administration, which include salivary clearance as well as differing epithelia in sublingual, cheek, and palate mucosae, Generex Biotechnology, Inc. introduced Oralgen, an insulin spray targeting the buccal cavity using the Rapidmist technology (Heinemann and Jacques, 2009; Palermo et al., 2012). Oralgen has shown some success in achieving glycemic control among type 2 diabetic patients; however, clinical use of Oralgen is limited due to high cost and lack of strong evidence showing efficacy. The transdermal route has been explored but showed little success because poor skin permeability to macromolecular therapeutics leads to unacceptably low insulin bioavailability (Zhang et al., 2019). The applications of microneedles and microwave radiation to promote transdermal insulin delivery have recently been introduced, and these have met with varying degrees of success (Harjoh et al., 2020; Ahad et al., 2021; Zhang et al., 2021; Liu et al., 2022).
Peroral delivery of insulin is highly desirable from a patient’s perspective, but this route has its fair share of barriers. Insulin is susceptible to degradation by enzymes in the gastrointestinal tract (GIT), such as pepsin, chymotrypsin, elastase, carboxypeptidases and, at the brush border membrane, aminopeptidase, and the extreme pH range along the GIT inducing oxidation and deamination of insulin (Gedawy et al., 2018; Xiao et al., 2020). The mucosa and mucus layers in the GIT present a physical barrier to insulin absorption (Wong et al., 2016; Gedawy et al., 2018; Xiao et al., 2020). Insulin has to avoid being trapped in the mucus and being lost to mucus turnover to maintain high bioavailability (Tan et al., 2020). Paracellular transit across the mucosal layer is not naturally accessible to insulin, and transcellular passage across the intestinal enterocytes, achievable via nanoformulations, poses risks of intracellular enzymatic degradation by endosomal lysosomes (Fan et al., 2018).
Nonetheless, the oral route remains an attractive option as it provides insulin availability that closely mimics the physiologic pathway. Insulin absorbed across the intestinal mucosa is transported to the liver via the portal vein, setting up a portal-peripheral gradient (Arbit and Kidron, 2009; Gedawy et al., 2018) that results in lower levels of systemic insulin and reducing the risk of hypoglycemia and weight gain (Gedawy et al., 2018). Consequently, substantial resources have been invested into formulating insulin to enable it to withstand degradation and to undergo transmucosal absorption in the GIT. For more than two decades, insulin nanoparticles (NPs), or interchangeably known as nanoinsulin, have been formulated, most of which incorporated with functional excipients, such as protease inhibitors and tight junction modulators to enhance insulin absorption (Cárdenas-Bailón et al., 2013; Wong et al., 2017; Gedawy et al., 2018; Xiao et al., 2020). The paracellular pathway is, however, not ideal as it makes up less than 1% of the intestinal mucosal surface, limiting the amount of insulin that can be transported. More recent studies explore the transcellular pathway, utilizing specific intestinal receptors, to mediate the delivery of nanoinsulin.
In diabetic patients, the microenvironment of the intestine, specifically receptors, undergoes pathophysiological changes in response to the progression of the disease (Chen et al., 2012; Young et al., 2013). Sha et al. (2016) demonstrated that receptors for advanced glycation endproducts and transforming growth factor β1 receptors are more abundant in the mucosa, submucosa, and muscular layer of the colonic wall of diabetic rats, whereas higher concentrations of receptors for advanced glycation endproducts in the colon as well as small intestine of the diabetic rats are reported elsewhere (Chen et al., 2012; Zhao et al., 2017). This review aims to profile and critically review the potential of published intestinal receptors that have been exploited, as well as apical membrane receptors of intestinal enterocytes that hold potential to equip researchers with relevant information for nanoinsulin formulations that facilitate oral absorption in diabetic individuals.
II. Intestinal Receptors Targeted for Nanoinsulin Delivery
Table 1 summarizes the physicochemical and physiologic characteristics of intestinal receptors used in oral nanoinsulin targeting. Apical sodium-dependent bile acid transporter (ASBT), folate receptor (FR), heparan sulfate proteoglycan receptor (HSPG), human neonatal fragment crystallizable receptor (FcRn), transferrin receptor (TfR), intrinsic factor-vitamin B12 receptor (IF-VB12) receptor, human proton-coupled amino acid transporter (PAT1), and Niemann-Pick C1-like protein 1 (NPC1L1) are available in the duodenum, jejunum, terminal ileum, or throughout the intestinal tract. They exhibit a high affinity for specific substrates such as bile acid, folic acid, cell-penetrating peptide, antibody, transferrin (Tf)-iron, vitamin B12, amino acid, and cholesterol, respectively. Ligands with similar physicochemical attributes as these receptor substrates, as well as the substrates themselves, can be used as the targeting moiety of nanoparticles for oral insulin delivery. Broadly, the ligands promote endocytosis and cellular uptake of nanoinsulin, protect the nanoinsulin from lysosomal degradation, and/or facilitate its exocytosis from the enterocytes into the blood stream and target site of action (Fig. 1).
A. Apical Sodium-Dependent Bile Acid Transporter
Orally administering the diabetic rats with deoxycholic acid–decorated nanoparticles (DNPs) and deoxycholic acid-chitosan conjugate-decorated liposomes (DC-LIPs) was characterized by an insulin bioavailability of about 16%, with reference to subcutaneous insulin (Fan et al., 2018; Wu et al., 2019). The significant in vivo hypoglycemic effects were attributed to ASBT targeting. DNPs were found to evade lysosomal degradation, whereas the undecorated nanoparticles were trapped in lysosomes, leading to enzymatic degradation of their insulin load (Fan et al., 2018). DC-LIPs similarly escaped from the lysosomal digestion; however, the lysosomal escape studies were not conducted with the nondecorated liposomes as control, making it difficult to conclusively attribute the lysosomal escape to deoxycholic acid (Wu et al., 2019).
B. Folate Receptor
The successful development of anticancer drugs through FRα targeting has spawned a widespread interest in reduced folate carrier (RFC) and proton-coupled folic acid transporter (PCFT) as potential targets for nanoparticle drug delivery system. In 2012, Jain et al. (2012) designed folate (FA)-coupled polyethylene glycol (PEG)ylated polylactide-co-glycolide (PLGA) nanoparticles as oral insulin carrier. More recently, El Leithy et al. (2019) encapsulated insulin for peroral delivery in folate-chitosan conjugate nanoparticles (FA-CS NPs), and Yazdi et al. (2020) formulated PEGylated liposomes with folate moieties. Liposomes are developed as they are elastic vesicles and deemed to be able to interact with the intestinal mucosa to a greater extent than the relatively rigid nanoparticles such as folate-coupled polyethylene glycolylated polylactide-co-glycolide nanoparticles. The introduction of PEG to liposomes helped circumvent liposomal instability in the harsh environment of the GIT (Iwanaga et al., 1997; Yazdi et al., 2020). PEGylation, however, may hinder mucosal permeation of liposomes and their uptake across the enterocytes (Suk et al., 2016; El Leithy et al., 2019). The impediment by PEGylation was found to be mitigated via FR targeting (Jain et al., 2012; Tan et al., 2020). Administering a fixed dose of 50 IU/kg insulin orally in the form of PLGA NPs to diabetic rats was characterized by a Cmax of plasma insulin of 23.25 µIU/ml. PEGylation of the nanoparticles reduced the Cmax to 14.90 µIU/ml. Decorating the PEGylated nanoparticles with FA raised the Cmax to 26.78 µIU/ml. FA promoted folate receptor–mediated endocytosis and its translation into nanocarrier has the potential to raise drug bioavailability (El Leithy et al., 2019).
FA has also been employed in the development of virus-mimicking oral insulin nanoparticles made of insulin-loaded poly(n-butylcyanoacrylate) core and hybrid coat consisting of positively charged chitosan-folate and negatively charged hyaluronic acid (Cheng et al., 2021). The coat electroneutrality enables the nanoparticles to penetrate through the intestinal mucus without excessive mucus retention or repulsion, and FA enhances the intestinal mucosal binding affinity of the nanoparticles. In vivo study in diabetic rats indicated that a FA graft content of 12.51% is met with a pharmacological bioavailability of 9.8%, with a given oral insulin dose at 50 IU/kg.
C. Heparan Sulfate Proteoglycan Receptor
On the basis of its hepatic association, the HSPG was investigated by Tan et al. (2020) for its role in the uptake of nanoinsulin from the intestine to the portal circulation. Tan et al. (2020) designed mesoporous silica nanoparticles (MSNs) modified with poly(lactic acid)−methoxy poly(ethylene glycol) and decorated with cysteine-modified low molecular weight protamine as the cell-penetrating peptide (CPP) (MSN@PLA−mPEG-CPP). Low molecular weight protamine is relatively safe and able to deliver insulin, and other peptide drugs, in the form of nanoparticles across the intestine without damaging cell membranes or causing immune responses (Sheng et al., 2016; Zhang et al., 2018a). MSN@PLA−mPEG-CPP was designed with CPP modulation to achieve net particle electroneutrality and mucopenetrating behavior to improve accessibility to receptor (Fig. 2), and its oral insulin bioavailability was about 2.7 times higher than that of MSN@PLA−mPEG (Tan et al., 2020).
A reduction in MSN@PLA-mPEG-CPP uptake by human colorectal adenocarcinoma HT29 cells in vitro was observed in the presence of sodium heparin, a competitive HSPG inhibitor, suggesting that MSN@PLA-mPEG-CPP cellular uptake was mediated by HSPG. PEG is hydrophilic and generally known to be mucus inert and this aids nanoparticles in mucus penetration (Fig. 2) despite the claim of Yazdi et al. (2020) that it “impedes mucus penetration and weakens cellular uptake.” Reduced hydrophobic and electrostatic interactions of MSN@PLA−mPEG-CPP with mucus decreased its mucus trapping by 36% and promoted its mucus penetration capacity (Fig. 2). The HSPG- and caveolae-mediated endocytosis, and the favorable electrostatic interactions, enhanced the intestinal cellular uptake of the nanoparticles by two- to threefold.
D. Neonatal Fragment Crystallizable Receptor
To target the FcRn, Pridgen et al. (2013) conjugated PLA-PEG nanoparticles with the Fc fragment of IgG, whereas Azevedo et al. (2020) opted for albumin, for its nontoxic, nonimmunogenic and biodegradable nature, to be conjugated to PLGA-PEG nanoparticles. Oral administration of the Fc-conjugated PLA-PEG nanoparticles to wild-type mice elicited a prolonged hypoglycemic response at a notably low dose of insulin (1.1 U/kg), compared with doses typically employed in other nanoparticles (10–100 U/kg) (Pridgen et al., 2013). The same observation was not found when the nanoparticles were administered to FcRn knockout mice, which ascertained the role of FcRn in mediating the improved cellular uptake and insulin delivery of the Fc-conjugated PLA-PEG nanoparticles.
FcRn was shown to be localized at the duodenum of the wild-type mice; however, it is expressed throughout the human intestine at an expression level above 30 pmol/g of tissue (Pridgen et al., 2013; Fan et al., 2019). Given the differences between rodents and humans, Azevedo et al. (2020) developed a mouse model to express human FcRn to enhance the receptor-binding propensity for albumin. Oral administration of PLGA-PEG nanoparticles, decorated with human albumin genetically engineered to improve binding to human FcRn, gave rise to the steepest reduction of blood glucose level at 1 hour and the lowest blood glucose level at 4 hours against controls, including the nanoparticles decorated with an albumin variant, which rejected FcRn binding. However, and unexpectedly, the minimum blood glucose concentration was achieved with free insulin orally administered rather than any of the insulin-loaded nanoparticles.
E. Transferrin Receptor
TfR has been exploited by many researchers for the transcytosis of insulin and other peptide drugs, such as exenatide, across the intestinal mucosa (Zhu et al., 2016; Liu et al., 2018; Zhang et al., 2018b). A limiting factor is the presence of the endogenous ligands ferritin and Tf (Liu et al., 2018). In humans, serum concentration of Tf is approximately 200–300 mg/dL (Kawabata, 2019). Tf has a high binding affinity with TfR and can competitively inhibit the binding of Tf-decorated nanoparticles to TfR and hinder their absorption. Liu et al. (2018) circumvented this hurdle by developing nanoparticles that targeted the Tf-TfR complex instead of TfR. Trimethyl chitosan (TMC) nanoparticles were conjugated with CRTIGPSVC (CRT), a ligand that binds to the Tf-TfR complex. A control sample was similarly prepared using HAIYPRH (HAI)—a ligand that binds to TfR. Tf-TfR complex targeting via TMC-CRT NPs provided the best hypoglycemic effect in vivo (41.5% blood glucose level reduction at 4 hours of administration) compared with the other orally administered insulin formulations in the study. TMC-CRT NPs displayed the highest bioavailability relative to subcutaneous insulin at 6.84%, whereas TMC-HAI NPs provided a bioavailability of only 3.59%, similar to the undecorated nanoparticles at 3.31%.
In Caco-2 cells where apo-Tf was absent, TMC-HAI NPs exhibited the highest cellular uptake, 1.5 times that of TMC-CRT NPs and the undecorated nanoparticles (Liu et al., 2018). With the addition of apo-Tf and the use of a coculture model of Caco-2/E12 to mimic mucus impediment, the transepithelial transport of TMC-HAI NPs was greatly reduced. Conversely, the cellular internalization of TMC-CRT NPs was 4.3 times higher when apo-Tf was present. The Tf was required to generate the Tf-TfR complex, the target of CRT, and therefore, the presence of Tf increased the effectiveness of TMC-CRT NPs to facilitate cellular insulin uptake. The transport of nanoparticles via TfR is mediated in apical-to-basolateral direction (Lim et al., 2007; Norouziyan et al., 2008; Liu et al., 2018). This study demonstrates that a judiciously selected ligand can reverse the antagonistic effect of endogenous ligands in receptor-mediated cellular uptake of exogenous therapeutic compounds. It is a more commendable approach in achieving therapeutic insulin levels than simply increasing the insulin dose, which translates to increased manufacturing costs and medication expenditure (Heinemann and Jacques, 2009).
F. Intrinsic Factor-Vitamin B12 Receptor
Chalasani et al. (2007b) conjugated VB12 to dextrans (Dex) and epichlorohydrin (Epi) in the form of nanoparticles in an effort to target the IF-VB12 receptor, also known as cubilin. Targeting cubilin produced an enhanced hypoglycemic effect. An insulin dose of 20 IU/kg administered through the VB12-Dex-Epi-NPs (3% w/w insulin loading) produced a bioavailability of 26.5% relative to subcutaneous insulin in diabetic rats, which was significantly greater than the 10.3% achieved using undecorated nanoparticles. The positive outcomes of cubilin targeting were observable in both streptozotocin-induced diabetic rats, used in most studies, as well as in non-obese diabetic mice, a congenital rodent diabetic model (Chalasani et al., 2007a). Cubilin targeting also allowed for relatively low doses of insulin to be administered. VB12-Dex-Epi-NPs were able to produce a relative bioavailability of 26.5% at a dose of 20 IU/kg.
With the success of VB12 receptor targeting, a recent study explored the vitamin B7 (biotin) receptor for oral nanoinsulin delivery (Cui et al., 2021). The biotinylated chitosan was synthesized and complexed with insulin followed by hyaluronic acid coat decoration into a virus-mimicking polyelectrolyte nanocomplex. Its relative bioavailability was nonetheless low (<5%) at an oral dose of 50 IU/kg.
G. Proton-Coupled Amino Acid Transporter
Han et al. (2020) encapsulated insulin in a micellar system (DSPE-PCB), which conjugated the zwitterionic betaine polymer polycarboxybetaine (PCB), a PAT1 targeting ligand, with the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) lipid. The zwitterionic carrier effectively navigated through the mucus and epithelial barriers to improve insulin uptake without inducing significant tight junction opening, which may lead to autoimmune disease, bacterial infection, and inflammatory bowel diseases over long-term applications (Lerner and Matthias, 2015; Han et al., 2020). The DSPE-PCB possessed an extremely low critical micelle concentration and was therefore unlikely to have dissociated into its constituents that can potentially modulate intestinal cell membranes and tight junctions.
The lyophilized insulin-containing DSPE-PCB was packed into a size M porcine gelatin capsule and enteric-coated to protect against the acidic environment of the stomach for oral administration to streptozotocin-induced diabetic mice at an insulin dose of 20 IU/kg (Han et al., 2020). A greater bioavailability was obtained with the DSPE-PCB capsule (42.6%) than betaine-free formulation (8.35%). The DSPE-PCB capsule lowered the rodent blood glucose level by more than fivefold of the control capsule.
H. Niemann-Pick C1-Like Protein 1
Ding et al. (2020) designed NPC1L1-targeting amphiphilic nanoparticles (Hc@CP-Ca), where cholesterol as hydrophobic targeting moiety was conjugated to phosphate groups, chosen for their hydrophilicity and biocompatibility, via hexane linkers and stabilized by calcium ion chelation. In vitro studies using the Caco-2 cell model revealed a significant 31.4-fold increase in insulin uptake with Hc@CP-Ca compared with free insulin at 1 hour of incubation, indicating the involvement of NPC1L1 and cholesterol-mediated endocytosis. This was further confirmed by a reduction in the insulin uptake efficiency of Hc@CP-Ca when ezetimibe, which blocks NPC1L1, was added. The benefit of surface modification with amphiphilic phospholipid and glycocholate was evident from in vivo studies where a dose of 50 IU/kg of insulin administered to diabetic rats via Hc@CP-Ca achieved a Cmax of 49.1 µIU/ml at 2 hours of administration, whereas nonsurface decorated nanoparticles displayed a lower Cmax (32.4 µIU/ml) at 3 hours.
III. Limitations, Challenges, and Future Perspectives
Overall, the in vitro cell culture and in vivo small animal studies suggested that intestinal receptor targeting promotes cellular uptake of oral nanoinsulin and enhances insulin bioavailability and its antihyperglycemic activity. Nonetheless, only a limited number of studies have been conducted for each of the intestinal receptors targeted in oral nanoinsulin delivery research. The physiologic functionality of the target intestinal receptors, the optimal physicochemical designs of the nanoparticles, and the level of specificity of targeting ligands have not been comprehensively understood. All in vivo studies reported to date are conducted in the small animal models, which may have different intestinal receptor profiles from that of the human. An example is the FcRn, which is expressed throughout the human intestine but is only detected in the duodenum of the wild-type mice (Pridgen et al., 2013). The research outcomes derived from the small animal may not be applicable in the clinical setting for humans. In vitro gut models, as a prescreening tool, may have underestimated the level of drug transport in vivo due to lack of peristalsis, fluid flow, microbial flora, and receptor-active cell line (Kim et al., 2012).
With reference to drug release study, the targeting ligand-decorated nanoparticles are found to be characterized with a lower insulin release propensity than undecorated nanoparticles and/or free insulin (Jain et al., 2012; Ding et al., 2020; Tan et al., 2020; Yazdi et al., 2020). A large number of investigations, however, reflect that 20%–40% of the insulin load is released in the gastric phase (Chalasani et al., 2007a; Jain et al., 2012; Wu et al., 2019; Azevedo et al., 2020; Han et al., 2020; Yazdi et al., 2020). The nanoparticles reaching the intestinal region are characterized by lower insulin loads. Even when the intestinal targeting formulation strategies are used, the effective dose of insulin transported across the mucosa may be far from satisfactory. Such complication is further exacerbated by oral nanoinsulin development studies that presented no drug release investigation in the gastric medium (Pridgen et al., 2013; Fan et al., 2018; Liu et al., 2018).
Given that the oral nanoinsulin has its drug release profiles optimized, the pathophysiological changes of intestinal receptors as a function of diabetes progression may complicate its insulin delivery performance. Annaba et at. (2010) reported that ASBT expression and function were increased following streptozotocin-induced diabetes in rats. The expression of NPC1L1 in diabetic rats was also found to double that of healthy rats (Ding et al., 2020). Thus far, there is no known report investigating the genotype-phenotype relationship of ASBT, FR, HSPG, FcRn, TfR, IF-VB12 receptor, PAT1, and NPC1L1 with reference to the progression of diabetes in humans and the interplay of diabetes type, age, gender, race/ethnic group, and geographical distribution of these receptors. The relationship between oral nanoinsulin delivery and pathophysiological changes of intestinal receptors has yet to be investigated in vivo using small animal models, and their clinical implications are not known.
Another risk factor is the long-term effects of oral nanoinsulin administration. Insulin is a growth factor (Chen et al., 2011; Gedawy et al., 2018). Its long-term oral administration at high doses raises concerns of mitogenic changes in the gastrointestinal epithelium. The associated risks and benefits of nanoinsulin, with their inherent requirement for higher oral doses to be administered, will need to be weighed against the current mode of management—that is, subcutaneous insulin administration.
A reduction in oral insulin dose relative to the doses reported in the current literature is generally preferred. To realize this goal, it is imperative to develop an efficient nanoinsulin delivery system capable of achieving high oral insulin bioavailability and minimizing the availability of nonabsorbed or degraded insulin in the gastrointestinal tract. The capability is associated with the physicochemical properties of the nanoinsulin formulations. Table 2 shows the primary physicochemical attributes of oral nanoinsulin that have been developed for intestinal receptor-specific delivery. The uptake of nanoparticles by mammalian cells is broadly governed by their size, shape, surface roughness, crystallinity, and surface chemistry (Oh and Park, 2014; Musalli et al., 2020). The aggregative behavior of nanoparticles may affect their biologic performances at the cellular interface via changes in the primary particulate physicochemical properties in response to physiologic milieu (Shaedi et al., 2021). Currently, there is little consensus on an optimized design for nanoinsulin. The oral nanoinsulin formulations reported in the literature consisted of a wide range of matrix materials of differing polarities (Table 2). In most of these studies, parameters such as particulate surface roughness and shape are not quantified, and the matrix crystallinity and particle aggregation tendency are not examined. In addition, the oral nanoinsulin bioavailability has not been determined in some studies. Both mice and rats, with expected physiologic and pharmacokinetic variations, are used as the animal model. The summative observations, within a small number of studies, do not allow one to generalize the desired physicochemical attributes of oral nanoinsulin that are possibly favorable in diabetes treatment. Moreover, different intestinal receptors may have affinity for nanoparticles of different size, shape, surface roughness, and crystallinity characteristics, though this has yet to be determined.
Optimization of nanoinsulin design should therefore be target-specific, requiring intestinal receptor-specific studies instead of generalization of findings obtained from investigations involving different intestinal receptors. This complication is further exacerbated by endogenous and exogenous substrates that compete with the targeting ligand of nanoinsulin for the same receptor (Liu et al., 2018). Metformin, a drug that is widely prescribed to treat type 2 diabetes, has been shown to inhibit calcium-dependent vitamin B12 absorption by interfering with intestinal calcium metabolism and uptake (Kibirige and Mwebaze, 2013; Wong et al., 2018). Metformin-induced vitamin B12 deficiency is relatively common in type 2 patients with diabetes (Aroda et al., 2016; Kim et al., 2019). As these patients may also require insulin, the impact of metformin on the intestinal uptake of vitamin B12-conjugated nanoparticles and their blood glucose–lowering performance warrants further investigation.
Thus far, only the DSPE-PLB provides a substantial improvement in oral insulin bioavailability (>40%) (Table 2). In terms of blood glucose reduction, a reduction of more than 70% blood glucose is only attained with DSPE-PLB, VB12-Dex-Epi NP and Hc@CP-Ca NP. The underlying reasons for why some formulations are more successful than others may be complex and possibly due to a lack of nanoinsulin formulation optimization and in-depth knowledge about the population density of intestinal receptors and their physiologic behaviors in association with nanoinsulin delivery.
Alongside efficient binding to the target receptor, the intracellular translocation of nanoinsulin also affects its bioavailability and efficacy. The intracellular lysosomal insulin degradation can be minimized by enabling rapid endosomal escape and exocytosis of the nanodelivery system. Influenza virus–derived hemagglutinin-2 is a pH-responsive membrane fusogenic peptide (Xu et al., 2018). In endosomes, the protonation of the influenza virus–derived hemagglutinin-2 induces a conformational change to α-helixes. This enables the peptide to promote endosome-cellular membrane fusion and subsequent exocytosis, a mechanism that was inferred from a recent study on insulin-loaded solid lipid nanoparticles. Similar observations were obtained with bile acids and engrailed secretion peptide (Fan et al., 2018; Cohen et al., 2020). Apart from these few reports, the intracellular destiny of the majority of oral nanoinsulin systems as a function of particle composition remains unknown. Advancing delivery science at the subcellular level is therefore necessary to further optimize particle design for nanoinsulin.
Recently, apple-derived nanoparticles containing the fruit microRNA were developed and found to downregulate, via the matrix materials, the mRNA expression of the human intestinal transporter OATP2B1/SLCO2B1, the apical sodium dependent bile acid transporter ASBT/SLC10A2, and the carnitine transporter OCTN2/SLC22A5 in the Caco-2 cells (Arai et al., 2021). This suggests that the composition of nanoparticles, through regulating intestinal transporter expression, may facilitate or hinder cellular uptake of oral nanoinsulin. It is imperative to examine the molecular biology aspects of the oral nanoinsulin systems to enable one to establish the complex relationship between nanoparticle design and cellular trafficking.
Table 3 highlights intestinal receptors with potential for oral nanoinsulin targeting. Among these, the sweet taste receptors hold particularly strong promise. The expression of sweet taste receptors was enhanced in patients with type 2 diabetes (Greenfield and Chisholm, 2013; Young et al., 2013). Although there have been no studies conducted among humans thus far, sweet taste receptors have been reported to upregulate sodium-glucose cotransporter 1 (SGLT-1) in species such as rats and mice (Margolskee et al., 2007; Stearns et al., 2010). Sweet taste receptors appear to exert opposing influences in diabetes control. They promote intestinal glucose absorption (Greenfield and Chisholm, 2013; Young et al., 2013); on the other hand, they induce the satiety hormone release to control the state of hyperglycemia. With reference to oral nanoinsulin delivery, the targeting ligand for sweet taste receptors as such should preferentially be an agonist for satiety hormone release only, apart from being the homing device for oral insulin nanoparticles. Should SGLT-1 be a feasible transport pathway for oral nanoinsulin, dual targeting of sweet taste receptors and SGLT-1 can be an alternative strategy to promote oral nanoinsulin delivery using targeting ligands that stimulate the expression of sweet taste receptor, which in turn leads to the upregulation of SGLT-1 and have binding affinity for both the sweet taste receptor and SGLT-1. A synergistic action between the sweet taste receptor and SGLT-1 in nanoparticle binding is envisaged to lead to enhanced insulin absorption and its bioavailability.
IV. Final Remarks
Receptor targeting has been exploited for oral nanoinsulin delivery to improve oral insulin bioavailability and blood glucose–lowering response. Despite lower bioavailability when compared with subcutaneous insulin, a more sustained glucose reduction was observed for most of the orally administered nanoinsulin systems examined in this review. Receptor targeting in oral nanoinsulin delivery has not been extensively explored. The relationships of insulin dose, targeting ligand type and content, physicochemical, and molecular biologic characteristics of nanoparticles with in vivo or clinical diabetes responses require further investigation. The designs of future oral nanoinsulin delivery systems should consider the population characteristics of intestinal receptors with the progression of diabetes, albeit the latter is still poorly defined. Additionally, the oral nanoinsulin delivery must effectively overcome both mucus and mucosa barriers. Of the intestinal receptors reviewed in this paper, PAT1 appears to be a promising intestinal target receptor for the transmucosal transport of nanoinsulin, with a bioavailability greater than 40% reported.
Acknowledgment
The authors would like to express their heart-felt thanks to National University of Singapore for facility support.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: Choy, Lim, Chan, Cui, Mao, Wong.
Footnotes
- Received April 13, 2022.
- Revision received June 1, 2022.
- Accepted June 24, 2022.
No author has an actual or perceived conflict of interest with the contents of this article.
Abbreviations
- Apo-Tf
- apo-transferrin
- ASBT
- apical sodium-dependent bile acid transporter
- CPP
- cell-penetrating peptide
- CRT
- CRTIGPSVC
- CUB
- complement C1r/C1s, Uegf, BMP1
- DC-LIP
- deoxycholic acid-chitosan conjugate-decorated liposome
- Dex
- dextran
- DNP
- deoxycholic acid–decorated nanoparticle
- DSPE
- 1,2-distearoyl-sn-glycero-3-phosphoethanolamine
- Epi
- epichlorohydrin
- FA
- folate
- FA-CS
- folate-chitosan
- FA-CS NP
- folate-chitosan conjugate nanoparticle
- FcRn
- human neonatal fragment crystallizable receptor
- FR
- folate receptor
- GIT
- gastrointestinal tract
- GLP-2R
- glucagon-like peptide 2 receptor
- HAI
- HAIYPRH
- Hc@CP-Ca
- NPC1L1-targeting amphiphilic nanoparticle
- HSPG
- heparan sulfate proteoglycan receptor
- IF-VB12
- intrinsic factor-vitamin B12 receptor
- MSN
- mesoporous silica nanoparticle
- MSN@PLA-mPEG-CPP
- mesoporous silica nanoparticles modified with poly(lactic acid)-methoxy ply(ethylene glycol) and decorated with cysteine-modified low molecular weight protamine cell penetrating peptide
- NP
- nanoparticle
- NPC1L1
- Niemann-Pick C1-like protein 1
- PAT1
- human proton-coupled amino acid transporter
- PCB
- polycarboxybetaine
- PCFT
- proton-coupled folic acid transporter
- PEG
- polyethylene glycol
- PLGA
- polylactide-co-glycolide
- RFC
- reduced folate carrier
- SGLT-1
- sodium-glucose cotransporter 1
- T1R2
- taste 1 receptor member 2
- T1R3
- taste 1 receptor member 3
- Tf
- transferrin
- TfR
- transferrin receptor
- TMC
- trimethyl chitosan.
- Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics