Glucose-responsive oral insulin delivery platform for one treatment a day in diabetes

Abstract

•Glucose-responsive oral insulin platform to achieve one treatment a day•Protect the integrity and bioavailability of insulin against the harsh GI tract•Overcome the intestinal epithelial barriers via FcRn-mediated epithelial transport•On-demand release of insulin dependent solely on blood glucose levels Insulin delivery systems have gained much attention in the treatment of type 1 and advanced type 2 diabetes mellitus. Among them, non-invasive delivery, such as oral delivery, has the intrinsic affinity to patient compliance and convenience. In this work, we present a closed-loop oral insulin platform not only to overcome the harsh GI tract, but also to provide patients with improved compliance and reduced risk of hypoglycemia. This glucose-responsive FcRn-targeted oral insulin platform was capable of regulating BGLs with extended efficacy to one treatment a day, which exhibited potential for clinical translation and broadened the design strategy of oral delivery of biologics in diabetes mellitus and related complications treatment. Oral delivery of insulin is of great convenience in treating diabetes but is still subject to the harsh gastrointestinal tract. Herein, we developed an intelligent oral insulin platform based on glucose-responsive polymeric nanoparticles (NPs), which possess the following advantages: (1) protect the integrity and bioavailability of loaded insulin against gastrointestinal tract, (2) overcome the intestinal epithelial barriers via neonatal Fc receptor-mediated transport, and (3) on-demand release of insulin dependent solely on blood glucose levels to avoid hyperglycemia and hypoglycemia caused by unsafe doses of insulin. Notably, our oral NPs extended the therapeutic effect to up to 16 h in type 1 diabetic mice. To our knowledge, this is the longest effective time among such types of oral insulin platforms reported to date and may reduce the frequency of insulin administration to once daily. This platform might be useful for the oral delivery of biologics in treating diabetes and related complications. Oral delivery of insulin is of great convenience in treating diabetes but is still subject to the harsh gastrointestinal tract. Herein, we developed an intelligent oral insulin platform based on glucose-responsive polymeric nanoparticles (NPs), which possess the following advantages: (1) protect the integrity and bioavailability of loaded insulin against gastrointestinal tract, (2) overcome the intestinal epithelial barriers via neonatal Fc receptor-mediated transport, and (3) on-demand release of insulin dependent solely on blood glucose levels to avoid hyperglycemia and hypoglycemia caused by unsafe doses of insulin. Notably, our oral NPs extended the therapeutic effect to up to 16 h in type 1 diabetic mice. To our knowledge, this is the longest effective time among such types of oral insulin platforms reported to date and may reduce the frequency of insulin administration to once daily. This platform might be useful for the oral delivery of biologics in treating diabetes and related complications. Diabetes mellitus (DM), a life-long metabolic disease, is generally characterized by high blood glucose levels (BGLs).1Xiao Y. Tang Z. Wang J. Liu C. Kong N. Farokhzad O.C. Tao W. Oral insulin delivery platforms: strategies to address the biological barriers.Angew. Chem. Int. Ed. 2020; 59: 19787-19795Crossref PubMed Scopus (28) Google Scholar,2Shi S. Kong N. Feng C. Shajii A. Bejgrowicz C. Tao W. Farokhzad O.C. Drug delivery strategies for the treatment of metabolic diseases.Adv. Healthc. Mater. 2019; 8: 1801655Crossref PubMed Scopus (24) Google Scholar With the rapid growth in overall incidence and a trend toward increasing incidence in younger patients, DM has become a major human health problem. There are about 463 million patients worldwide and 4 million deaths every year due to severe complications related to DM.3Saeedi P. Petersohn I. Salpea P. Malanda B. Karuranga S. Unwin N. Colagiuri S. Guariguata L. Motala A.A. 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Eng. 2018; 4: 2007-2017Crossref PubMed Scopus (20) Google Scholar Nevertheless, the study on developing glucose-responsive platforms for oral insulin delivery is still in its infancy. Herein, we design an intelligent oral insulin platform based on a glucose-responsive polymer poly(l-glutamic acid-co-l-glutamyl phenylboronic acid pinacol ester) (P(GA-co-GAPBAPE)) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] (DSPE-PEG-Mal), which can be self-assembled into NPs directly in water without any involvement of organic solvents, and which protect loaded insulin against the harsh GI tract. Immunoglobulin G (IgG) Fc was further conjugated with DSPE-PEG-Mal, assisting in epithelial transport in the GI tract via specific Fc receptors expressed throughout the intestine (Scheme 1). In vitro transepithelial transport experiments demonstrated 2-fold higher transmembrane transport of insulin-loaded Fc-conjugated NPs (NP-Fc) compared with NPs without Fc modification in a Caco-2 cell monolayer model. In vivo antidiabetic tests showed excellent hypoglycemia effects of insulin-loaded NP-Fc for at least 16 h, whereas free insulin can maintain normal BGLs for only a maximum of 4 h. As drastic changes in BGLs happen mainly during the daytime after meals, 16 h of effective time is potentially long enough for one treatment a day in diabetes treatment. Overall, this glucose-responsive oral insulin platform, with the longest effective time reported to date, holds great promise in clinical applications, and might also open new horizons for the oral delivery of biologics (e.g., various DNAs, RNAs, peptides, proteins) in the treatment of DM and related complications. The glucose-responsive copolymer P(GA-co-GAPBAPE) was obtained by grafting 4-aminophenylboronic acid pinacol ester onto poly-l-glutamic acid (PGA) polymer via amidation (Figure S1). In brief, the carboxyl groups of PGA were first activated by N-hydroxysuccinimide (NHS), followed by adding N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 4-aminophenylboronic acid pinacol ester. The mixture was then reacted under vigorous stirring at room temperature (RT) for 24 h and the excess monomer was removed by dialysis for 3 days. The chemical structure was characterized by 1H NMR spectra (Figure S2) both in DMSO-d6 and D2O. The peaks at 7.20 and 7.62 ppm indicated the successful modification with PBAPE and the degree of PBAPE was calculated to be 38, finally yielding P(GA62-co-GAPBAPE38). The NPs were prepared by self-assembly of P(GA-co-GAPBAPE) and DSPE-PEG-Mal directly in water, without any organic solvents. During the co-self-assembly process, the hydrophobic DSPE tails were inserted into the hydrophobic polymer core, while the hydrophilic PEGs were distributed outside the NP surface (Scheme 1). Dynamic light scattering (DLS) was used to assess the size of NPs, and transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to visualize the morphology of NPs. As shown in Figures 1A and S3A, DLS showed an average diameter of 79 nm with a narrow polydispersity of 0.280. The vesicular structure was investigated by TEM, which revealed typical collapsed NPs, indicating a hollow vesicular structure (Figure S3B). SEM images in Figure S3C also confirm the vesicular structure, where partially collapsed NPs were observed. The phenylboronic acid derivate PBAPE block acts as a glucose-responsive compound, and can prevent the leakage and enzymatic hydrolysis of payload in the GI tract. The stability of NPs under both acidic and neutral pH was tested, as they mimic the extremely acidic environment in the stomach and physiological environment in the human blood circulation. As shown in Figure 1B, the DLS study exhibited excellent stability of the NPs at both pH 1.0 and 5.5 with negligible changes in diameter, which indicated that the NP could protect the payload from leakage caused by gastric acid and digestion by physiological enzymes. The TEM image in Figure 1C demonstrates the solid structure of NPs in pH 1.0 PBS. In contrast, the NP exhibited obvious size change under pH 7.4 resulting from the deprotonation of PGA, which assisted in the release of payload under systemic circulation. TEM images of NPs with fused membranes and larger size were observed, further demonstrating the change of NPs under pH 7.4 (Figure 1D). PBAPE can covalently bind with 1,2- and 1,3-diols, such as glucose, with an increase in hydrophilicity, which in turn affects the structure of NPs and contributes to glucose responsiveness.47Liu X. Li C. Lv J. Huang F. An Y. Shi L. Ma R. Glucose and H2O2 dual-responsive polymeric micelles for the self-regulated release of insulin.ACS Appl. Bio Mater. 2020; 3: 1598-1606Crossref Scopus (17) Google Scholar The glucose responsiveness was first confirmed by TEM, where unsolid NPs with fused membrane were observed (Figure 1E). Then, the responsive release of the payload was further performed. The commercial dye Alexa Fluor 488 (AF488) was used to study the release profile. TEM and DLS studies were performed to show that loading of AF488 had little influence on the morphology or size of NPs (Figures 1A and S4). The standard curve of AF488 was first measured by fluorescence spectrum (Figure S5). The release profile was assessed in different glucose solutions, i.e., 100, 500, and 1,000 mg/dL. As presented in Figure S6, both the release content and rate increased as the external glucose concentration increased, indicating the glucose responsiveness of these NPs. In addition, the release content in 1,000 mg/dL glucose solution (extremely high BGL) was almost 2-fold higher than that in 100 mg/dL glucose solution (normal BGLs), further demonstrating the glucose-responsive release of AF488. Furthermore, insulin was loaded into NPs at a drug loading content of 32.8% and drug encapsulation efficiency of 49.2%, as calculated by UV-vis spectrum (Figure S7). The high loading efficiency was attributed to the vesicular structure of polymersome, where hydrophilic insulin can be encapsulated both inside and outside the NP core (structure shown in Scheme 1). Loading of macromolecules only increased the size of NPs to 261 nm without any change in morphology (Figures 1A and S8) as the vesicular structure was also observed in the TEM images. Cumulative release of insulin was also assessed in 100, 500, and 1,000 mg/dL glucose solutions, representing normal and high glucose levels. Rapid insulin release was noted in the 1,000 mg/dL glucose solution. As shown in Figure 1F, around 40% of insulin was released in 1,000 mg/dL glucose solution (extremely high BGL) within 1 h, whereas less than 20% of insulin was released in 100 mg/dL glucose (normal BGL), confirming glucose-triggered insulin release. In addition, continuous insulin release was also observed at high glucose levels, with more than 80% and 50% of insulin released in 1,000 and 500 mg/dL glucose solutions (hyperglycemia condition), respectively. However, in a low-glucose environment, release tended to level off after 1 h and only 30% of insulin had been released after 24 h. Furthermore, the "on-off" insulin release experiment was carried out to investigate the closed-loop insulin delivery. The Ins/NP was firstly immersed in 500 mg/dL glucose solutions for 1 h ("on" state) and then switched to PBS for 1 h ("off" state). As indicated in Figure 1G, the release was fast in the presence of glucose, and slowed down without glucose. The on-off release was repeated for three cycles, indicating a self-regulated insulin release behavior in response to the glucose levels. Overall, both the AF488 and insulin release experiments indicated the glucose-responsive behavior of NPs and the controlled release of payload. The intestinal mucosal barrier protects immunity by preventing pathogens from passing into the systemic circulation. Unfortunately, NPs are also stopped by the intestinal mucosal barrier. Thus, the use of targeted molecules is being considered to modify NPs to overcome this barrier and achieve better permeability. Neonatal Fc receptor (FcRn) is an Fc receptor that binds IgG under acidic pH via electrostatic interaction and mediates the transcytosis pathway across the intestinal mucosal barrier.48Sockolosky J.T. Szoka F.C. The neonatal Fc receptor, FcRn, as a target for drug delivery and therapy.Adv. Drug Deliv. Rev. 2015; 91: 109-124Crossref PubMed Scopus (124) Google Scholar Therefore, a polyclonal IgG Fc fragment was covalently conjugated with DSPE-PEG-Mal via maleimide-thiol chemistry to improve the intestinal transport efficiency of NPs. The amount of conjugated Fc on the surface of NPs (NP-Fc) was measured by bicinchoninic acid (BCA) assay (Figure S9), confirming 72.7% modification and a 3.125% weight ratio to NP. In addition, modification of Fc barely changed the morphology (Figure S10). The average size of NP-Fc was measured to be 84 nm. The stability of NP-Fc in Dulbecco's modified Eagle's medium (DMEM) was accessed. As displayed in Figure S11, NP-Fc remained almost stable in pH 1.0 DMEM, whereas larger size and loose structure were observed in pH 7.4 DMEM, further conforming the stability of NP-Fc at low pH. The cellular internalization of NP-Fc was tested using Caco-2 cells, a human colon adenocarcinoma cell line commonly used to simulate the intestinal mucosal barrier in oral drug permeability tests. Both AF488- and green fluorescent protein (GFP)-loaded NP-Fcs (i.e., AF488/NP-Fc and GFP/NP-Fc) were used to study the cellular uptake behavior. AF488- and GFP-loaded NPs without Fc conjugation (i.e., AF488/NP and GFP/NP) served as controls. As presented in Figure S12, no obvious cell uptake of AF488 was seen in the AF488/NP group even after 24 h of incubation. However, for AF488/NP-Fc, the intracellular green fluorescence signals were observed in 2 h, indicating fast NP internalization. After 24 h, distinct fluorescent signals in the cells were observed, showing a significant difference from that of AF488/NP. The cellular uptake and intracellular release of macromolecule-loaded NPs were further studied using GFP/NP-Fc. DLS study and TEM images also indicated an increased size of NP after loading with GFP (Figure S13), which was similar to Ins/NP. Cells were imaged by both inverted fluorescence microscopy and confocal laser scanning microscopy (CLSM) after incubation with either GFP/NP or GFP/NP-Fc for a predetermined time. As presented in Figure S14, there was no obvious fluorescent signal in either group after 2 h. Internalization occurred after 6 h incubation with GFP/NP-Fc, and intense signals were widely dispersed after 24 h incubation. However, little fluorescent signal of GFP/NP was observed after 10 h incubation and the signals remained weak after 24 h. CLSM was further employed to investigate Fc-mediated cellular uptake. As shown in Figure 2A , GFP/NP-Fc was successfully internalized by Caco-2 cells, as indicated by the bright green fluorescence. In contrast, slight green fluorescence was detected in GFP/NP-treated Caco-2 cells, consistent with the results using AF488-loaded NPs. The relative green fluorescence was calculated, and the