How Can Biomolecules Improve Mucoadhesion of Oral Insulin? A Comprehensive Insight using Ex-Vivo, In Silico, and In Vivo Models

biomolecules Article How Can Biomolecules Improve Mucoadhesion of Oral Insulin? A Comprehensive Insight using Ex-Vivo, In Silico, and In Vivo Models Mariana Amaral 1,†, Ana Sofia Martins 1,†, JoséCatarino 2, Pedro Faísca 2, Pradeep Kumar 3, João F. Pinto 1, Rui Pinto 1,4, Isabel Correia 5, Lia Ascensão6, Ricardo A. Afonso 7,8,9, M. Manuela Gaspar 1, Adília J. Charmier 10 , Isabel Vitória Figueiredo 11,12 and Catarina Pinto Reis 1,13,* 1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal; [email protected] (M.A.); [email protected] (A.S.M.); [email protected] (J.F.P.); [email protected] (R.P.); [email protected] (M.M.G.) 2Faculdade de Medicina Veterinária, Universidade Lusófona de Humanidades e Tecnologias/DNAtech Laboratório Veterinário, 1749-024 Lisboa, Portugal; [email protected] (J.C.); [email protected] (P.F.) 3Department of Pharmacy and Pharmacology, School of Therapeutic Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2193, South Africa; [email protected] 4JCS. Dr. Joaquim Chaves, Laboratório de Análises Clínicas, 1495-068 Miraflores-Algés, Portugal 5Centro de Química Estrutural, Departamento de Engenharia Química, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal; [email protected] 6Centro de Estudos do Ambiente e do Mar (CESAM), Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal; [email protected] 7 CEDOC, NOVA Medical School/Faculdade de Ci ê ncias M é dicas (NMS/FCM), Universidade Nova de Lisboa, 1150-082 Lisboa, Portugal; [email protected] 8Ciências Funcionais e Alvos Terapêuticos, NOVA Medical School, Faculdade de Ciências Médicas (NMS|FCM), Universidade Nova de Lisboa, 1169-056 Lisboa, Portugal 9Departamento de Física, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 10 DREAMS, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749-024 Lisboa, Portugal; [email protected] 11 Pharmacology & Pharmaceutical Care, Faculty of Pharmacy, Universidade de Coimbra, 3000-548 Coimbra, Portugal; [email protected] 12 Coimbra Institute for Clinical and Biomedical Research (iCBR), University de Coimbra, 3000-370 Coimbra, Portugal 13 IBEB, Biophysics & Biomedical Engineering, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal *Correspondence: [email protected]; Tel.: +351-217-946-429 (ext. 14244) †These authors contributed equally to this work. Received: 30 March 2020; Accepted: 22 April 2020; Published: 27 April 2020   Abstract: Currently, insulin can only be administered through the subcutaneous route. Due to the flaws associated with this route, it is of interest to orally deliver this drug. However, insulin delivered orally has several barriers to overcome as it is degraded by the stomach’s low pH, enzymatic content, and poor absorption in the gastrointestinal tract. Polymers with marine source like chitosan are commonly used in nanotechnology and drug delivery due to their biocompatibility and special features. This work focuses on the preparation and characterization of mucoadhesive insulin-loaded polymeric nanoparticles. Results showed a suitable mean size for oral administration (<600 nm by dynamic laser scattering), spherical shape, encapsulation efficiency (59.8%), and high recovery yield (80.6%). Circular dichroism spectroscopy demonstrated that protein retained its secondary structure after encapsulation. Moreover, the mucoadhesive potential of the nanoparticles was assessed in Biomolecules 2020,10, 675; doi:10.3390/biom10050675 www.mdpi.com/journal/biomolecules Biomolecules 2020,10, 675 2 of 20 silico and the results, corroborated with ex-vivo experiments, showed that using chitosan strongly increases mucoadhesion. Besides, in vitro and in vivo safety assessment of the final formulation were performed, showing no toxicity. Lastly, the insulin-loaded nanoparticles were effective in reducing diabetic rats’ glycemia. Overall, the coating of insulin-loaded nanoparticles with chitosan represents a potentially safe and promising approach to protect insulin and enhance peroral delivery. Keywords: marine-derived biomolecules; diabetes mellitus; insulin; mucoadhesion; nanoparticle; oral delivery 1. Introduction Diabetes mellitus is a group of metabolic disorders that arise from defective action and/or secretion of insulin, resulting in hyperglycemia [ 1 ]. Type 1 diabetes is characterized by the life-long need of exogenous insulin replacement. These patients have the need to self-administer long-acting insulin in order to establish basal levels, and short-acting insulin before meals [ 2 , 3 ]. Insulin administration is done by subcutaneous injection or by constant subcutaneous infusions [ 3 ]. Although this route is considered the only option for insulin therapy, and very efficient and broadly used, there are drawbacks and disadvantages associated with its usage [4,5]. Some of these include lipoatrophy and lipohypertrophy on the injection site and discomfort [ 6 ]. In addition, all tissues are being exposed to equal quantities of insulin [ 7 ], being that insulin reaches the muscles and adipocytes prior to the liver [ 8 ], with only around 20% of the administered insulin reaching this target organ [ 9 ]. The peripheral hyperglycemia might lead to unwanted overstimulation of the metabolic responses [ 6 ]. When insulin is administered subcutaneously, it is first distributed to the peripheral tissues [ 10 ]. Thus, this route of administration does not mimic the endogenous insulin produced by the islets of Langerhans by non-diabetics. When administered orally, exogenous insulin is absorbed in the intestine, reaching the liver through the portal vein and inhibiting hepatic glucose output, mimicking the physiological pathway by undergoing hepatic first passage [ 10 , 11 ]. Although oral delivery of insulin is the most comfortable and convenient route for the patient, it also has difficulties associated with its usage due to its protein nature [ 12 ]. Such difficulties include poor absorption by the intestine epithelium due to insulin’s hydrophilicity and large dimensions, as well as insulin degradation due to the pH and enzymes in the stomach and small intestine [10,13], leading to low bioavailability [14]. To this date, many efforts have been done to try to improve oral administration of insulin, including the use of nanoparticles (NPs) [ 15 , 16 ]. NPs may overcome the mentioned difficulties by protecting the protein drug from the hostile conditions of the gastrointestinal tract (GIT) and improving its absorption [ 14 ]. This can be achieved by adjusting the surface charge, shape, size, and hydrophobicity of the NPs, and other characteristics [ 17 ]. Furthermore, the use of NPs allows control over the drug release [ 17 , 18 ]. Previous reports have shown that using synthetic and/or natural polymers to nanoencapsulate insulin improves its absorption in the intestine [ 17 , 19 ]. Moreover, by choosing the correct materials to prepare the nanoformulation, certain beneficial characteristics can be achieved. The developed insulin formulation for oral delivery entails insulin-loaded poly (D, L-lactic-co-glycolic acid) (PLGA) NPs coated with chitosan and polyethylene glycol (PEG), with an external coating composed of bovine serum albumin (BSA). PLGA is widely used as a nanocarrier, because it is biodegradable and, when in combination with polyethylene glycol (PEG), allows for longer plasmatic circulation time [ 20 ]. The addition of (BSA), as an outer coat of the NPs, acts as a protective layer against proteolytic enzymes of the GIT, allowing insulin to reach systemic circulation intact and increasing its bioavailability at its absorption site [ 21 – 23 ], i.e., the intestine. But one of the most important biomolecules in this work comes from the sea. In this case, the ocean has been shown to provide a rich place with great biodiversity and chemical entities with proven bioactivities. Chitosan is generally derived from the shells of shrimp and other sea crustaceans and it acts as a permeability enhancer Biomolecules 2020,10, 675 3 of 20 by opening the tight junctions of the intestinal epithelium, facilitating paracellular and transcellular transport [ 23 – 25 ]. Moreover, chitosan prolongs the residence of time of chitosan-coated formulations in mucosae, through its interactions with mucins [26,27]. This work focuses on preparation and characterization of double-coated insulin-loaded NPs by using the following techniques: dynamic light scattering and electrophoretic mobility for size and surface charge analysis, respectively; scanning electron microscopy to assess the NPs’ surface; HPLC for determination of encapsulation efficiency; circular dichroism to evaluate insulin’s activity after the encapsulation and ex-vivo and in silico studies to access the mucoadhesion. Safety assessment was in vitro preliminarily assessed using cells and then by in vivo using animal models. Finally, the efficacy of the formulation was in vivo assessed by evaluating the effect of glycemia in diabetic rats following oral administration of the double-coated insulin-loaded NPs. 2. Materials and Methods 2.1. Materials 2.1.1. Chemicals Pluronic ® F167 (POLX), pepsin (250 IU/mL), BSA (MW 66 kDa) and PEG 4000 were acquired from Sigma-Aldrich (St. Louis, MO, USA). PURASORB ® PDLG 5002PLGA Ratio L/G% 50:50 (MW 45,000–75,000 Da) was purchased from Purac (Gorinchem, The Netherlands). Chitosan from crab shells with low molecular weight (Aldrich), 75–85% deacetylated, was used [ 27 ]. The insulin used was Insuman Rapid (Sanofi, Paris, France), a fast-acting insulin, at a concentration of 100 IU/mL. Water MiliQ by Millipore Corporation (Burlington, MA, USA). All the chemical products and solvents used are of analytic purity grade. 2.1.2. Animals Male Wistar rats 8–10-weeks old, with an average weight of 200 g, were purchased from Charles River (Barcelona, Spain). Males were chosen over female Wistar rats due to the potential influence of female hormones over insulin sensitivity [ 28 ]. The animal housing was kept at the controlled temperature of 22.0 ± 1.0 ◦ C, humidity at 50.0 ± 15.0% and a cycle of light of 12 h. Animals were kept under standard hygiene conditions, fed with commercial chow and given acidified drinking water ad libitum. The feed was removed 12 h prior to the day of treatment. All animal experiments (Protocol title: Improvement of insulin oral availability through encapsulation in polyelectrolyte complex nanoparticles, n. ◦ POCI/SAU-FCF//59940) was conducted in accordance with the EU Directive (2010/63/UE), the Portuguese law (DR 113/2013, 2880/2015 and 260/2016) and the Animal Welfare Commission of the Faculty of Pharmacy, University of Coimbra, approved by Ethics Committee of the Faculty of Pharmacy, University of Coimbra and by the competent national authority Direcção-Geral de Alimentação e Veterinária (DGAV). 2.2. Methods 2.2.1. Preparation of NPs The NPs were prepared according to the modified-spontaneous emulsification solvent diffusion method [ 29 ]. An organic solution containing PLGA and insulin was prepared in a non-aqueous solvent mixture. The latter suspension was gradually added to an aqueous solution containing a surfactant at 0.1%, POLX, dissolved in water, at pH 4.5, at room temperature (25 ◦ C) and stirred at 800 rpm (Heidolph MR3001, Heidolph Instruments, Schwabach, Germany), for 15 min [ 30 ]. All parameters were considered based on previous studies reported [ 29 , 30 ]. Next, the NPs were coated with a chitosan aqueous solution (0.03% w/v, previously solubilized with glacial acetic acid at 1%) enriched with PEG (0.150%, w/v), by mixing the previous solution with an aqueous solution containing these compounds. This was done at room temperature, and the NPs were constantly stirred at a speed of 180 rpm, Biomolecules 2020,10, 675 4 of 20 for 30 min. Then, the coated NPs were re-coated with a BSA aqueous solution, at a concentration of 1 g/mL, by stirring the chitosan-NPs solutions with this aqueous solution at a speed of 100 rpm, for 30 min. After the final coating, the formulation was centrifuged at 10.000 × g, for 15 min (Beckman Instruments centrifuge, Inc., Brea, CA, USA), in order to remove all the reagents that did not react. 2.2.2. NPs Characterization Mean Size, Polydispersity Index (PI), and Zeta Potential Analysis The uncoated NPs, chitosan-coated NPs and double-coated NPs were characterized regarding their mean particle size, polydispersity index (PdI) and surface charge as zeta potential. Particle size and PdI were measured in diluted samples with water MiliQ (1:10, v/v) using Dynamic Light Scattering (Zetasizer Nano S, Malvern Instruments, Malvern, Worcestershire, UK), and performed in triplicates. Zeta potential was analyzed using an electrophoretic mobility assay using the same equipment, using NPs diluted in water MiliQ (same dilution). Surface and Morphological Analysis The morphology of single-coated and double-coated NPs was observed by Scanning Electron Microscopy (SEM). Aliquots (10 µ L) of particle suspensions were scattered over round glass coverslips coated with poly L-lysine, that were previously attached with a double face tape to the microscope stubs. The samples, after dying in a desiccator, were coated with a thin layer of gold and observed on a JEOL 5200 LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) at 20 kV. The images were digitally recorded. 2.2.3. Determination of Encapsulation Efficiency (EE) The percentage of insulin encapsulated was determined indirectly by quantifying the amount of insulin present in the supernatant after centrifugation of samples (10.000 × g, 15 min; using a Beckman Instruments centrifuge, Inc., Brea, CA, USA). This quantification was done by HPLC (Hitachi System LaCrom Elite, Column oven, Diode Array Detector Uv-vis and Pump, Tokyo, Japan), using a Column Waters Symmetry C18, 5 µ m 4.6 × 150 mm, with an isocratic flow of 0.7 mL/min. The mobile phase was composed by acetonitrile:TFA water (60:40) (v/v). The measurements were performed in triplicates and the calibration was done with a standardized solution of insulin, at 220 nm wavelength. The linearity range was established in the 1.09–70 µ g/mL range and the detection limit was 0.359 µ g/mL and the quantification limit was 1.087 µ g/mL. The retention time was equal to 3.1 min. Encapsulation efficiency (EE, %) was then determined by using Equation (1): EE(%)=(Initially added insulin −insulin present in supernatant) Initially added insulin ×100 (1) 2.2.4. Determination of Recovery Yield (RY) NPs were recovered after being centrifuged and lyophilized at − 49 ◦ C for at least 48 h (Freezone 2.5 L, Freeze-dryer Labconco, Kansas City, MO, USA). Next, NPs were stored at 4 ◦ C, according to previous works [29]. The recovery yield (RY, %) was determined using Equation (2): RY (%)=final mass of nanoparticles mass of components used in formulation ×100 (2) Biomolecules 2020,10, 675 5 of 20 2.2.5. Insulin Activity Circular Dichroism (CD) The secondary structure of insulin was analyzed by CD spectroscopy. Circular dichroism (CD) spectra were recorded on a JASCO J-720 spectropolarimeter (JASCO, Hiroshima, Japan) with a 180–700 nm photomultiplier (EXEL-308). CD spectra were recorded in the far UV range from 260 to 200 nm with quartz Suprasil ® CD cuvettes (0.1 cm). The measurements were done at ~23 ◦ C in a room with controlled temperature. Each CD spectrum is the result of six accumulations recorded in degrees. The following acquisition parameters were used: data pitch, 0.5 nm; bandwidth, 2.0 nm; response, 2 s and scan speed, 50 nm/min. Samples for CD analysis were obtained after disrupting the chitosan-coated insulin-loaded NPs in PBS (USP 30), pH 7.4 and ultrasounds. The insulin concentration of every sample was normalized to 1 mg/mL and compared with equal concentrations of non-encapsulated insulin. The CD signal values obtained at 208 nm were used to estimate the α -helical (%) content of the protein [ 30 , 31 ]. CD measurements were expressed as the mean residue ellipticity (MRE in deg cm2dmol−1), calculated from Equation (3): MRE =CD (mdeg) Cp ×N×l(3) where N is the number of amino acid residues (51 for insulin), l is the length of the optical path (0.1 cm) and C p is the concentration of the protein. The α -helical content (%) is calculated from the MRE values at 208 nm, using Equation (4): α−helix (%)=−(MRE208nm −4000) (33000 −4000)×100 (4) 2.2.6. In Vitro Release Assay A specific amount (10 mg) of double-coated insulin-loaded NPs were placed in 50 mL HCl (pH 1.2), simulating gastrointestinal conditions, and always respecting sink conditions according to insulin’s solubility. The assay was performed at 37 ◦ C with continuous stirring (100 rpm), in a magnetic multiplate (Heidolph MR3001, Heidolph, Schwabach, Germany), for 2 h. Aliquots (1 mL) were collected at 0.25, 0.5, 1, and 2 h and replaced using fresh medium to have a constant final volume. After this time period, the NPs were centrifuged ( × g, 10 min) and the pellet was transferred to PBS at pH 6.8. Release assay continued at a speed of 100 rpm, at 37 ◦ C, for 6 h. Aliquots (1 mL) were collected at 0.25, 0.5, 1, 2, 4, and 6 h, when the assay experiment was stopped. At the determined time points, the aliquots were collected and centrifuged (1500 × g, 10 min), and the pellet was resuspended in the medium solution and returned to the release medium. The aliquots were analyzed and the concentration of insulin was determined by HPLC following the method previously described, in triplicate, and according to Equation (5) [29,31,32]: Released Insulin (%)=Cn V +Vi Pn−1 i=0Ci Total mass o f the particles X drug content ×100 (5) where Cn was insulin concentration at time n (time points), V was total volume of medium, Vi was volume of sample collected at time i, and Ci was concentration of insulin of sample collected at time i (initial time point). 2.2.7. Ex-Vivo Mucoadhesion Study A TA-XTPlus Texture Analyser (Stable Micro Systems, Godalming, UK) equipped with a 5 k load cell was used for mucoadhesion tests [ 33 ]. A fresh Wistar chemically-induced diabetic rat (explained in section in vivo efficacy assay) small intestine was harvested and opened longitudinally, cleaned and Biomolecules 2020,10, 675 6 of 20 cut into pieces that fit the movable cylindrical probe with the lumen side facing outwards, attached by a double-face tape to the probe. The rat intestine was also placed in a static holder aligned with the probe, also attached by double-face tape. Between the intestine’s portions and in contact with the stationary part, the suspensions with NPs were placed: non-encapsulated insulin, uncoated NPs and double-coated NPs after digestion with pepsin, in equal concentrations of insulin. The double-coated NPs were incubated with pepsin, for 2 h at 37 ◦ C, in order for the albumin to be digested, according to Pharmacopeia USP. The simulated gastric fluid was composed of 3.2 g/L of pepsin (with activity of 800 to 2500 units per mg of protein), sodium chloride, and hydrochloric acid. During the experiment, the movable part was lowered, until coming into contact with the mucosa, up to a force of 20 gf, and then raised at a constant speed of 0.25 mm/s. The displacement and the forces of compression and detachment were recorded. A curve of force (gf) versus time (s) was obtained for each experiment and the peak force of displacement (F max , gf) and area of the peak (AUC, gf.s) were acquired from this data. The experiments were performed five times for each sample. 2.2.8. In Silico Mucoadhesion Analysis For mucoadhesion analysis of chitosan-coated NPs versus uncoated PLGA NPs, energetic and geometric stability of the polymer-mucin molecular complexes were determined using static lattice atomistic simulations (molecular mechanics simulations; Chemlite30, Hypercube Inc., Gainesville, FL, USA). The structures of PLGA and PEG were generated as natural bond angles while the ones of chitosan and glycosylated mucin (MUC) were generated using the saccharide building and sequence editor tools, respectively [ 34 ]. The individual molecules (PLGA, PEG, chitosan, and MUC) as well as the molecular complexes (PLGA-MUC and chitosan/PEG-MUC) were energy minimized and optimized using MM+Force Field algorithm. For geometrical optimization, a Polak–Ribiere Conjugate Gradient method was employed until an RMS gradient of 0.001 kcal/mol was achieved [35]. 2.2.9. Preliminary Safety Assessment In Vitro Assessment The safety of double-coated insulin-loaded NPs was assessed in vitro by a MTT assay performed in Caco2 cells, a human intestinal cell line commonly used for this type of assessment. These cells were kept in Dulbecco’s Modified Eagle’s medium (DMEM) high-glucose (4.5 g/L), supplemented with 10% fetal bovine serum and 100 IU/mL of penicillin and 100 µ g/mL streptomycin (hereafter complete medium). Cells were maintained at 37 ◦ C, with a 5% CO 2 atmosphere, and checked every 2 to 3 days, until a confluence of 80% was reached. Then, the cells were seeded in 96-well plates, at a concentration of 5.0 × 10 4 cells/mL. The cells were incubated with double-coated insulin-loaded and empty NPs, as well as non-encapsulated insulin. The concentrations tested, for both free and nanoencapsulated insulin, ranged from 0.0625 to 1 IU/mL, and the equivalent was tested for double-coated empty NPs. After 24 h, complete medium was removed, the cells were washed with phosphate buffered saline (PBS) and the MTT solution in incomplete medium (0.5 mg/mL) was added. The cells were incubated for 4 h. After the incubation time, Dimethyl Sulfoxide (DMSO) was added in order to dissolve the formazan crystals. Absorbance was measured at 590 nm using a BioTek ELx800 Absorbance Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). In Vivo Preliminary Safety Assessment The in vivo preliminary safety assessment was performed in 18 male Wistar rats, weighing approximately 200 g. The animals were randomly separated into five groups: The test group (n =5), to which 50 IU/kg of double-coated insulin-loaded NPs was orally administered; the vehicle control group (n =5), which received empty double-coated NPs, by oral gavage; the negative control (n =2), which received PBS orally; the oral insulin control (n =3), that received 50 IU/kg of commercial insulin Biomolecules 2020,10, 675 7 of 20 not incorporated in NPs, orally; and finally, the control for insulin’s activity (n =3), to which 4 IU/kg of commercial insulin was subcutaneously administered. After 6 h, urine samples were collected from all animals. The animals were then euthanized, plasma was collected, for hematological and biochemical analyses, and spleen, stomach, liver, intestine and kidney were harvested for histologic analysis. The organs were fixed in 10% formalin and embedded in paraffin. Five micrometer sections of each organ were prepared for hematoxylin-eosin staining. The stained slices were examined under an Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan) and images were captured with NanoZoomer-SQ Digital slide scanner (Hamamatsu Photonics, Hamamatsu City, Japan). The urine samples collected were tested for leukocytes, urobilinogen, bilirubin, hematuria, nitrites, pH, density, proteinuria, glycosuria, and ketonic bodies, using Uritest 10 V Urinalysis Reagent Strips. Plasma samples were tested to quantify IL-6, ALT (to determine liver toxicity), creatine and urea (to determine kidney toxicity). 2.2.10. In Vivo Efficacy Assay Diabetes Mellitus Induction In order to chemically induce diabetes mellitus in the male Wistar rats, streptozotocin (STZ), prepared in citrate buffer 0.1 M at pH 4.5 (UPS 30), was administered intraperitoneally (i.p., 65 mg/kg of body weight). After this procedure, animals were exposed to a 5% glucose solution during the night, in order to avoid reactional hypoglycemia, caused by the STZ. The animals were classified as diabetic when glycemia was higher than 300 mg/dL, measured on the third day post-administration [ 31 , 36 ]. The experimental protocol was started 10 days after STZ administration [29,36]. Study Design For the in vivo efficiency assay (n =14), animals were randomly divided into three groups: The test group (n =5), in which 50 IU/kg of NPs formulation was orally administered; the negative control group (n =3), to which empty NPs were administered; in the third group, insulin was administered orally (n =6). The formulation efficacy is translated by decreasing glycemia, glycemia was measured at the following time points: 30 min, 1, 2, 4, 6, and 8 h. Glycaemia levels were determined by measuring glucose oxidase/peroxidase, using a glucosometer (OneTouch ® Verio ® IQ, Milpitas, CA, USA). The animals were then sacrificed. The plasma to which the double-coated insulin-loaded NPs were administered was collected and its insulinaemia was determined by electrochemiluminescence immunoassay/(Roche-Cobas®). 2.2.11. Statistical Analysis Each value is presented with a mean value ± SD. The statistical differences were evaluated with t-Student test and ANOVA. These tests allow to compare two or multiple groups, respectively. All analyses were conducted in GraphPad Prism Version 5.03 (GraphPad Software, San Diego, California, USA) and the differences were deemed significate at a p<0.05. 3. Results 3.1. NPs Characterization: Size, Surface Charge, PI, Morphology, EE, and RY Mean size and PdI are shown in Table 1. Insulin-loaded NPs have a larger mean particle size than empty NPs. It is assumed that the increase in particle size was related to the encapsulation of insulin. Besides the particle size, there are other important parameters that contribute to an increase absorption of the NPs in the intestinal mucosa, such as NPs charge. This property was evaluated by measuring the NPs zeta potential in all phases of production, also shown in Table 1. A representative scheme of insulin NPs is displayed in Figure 1. The uncoated empty and uncoated insulin-loaded NPs (Figure 1A) showed a negative surface charge. This value was coherent with the PLGA charge Biomolecules 2020,10, 675 8 of 20 at the considered pH, as previously reported [ 37 ]. The NPs charge was inverted to positive, in both empty NPs and insulin-loaded NPs by coating the NPs with chitosan, a cationic polymer, and PEG (Figure 1B). The final formulation was obtained after the BSA coating. The charge of the NPs coated with BSA, both empty and insulin-loaded double-coated NPs (Figure 1C), remained positive, although slightly less positive. These results were as expected and coherent with what was described in previous studies [23,38]. Table 1. Mean size, PdI and zeta potential throughout the different steps in formulation, for both empty and insulin-loaded NPs. All data is presented as mean ±SD (n =3). Sample Mean Size (nm) PdI Mean Zeta Potential (mV) Empty NPs Uncoated NPs 240 ±2 0.110 ±0.016 −39 ±6 Chitosan-coated NPs 328 ±2 0.231 ±0.015 +48 ±8 Double-coated NPs 233 ±2 0.184 ±0.016 +34 ±8 Insulin-loaded NPs Uncoated NPs 936 ±4 0.449 ±0.023 −49 ±5 Chitosan-coated NPs 819 ±7 0.527 ±0.028 +41 ±13 Double-coated NPs 560 ±9 0.546 ±0.030 +31 ±3 Figure 1. Double-coated insulin-loaded nanoparticles (NPs) throughout the formulation and coating processes: ( A ) uncoated insulin-loaded poly (D, L-lactic-co-glycolic acid) (PLGA) NPs; ( B ) chitosan-coated insulin-loaded PLGA NPs; and (C) double-coated insulin-loaded PLGA NPs. Figure 2shows the images obtained by scanning electron microscopy for both double-coated empty and insulin-loaded NPs, where NPs seem to have a well-defined spherical shape. Figure 2. SEM micrographs of double-coated NPs. ( A ) Empty NPs. ( B ) Insulin-loaded NPs. Note in both cases the well-defined NPs spherical shape. Scale bars =5µm. The EE was 59.8 ±2.6%, of the insulin used in the initial formulation. The RY was 80.6 ±1.1%. Biomolecules 2020,10, 675 9 of 20 3.2. Insulin Secondary Structure Circular dichroism (CD) was used to evaluate the secondary structure of nanoencapsulated insulin, after being released, which was shown to remain intact, as illustrated in Figure 3, where it is observed the characteristic spectra of a protein with an alpha helix structure. Figure 3presents CD spectra showing two peaks, which are characteristic of insulin’s CD spectrum, at 211 nm and 222 nm. An estimate of the α -helical content was done using the MRE at 208 nm. This yielded the following values 31%, 28%, and 27% for non-encapsulated insulin, uncoated NPs and chitosan-coated NPs, respectively. Thus, only a small decrease in the α -helical content was detected along the preparation and coating of NPs, suggesting no insulin fibrillation/aggregation among the conditions tested. Moreover, these findings were also supported by HPLC (observing the same retention time of insulin in all samples) and then after in vivo oral administration (Section 3.7). Figure 3. Circular dichroism (CD) spectra of non-encapsulated insulin ( ___ ), uncoated NPs (—) and chitosan-coated NPs ( . . . ) in the Far UV range. 3.3. In Vitro Release Assay This assay was performed in order to better understand insulin’s release profile in the gastrointestinal tract, and the results are shown in Figure 4. Temperature was kept at 37 ◦ C and stirring was always constant. Firstly, the nanoencapsulated insulin was kept in acidic medium and aliquots were collected up to 2 h. In the acidic medium simulating gastric conditions, chitosan is probably starting to be dissolved at acidic pH and insulin release was 25.6 ± 3.4% after 2 h. For the neutral medium (mimicking the intestine), NPs are less coated and insulin immediately started to release and it was 100% released after 4 h. Figure 4. Release assay for the double-coated insulin-loaded NPs in acidic medium until 2 h and conducted in neutral medium from 2 to 8 h, mimicking the pH range of the gastrointestinal tract (GIT). Biomolecules 2020,10, 675 16 of 20 stabilization was significantly higher for chitosan/PEG-MUC ( ∆ E ≈ − 54 kcal/mol) than for PLGA-MUC ( ∆ E ≈ − 7.5 kcal/mol). These results confirm the preferable mucoadhesive profile of chitosan/PEG-coated NPs as compared to the uncoated PLGA NPs, as predicted in the ex-vivo results. The development of suitable and biocompatible drug delivery systems is a prerequisite, especially for a chronic disease like diabetes [ 17 ]. MTT assay was done to assess cytotoxicity and exhibited good results as it demonstrated that double-coated insulin-loaded NPs did not alter cell viability. Regarding empty NPs, the lowest mean value of viability found was 85%. This value of cell viability of the empty NPs could be attributed to the NPs’ structure by itself as previously demonstrated [ 54 ], and it is not noticeable in insulin-loaded NPs possibly due to the insulin’s presence, since insulin can act as a growth factor, promoting cell growth. In vitro assays are extremely useful tools, but they cannot accurately predict the in vivo behavior of all formulations. Therefore, an in vivo preliminary safety assessment study was also performed. To assess NPs formulation safety when orally administered, animals’ behavior was closely monitored during the test. No death occurred after the treatment. When comparing the urine tests results, biochemical analysis and histological images of insulin-loaded NPs and negative control group (PBS group), it is displayed that there was no obvious damage to animals. Thus, in vivo studies results were in agreement with in vitro MTT assay, i.e., our insulin-loaded NPs are safe for oral administration. The presented research entails to develop an insulin formulation suitable for oral delivery as an alternative treatment for the commercially available subcutaneous insulin. Oral administration is considered as the best route of administration because of its cost-effectiveness and well-established acceptability. In addition, it allows avoiding the use of injections. An additional advantage is related to a more physiological action by its direct effect on hepatic glucose production. If the insulin would be absorbed in the gut, insulin would be transferred directly toward the liver. At the liver, the exogenously insulin would control hepatic glucose production to the same extent similarly as this is induced by endogenously insulin in healthy subjects. This more physiological delivery would be associated with reduced peripheral hyperinsulinemia in contrast to SC administration. In terms of pharmacokinetics, as representative example, in a small study made by Cernea et al. 2004 [ 55 ], male subjects under euglycemic conditions, oral insulin spray was associated with a higher Cmax, shorter T max , and faster time to peak glucose uptake compared with SC insulin. The short T max and the 120-min duration of effect of oral insulin spray suggest it may be a promising alternative for fulfilling meal-related insulin requirements in persons with diabetes. Another encouraging study was done with a few number of subjects with 8 mg of oral insulin formulation [ 56 ]. Administration of an oral form of insulin in the fasted state demonstrated a significant effect on insulin absorption. This substantial effect was seen by a reduction of blood glucose (7%–37%), decline in C-peptide levels (13%–87%), as well as an elevation of insulin level (20%–120%). It was noticed that some subjects developed symptomatic hypoglycemia. Insulin formulations were well tolerated. No adverse or serious adverse events have been reported. Therefore, we aimed to develop a new oral insulin formation and to evaluate if the developed formulation is effective when administered orally. The results showed that the formulation was effective in reducing glucose levels of diabetic rats (Figure 9). A previous study comparing the subcutaneous administration of 4 IU/kg insulin with oral administration of 50 IU/kg of nanoencapsulated insulin coated with chitosan and albumin in Wistar diabetic rats has shown that the NPs formulation achieved a reduction in glycemia of 28% between the 2nd and 4th hour, and 48% between the 8th and 12th hour [ 29 ]. When compared to other studied formulations, the formulation reported herein has shown preferable characteristics. Many of the previously developed formulations for oral delivery of insulin have negative surface charge [ 50 – 54 ], not favoring the NPs’ interactions with mucins, and thus not promoting adherence to the intestine’s mucosa [ 10 , 22 , 23 ]. Besides having a positive surface charge, the double-coated insulin-loaded NPs showed to interact with mucin, therefore proving its mucoadhesive properties, as observed both by the in silico simulations of molecular interaction conducted in vacuum, and by the ex vivo experiments. The double-coated formulation also showed lower insulin release in gastric-like conditions after 2 h (22.6%) than other formulations Biomolecules 2020,10, 675 17 of 20 intended for oral delivery of insulin with half and more than half of the encapsulated insulin being released in stomach-like conditions after two hours (~50% [ 57 ], ~60% [ 58 ]), suggesting that, unlike other formulations, our double-coated insulin-loaded NPs formulation has the potential to deliver insulin in the proper target—the gut. Moreover, our formulation was more efficient in lowering diabetic-rats than others: while our double-coated insulin loaded NPs induced a 50% glycemia decrease 8 h post-oral administration, other studied formulations reported smaller and later hypoglycemic effects, to less than 60% after 12 h [ 59 ] or 24 h [ 60 ] post-oral administration. Furthermore, when compared to other chitosan-based insulin-loaded NPs, the developed formulation had higher encapsulated efficiency, was more resistant to gastric-like conditions or/and achieved higher decrease in glycemia, at 4 h, than what was seen for other chitosan-based formulations of insulin nanoparticles [61–63]. 5. Conclusions Oral insulin replacement therapy remains a very appealing alternative to subcutaneous injections for patients with diabetes mellitus. However, it seems that the search for an acceptable insulin oral formulation is much more difficult than initially thought. After decades of failed attempts to produce an oral insulin formulation, the number of published clinical trial reports so far is limited. The hope clearly is to see more clinical data. A suitable drug carrier is important to ensure site-specific sustained drug delivery. As described herein, our double-coated insulin-loaded NPs formulation showed to be efficient in reducing the glycemia up to 50% in chemically-induced diabetic rats and it is safe. Additionally, the encapsulation was effective and the method of encapsulation did not alter the insulin’s secondary structure. So, it is expected that the drug maintains its integrity when it reaches its biological target. Furthermore, it was proved in the in vitro assays that by encapsulating insulin and coating the NPs with two different layers, insulin was protected from the hostile environment of the GIT. Moreover, the ex-vivo study showed that the chitosan coating exerts its mucoadhesiveness, correlating with the in silico analysis. Thus, these double-coated insulin-loaded NPs might provide a more efficient and safer platform for delivering insulin by mimicking physiologic processes and directly deliver insulin to the liver rather than via the bloodstream. A great amount of work still remains to be done, but like many other examples in other therapeutic areas, nanomedicine brings new hope to succeed in obtaining an oral treatment of insulin available to diabetic patients. Author Contributions: Conceptualization, C.P.R.; Methodology: C.P.R., M.A., A.S.M.; Formal Analysis, M.A., A.S.M., P.F., P.K., J.F.P., R.P., I.C., L.A., M.M.G., R.A.A., I.V.F., R.P., C.P.R.; Investigation, M.A., A.S.M., J.C., P.F., P.K., R.P., I.C., L.A., M.M.G., I.V.F., C.P.R.; Writing – Original Draft Preparation, M.A., A.S.M.; Writing – Review & Editing, P.F., P.K., J.F.P., R.P., I.C., L.A., M.M.G., R.A.A., I.V.F., C.P.R.; Supervision, C.P.R.; Project Administration, C.P.R.; Funding Acquisition, A.J.C. All authors have read and agreed to the published version of the manuscript. Funding: Supported in part by UID/DTP/04138/2019 from FCT, Portugal and DREAMS (ULHT). 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