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Preparation and characterization of hypoglycemic nanoparticles for oral insulin delivery Li Zhang, Yu-Xiao Zhang, Jia-Ni Qiu, Jian Li, Wuya Chen, and Yan-Qing Guan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01322 • Publication Date (Web): 23 Oct 2017 Downloaded from http://pubs.acs.org on October 26, 2017

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Manuscript submitted to Biomacromolecules （based on an earlier manuscript ID:bm-2017-008369）

Preparation and characterization of hypoglycemic nanoparticles for oral insulin delivery Li Zhang1, Yu-Xiao Zhang1, Jia-Ni Qiu1, Jian Li2, Wuya Chen1 and Yan-Qing Guan1,2,3 a)

1 2

School of Life Science, South China Normal University, Guangzhou 510631, China

MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China

3

Joint Laboratory of Laser Oncology with Cancer Center of Sun Yet-sen University, South China Normal University, Guangzhou 510631, China

corresponding author at: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, & Joint Laboratory of Laser Oncology with Cancer Center of Sun Yet-sen University, South China Normal University, Guangzhou 510631, P. R. China. Tel.: (+86-20)85211241; E-mail address: [email protected] (Y. Q. Guan)

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ABSTRACT Polymeric nanoparticles have been widely investigated as insulin delivery systems for oral administration. However, the toxic nature of many artificial polymers hampers their effective application, creating a demand for the further exploration of alternative natural polymers. In addition, ethnobotanical research has reported that over 800 plant species have a hyperglycemic function, some of which are polymers. To combine the advantages of both areas, the aim of this work was to choose an organic hypoglycemic polymer and prepare it into an insulin carrier, in order to build a dual-functional oral insulin delivery system. We found that the insulin loading rate, release mode, thermo stability, and both in vitro and in vivo absorption and efficacy varied with the different modifications of polygalacturonic acid (PGLA) nanoparticulate backbones. By in vivo pharmaceutical testing and constantly monitoring the symptoms of type 1 diabetic (T1D) rats, we ascertained the hypoglycemic function of the nanoparticles, and showed that overall diabetic symptoms were ameliorated after the long-term daily administration of nanoparticles, with no significant damage to organ structure and cell viability.

KEY WORDS: polygalacturonic acid (PGLA), Chitosan, Genipin, Insulin, Oral delivery


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Introduction The first scientific publication addressing oral insulin was printed only 2 years after insulin was introduced into clinical use in 1922, but the first clinical oral insulin trial did not begin until in 2000. Before 2014, only one oral insulin delivery system had been approved for phase III clinical trials by the FDA.1 The intense work of oral insulin research, spanning nearly one century, still attracts the attention of scientists, because it might provide a more natural physical circulation mode and better glucose homeostasis, as a remedy for the current low compliance of diabetic patients and peripheral hyperinsulinemia.2–5 Nevertheless, due to low insulin bioavailability, the toxicity of delivery materials and the lack of dose stability, most of the finely designed oral insulin delivery systems are still far away from clinical application. Prolongation of the intestinal residence time, protection in the harsh environment of the gastrointestinal tract, targeting of specific cells or proteins on the intestinal surface, and the enhancement of cell penetration are mainstream strategies utilized by recently developed oral insulin delivery systems to improve the efficacy of insulin delivery and blood glucose control.6–9 Many oral insulin polymer delivery systems and different formulations of interpolymer complexes have been developed.10,11 Among these, natural polymers are popular because of advantages like low toxicity, good biocompatibility, and acquirability.12–16 Most of the natural polymers adopted in delivery systems are polysaccharides, useful for their high degradability, biocompatibility, and other special characteristics.17 For example, chitosan, a cationic nature polymer, can not only adhere to the mucus on the intestinal surface, thereby prolonging intestinal residence time, but is also able to open the tight junctions between cells to provide an extra tunnel for micro- and nano- delivery systems to enter the circulation.18–22 Nevertheless, to the best of our knowledge, no oral insulin research has ever been intentionally used a polymer for a medical function. In fact, over 800 species of plants have hypoglycemic function according to ethnobotanical research,23 and many exact polymers have been identified.24–28 For instance, polygalacturonic acid (PGLA), the main constituent of pectin, can help to control blood glucose. PGLA is believed to slow the sugar absorption speed of the intestine, and the daily administration of a 200 mg dose could significantly reduce blood glucose and increase liver glycogen.29 In this study, we developed a co-polymer insulin oral delivery system from a different angle, which was to use a medical functional material and control blood glucose with dual mechanisms. Many types of carriers, including hydrogel, liposomes and nanoparticles, have been used in oral protein delivery.30–35 Nano-sized structures have always been preferred in oral delivery 3 Environment ACS Paragon Plus

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systems for their advanced cell absorption and epithelial penetration.36–38 Among the many methods of nanoparticle fabrication, the emulsion system is common, due to its relatively controlled particle formation.39 Several studies have been conducted using emulsion systems to create a separated water phase, in order to limit the size of the nanoparticles.40,41 A traditional water-in oil-in water (W/O/W) double emulsion system is maintained by oil dissolved polymers coating the elements dissolved in the inner water phase.42–44 A double emulsion system is more stable and the nanoparticle size is smaller than those fabricated with water-in oil (W/O) or oil-in water (O/W) emulsion systems. The aim of this study was to prepare an oral insulin delivery system based on a natural and dual-functional delivery device, with a hypoglycemic polymer and a modified W/O/W double emulsion system to fabricate smaller nanoparticles. We developed a mucoadhesive and hyperglycemic nanoparticle based on chitosan and PGLA. The basic characteristics such as particle size, zeta potential, morphology, and release mode were exploited. The system was tested in vitro regarding the cellular absorption of delivered insulin and its cytotoxicity. Moreover, ex vivo and in vivo tests showed the intestinal absorption, pharmacology, toxicity and long term efficacy of the delivery systems.

Materials and methods Materials. Human insulin was provided by Biosharp (Shanghai, China), PGLA (>90%, and molecular weight of 25-50 kDa), chitosan and pluronic 188 were provided by Sigma Aldrich Co., Ltd. (Shanghai, China). Genipin was provided by Xi Bao Biotech Co., Ltd. (Shanghai, China)

Preparation of insulin-loaded nano-particles. The three types of nanoparticles were prepared using a modified W/O/W solvent evaporation technique.45 Briefly, an appropriate amount of insulin (1 mg) dissolved in 20 µl of 5% (w/v) aqueous solution of pluronic 188 containing 5 mg/ml PGLA and 5 mmol/ml NaHCO3 was emulsified with 1 ml ethyl acetate by ultra-sonication for 60 s, in order to produce the primary emulsion (w/o). After addition of 5 ml of a 20 % (w/v) aqueous solution of pluronic 188, the emulsion was ultra-sonicated again for 60 s, forming a double emulsion (w/o/w). Then the resulting double emulsion was poured into 40 ml of a 2 % (w/v) aqueous pluronic 188 solution (pH 5.4) and maintained under mechanical stirring for 0.5 h at 1000 rpm. The residual ethyl acetate was evaporated under vacuum. Aliquots of the nanoparticle suspension (1.8 ml) were washed twice with 20 mM HEPES pH 7.0 by centrifugation (8000 rpm, 10 min, 4 °C) and resuspended. 4 Environment ACS Paragon Plus

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Particle characterization. FT-IR, Raman, TEM, DLS, and TGA were employed to characterize the three types of fabricated nanoparticle.46 Frozen dried powder of each material and each type of nanoparticles were examined on a Bruker Vertex 70 FT-IR spectrometer and H.J.Y. LabRAM Aramis for FT-IR and Raman spectra, respectively. The morphology of the particles was examined by transmission electron microscopy (H-7650, HITECHI Co., Japan) with an accelerating voltage of 300 kV. Suspensions of different nanoparticles were dropped onto a carbon coated copper grid, and after complete drying, the grids were observed. The particle size and zeta potential of the freshly prepared nanoparticles and freeze-dried nanoparticles resuspended in ultrapure water were determined by dynamic light scattering (DLS) using a Zeta Sizer (Nano ZS, Malvern Co., UK). Thermo gravimetric analysis (TGA) was performed by a thermo gravimetric analyzer (TGA/DSC 1, Mettler Co., Switzerland) under a steady flow of nitrogen at a heating rate of 10 °C /min at the temperature range between 20 and 800 °C.

Determination of encapsulation efficiency. The insulin loaded into the nanoparticles was determined by measuring the insulin released from the nanoparticles after 24 h. The insulin concentration in the supernatants recovered after the centrifugation of the released solution was quantitatively analyzed using high-performance liquid chromatography (HPLC), as described, with a SCL-6A instrument (Shimadzu, Japan). The HPLC column (Hypersil ODS, 5 µm, C18 150 mm × 4.60 mm) was kept at room temperature during the experiments. The mobile phase was composed of 18% acetonitrile in 0.30% trifluoroacetic acid (TFA) solution (v/v). The flow rate of the mobile phase was set at 1.5 mL/min, where a 10 µl sample was automatically injected. Insulin was detected spectrophotometrically at a 214 nm wavelength.47

Loading Capacity (%) = Loading Efficiency (%) =

total amount of drug - amount of drug in supernatant × 100% weight of nanoparticles total amount of drug - amount of drug in supernatant × 100% total amount of drug

(1) (2)

Circular dichroism. Circular dichroism (CD) was employed to evaluate the conformational changes of the insulin released from W/O/W nanoparticles.48 After 12 h of release in vitro, the dissolution media were subjected to ultracentrifugation and the supernatant containing insulin were collected for CD analysis. All CD measurements were 5 Environment ACS Paragon Plus

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conducted by scanning from 190 nm to 260 nm on a CD spectropolarimeter (TENSOR 27, Bruker, German). The generated ellipticity values were subsequently converted to molar ellipticities using the equation [θ]λ =θλ·M/C·L, where θλ is the observed ellipticity at the wave length λ, M is the mean residue molecular weight (g/mol), C is the insulin concentration (g/ml) and the L is the optical path length (cm). To eliminate possible contributions from EPL that were also present in solution, their CD spectra were recorded and subtracted from the spectra of the supernatants.

Release in vitro. In order to determine the amount of insulin released from the nanoparticles in the stomach and intestine, buffers with different pH (1.1 and 6.8) levels were used. Each 200 µl solution of PGLA, CS-PGLA, or Gen-CS-PGLA nanoparticles was added into 5 ml of a pH=1.1 HCl solution and a pH=6.8 PBS buffer, and was shaken continuously at 50 rpm/min for 6 hours. Aliquots of 50 µl were taken at specified time points (0.5 h, 1 h, 2 h, 4 h, and 6 h). The insulin concentration in the aliquots was analyzed using HPLC, as described above. The cumulative amount of insulin released from the nanoparticles was calculated. After calibrating, the cumulative percentage of insulin released was plotted versus time. Each data point was calculated from 3 measurements.

Animal care Male Sprague-Dawley rats, 6-8 weeks old, were provided by the Animal Experimental Center of Southern Medical University, Guangzhou, China. The animals had free access to rat chow and tap water. All of the animal experiments were carried out according to the Institutional Animal Care and Use Committee (IACUC) guidelines.

T1D induction Diabetes was induced in Male Sprague-Dawley rats weighting 190-210 g by an injection of streptozotocin (50 mg/kg) dissolved in citrate buffer for 3 days.30 The blood glucose level was determined using a glucose meter (Active, Roche, China). Rats were considered to be diabetic when their fasting blood glucose level were higher than 13 mmol/L one week after streptozotocin treatment. During the week, blood glucose, body weight, and uptake of food and water of each rat were recorded.

Ex vivo absorption studies in ligated intestinal loops. The ex vivo uptake of PGLA, CS-PGLA, and Gen-CS-PGLA nanoparticles was evaluated using the ligated intestinal loops model following a previous described method47. Four T1D Sprague-Dawley rats were fasted overnight before the experiment, but were allowed free access to water. The rats were 6 Environment ACS Paragon Plus

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anesthetized with sodium pentobarbital (0.04 mg/kg). After the abdomen was exposed, 5 cm loops of the ileum were made by ligation at both ends and washed with physiological saline. Equal quantities of FITC-Insulin loaded PGLA, CS-PGLA, and Gen-CS-PGLA nanoparticles (0.5 ml of nanoparticle solution, containing 50 IU FITC-Insulin) were injected into the intestinal loop of each rat. Rats were sacrificed after 2 h and sections of each loop were removed. Subsequently, the removed loops were washed with PBS, fixed in 4% paraformaldehyde for 2 h and immersed overnight in 30% sucrose at 4 oC. Samples were embedded and frozen at -20 oC. Frozen ileum sections were cut (20 µm) using a cryostat (CM 1950, Leica, German) and stained with DAPI. The tissue-sections were then visualized using a fluorescence microscope (Eclipse Ti, Nikon, Japan).

Pharmacological study. T1D Sprague-Dawley rats were fasted overnight before the experiment, but allowed free access to water. Insulin was administered to the diabetic rats by gavage of insulin solution (50 IU/kg), insulin loaded PGLA, CS-PGLA, and Gen-CS-PGLA nanoparticles (50 IU/kg), and by subcutaneous injection of insulin (5 IU/kg). Each group contained 3 rats. Blood samples were collected from the tail veins of rats after drug administration and at various times (1, 2, 4, 6, and 8 h) after dosing. The blood glucose levels were analyzed. The area under the serum glucose concentration-time curve over 8 h was calculated using the integrate tool of Origin 8.5. The total decreases (D%) in serum were calculated using a modification method as follows49: D% =

AUC (insulin .oa ) − AUC (test ) × 100% AUC (insulin .oa )

where AUC is the total area under the curve of plasma glucose concentration vs. time. Long-term efficacy and in vivo toxicity. In order to study the long-term efficacy and toxicity of the nanoparticles, 4 group of T1D rats were set, as the control, PGLA, CS-PGLA, and Gen-CS-PGLA nanoparticle gavage groups. Rats in each group were gavaged with saline, PGLA, CS-PGLA, or Gen-CS-PGLA nanoparticles containing 50 IU insulin every morning for 14 days, monitoring the body weight, and the amount of daily food and water. After 14 days, rats were sacrificed, and their skeletal muscle, cardiac muscle, spleens, pancreases, kidneys and livers were prepared for sectional histology. Each of the organs was fixed in 10% phosphate buffered formalin, embedded in paraffin, cross sectioned at 5 µm and then stained with hematoxylin and eosin (H&E) and periodic acid-schiff (PAS) stain50. A microscope (Eclipse Ti, Nikon, Japan) was used to record the morphology and H&E or PAS tissue staining. The PAS stain pictures were analyzed by Photoshop software (8.0.1, Adobe, USA), 7 Environment ACS Paragon Plus

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in order to qualitatively compare the stain area, which represents the amount of glycogen, in each organ.

Cell culture The human intestinal Caco-2 cell were obtained from Medical University of Guangdong, and cultured with DMEM high glucose medium with 20 % FBS, amino acid and 1 % penicillin-streptomycin antibiotic solution. Cells were incubated in 37 oC, with 5 % CO2. MTT assay Cells were seeded in 96-well plates at a density of 7 × 104 cells per well and incubated for 12 h. Full medium of each well was replaced by non FBS medium with different concentrations of each nanoparticle (0 µg/ml, 125 µg/ml, 250 µg/ml, 500 µg/ml, 1000 µg/ml). After 24 h incubation, 20 µl MTT (5 mg/ml) was added into each well and keep incubated for another 4 h. Then medium were draw out before adding 100 µl DMSO in each well, gently shaking for 10 min, examined with 510 nm wave length.51

In vitro quantification of released and absorbed insulin In order to study the function of delivered insulin at a cellular level, caco-2 cells were seeded in four dishes at a density of 107 cells per dish. After the cells grew and spread, saline and a PGLA, CS-PGLA, or Gen-CSPGLA nanoparticle solution containing 5 IU loaded insulin, were added into cell culture and incubated for 4 h, whereupon cells were harvested. Cells were then fixed in 4 % paraformaldehyde. Samples from each dish were divided into 2 groups, and each group was incubated with rabbit polyclonal anti-human insulin, and anti-human glut4 (Beijing Biosynthesis Biotechnology, China) overnight at 4 oC. After incubation with the secondary antibody (Cy5 tagged second anti-body, Gene Copoeia, China) for 4 h at room temperature, insulin and cell membrane glut4 were quantified with flow cytometry (CytoFLEX, Beckman Coulter Inc., USA). Each test was repeated 3 times.

Absorption in vitro For the cellular uptake study, Caco-2 cells were seeded in glassbottom dishes at a density of 1 × 106 cells per dish. After incubation at 37 oC for 48 h, whereupon the cells had grown and spread to form a membrane, the growth medium was replaced with fresh medium contain PGLA, CS-PGLA, or Gen-CS-PGLA nanoparticles. The dishes were incubated at 37 oC for 4 hours, and fixed with 4 % paraformaldehyde. The cells were blocked for 2 h with 5% nonfat milk and then incubated with rabbit polyclonal antihuman insulin (anti-insulin, Beijing Biosynthesis Biotechnology, China) overnight at 4 oC. After incubation with the secondary antibody (Cy5 tagged second anti-body, Gene Copoeia, 8 Environment ACS Paragon Plus

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China) for 4 h at room temperature, the nuclei were stained with DAPI. Confocal laser scanning microscopy (CLSM) (SP5 II, Leica, Germany) was used to assess the intracellular location of insulin.52

Results Particle characterization. Three types of nanoparticles were synthesized according to the procedure in Figure 1. The infrared spectra (FT-IR) of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles are displayed in Figure 2A, revealing the characteristic polysaccharide and protein absorption bands. The peaks between 1000 cm-1 and 1160 cm-1 are relevant to units of polysaccharide. The C-C ring breathing and C-O-C symmetric glycosidic stretch contributed to the peaks at 1160 cm-1 and 1100 cm-1. The C-OH stretching vibration of the hexoses backbone caused the peak near 1050 cm-1.53 The polysaccharide showed a peak at 3440 cm-1 which corresponded to the -OH band within the standard range of 3400-3600 cm1 54

. The characteristic amide peaks of protein contained an amide I (-C=O stretching) at 1637

cm-1, amide II at 1567 cm-1(-NH bending and -CN stretching modes) and amide III around 1240 cm-1.55,56 The Gen-CS-PGLA peak was sharper than that of the CS-PGLA and PGLA nanoparticles, which might be caused by slight changes in the chemical bonds. Figure 2B shows the Raman spectra of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. In all spectra, the polysaccharide and protein peaks were very significant. The polysaccharides peaked at 2942 cm-1 (-CH/CH2/CH3), 1378cm-1(-CH2), 1339cm-1(-COH, CH), 1131cm-1(-CO, -CC, -COH, -COC), 1086cm-1(-COH, -CH), and 854 cm-1(COC). They are all ring breathing, and all of the glycosidic linkages were identified.57,58 Peaks within a standard range of amide bands (amide I band 1590-1720 cm-1 and amide III band 1200-1300 cm-1) and S-S bonds (490-550 cm−1) show the existence of insulin in each of the nanoparticle types.59 In the Gen-CS-PGLA spectrum, the peaks at 1730 cm-1 (-COOH) disappeared, and the peak at 1429 cm-1 (-CH2/CH3), 2054 cm-1 (OH…O) and 1655 cm-1 (C=C) increased.60 This phenomenon indicates that introduction of genipin may influence the vibration of COOH and lead to the formation of double and hydro bonds. The TEM morphology, particle size, and zeta potential of the three types of nanoparticles were all explored. The TEM morphology showed that nanoparticles were well separated and that their shape was nearly round. The particle size was found to be a little smaller than the DLS size results, which is common in other research (Figure 2C). We determined that the particle sizes of PGLA, CS-PGLA, and Gen-CS-PGLA nanoparticles were 66.17 ± 8.61 nm, 67.73 ± 7.89 nm, and 58.61 ± 10.36 nm, respectively (Figure 2D). The zeta potentials of 9 Environment ACS Paragon Plus

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PGLA, CS-PGLA, and Gen-CS-PGLA nanoparticles are -15.72 ± 3.03 mV, -7.35 ± 3.00 mV, and -4.87 ± 3.15 mV (Figure 2E). The thermal decomposition behavior of each type of nanoparticle was characterized by TG analysis. The weight loss at 30 °C to 150 °C might have be caused by free and bonded water evaporation.59 Comparing the weight loss curve between CS-PGLA and Gen-CS-PGLA nanoparticles, it seems that the bond between genipin and chitosan might strengthen the structure61, and prevent about 10% weight loss from 600 °C to 800 °C. All three nanoparticles have better thermo-stability than insulin alone since the temperature was higher than 300 °C (Figure 2F). The loading efficiency and capacity were tested and calculated. Loading capacities of 42 ± 7%, 47 ± 4%, and 55 ± 3% and loading efficiencies of 19 ± 3%, 21 ± 2%, and 25 ± 1% for PGLA, CS-PGLA, and Gen-CS-PGLA nanoparticles were observed. The loading capacity and efficiency of the nanoparticles gradually increased with each step of decoration (Figure 2G).

The structural integrity of insulin after fabrication and release The far UV CD spectra for insulin released from all three types of nanoparticles almost entirely overlapped with the CD spectrum for the control insulin solution (Figure 2H). The minimum points at 208 and 222 nm and the maximum point at 195 nm were typical of the predominant α-helix in the insulin secondary structure, and were very similar to previous CD results of insulin in other research.62

Insulin release in vitro. Figure 2I shows the release profiles of CS-PGLA at 0.1 M HCl (pH=1) and PBS (pH=6.8). Like most of nanoparticles, this delivery system presents controlled release mode with delayed release and pH-sensitivity, discovered by the incubation in simulated gastric fluid (SGF, pH=1.1) and simulated intestinal fluid (SIF, pH=6.8). All the three types of nanoparticles successfully delayed the released of insulin and released insulin more quickly in SGF.

T1D model. One week after STZ injection, the blood glucose of SD rats increased above 13 mmol/L, indicating that the T1D model was successfully induced. In accordance with medical consensus, disease symptoms were seen in the T1D group when they took in significantly more food, about 1.5 times which of the control group, and water, about 6 times that of the control group. Nevertheless, the body weight of the T1D group remained nearly 10 Environment ACS Paragon Plus

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unchanged for 7 days after STZ injection, instead of continuously increasing as control group (Figure 3A, Figure 3B).

In vivo hypoglycemic effects and bioavailability. The pharmacological effects of orally-administered PGLA, CS-PGLA, and Gen-CS-PGLA nanoparticles were evaluated in fasted diabetic rats. PGLA, CS-PGLA, and |Gen-CS-PGLA nanoparticle administered groups were all exhibited relatively strong hypoglycemic effects at 2, 4 or 6 hours after administration (P < 0.05) as compared with insulin solution group. Moreover, with the modification of chitosan and genipin, CS-PGLA showed a delayed and better hypoglycemic effect with the maximal blood glucose depression of 32% at 4 h, compared to PGLA nanoparticles 32% at 2 h, and Gen-CS-PGLA showed a much delayed trend with blood glucose lowered 30% at 6 h (Figure 3C). Furthermore, to comprehensively understand the hypoglycemic effect of each group, the D% of each formulation was calculated. It was obtained that the D% of PGLA, CS-PGLA, and |Gen-CS-PGLA nanoparticle (50 IU/kg) were 14.88%, 24.06% and 19.06%. The D% of SC insulin group (5 IU/kg) was 42.44%. Based on other previous reports that insulin should always been metabolized 8 h post-administration, bioavailability of each type of nanoparticle was calculated, according to the blood glucose depression data. The bioavailability of PGLA, CS-PGLA, and |Gen-CS-PGLA nanoparticle were 3.5 %, 5.6 %, and 4.4 %. During this experiment, the fasting blood glucose of CS-PGLA, Gen-CS-PGLA nanoparticle and SC insulin group did not return to the initial level after 8 h, same as other previous reports, which might be the consequence of the dual effects of hunger and hypoglycemic agents.

Intestinal ligation absorption of nanoparticles. The absorption of PGLA, CSPGLA, and Gen-CS-PGLA nanoparticles by the microvilli of ileum loops was observed. Figure 3D shows the intestinal absorption of FITC-insulin delivered by three types of nanoparticles after 2 h incubation. Scattered green fluorescence was observed in microvilli of loops treated with PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. The absorption of FITC-insulin delivered by the CS-PGLA and Gen-CS-PGLA nanoparticles permeated deeply into the middle and basal layers of the microvilli after 2 h incubation, indicating their advanced cell penetration.

Long-term efficacy and in vivo toxicity studies. The gastrointestinal tract is continually exposed to various potentially detrimental substances, including chemical and 11 Environment ACS Paragon Plus

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bacterial toxins.50 Since our CS-PGLA and Gen-CS-PGLA nanoparticles contained chitosan, which is capable of enhancing paracellular permeability by opening the epithelial tight junctions, it is therefore crucial to study whether this could lead to in vivo toxicity. Moreover, the effect of insulin is relevant, not only to changes in blood glucose, but it is also relevant to the long term amelioration of T1D symptoms, such as body weight, and the daily uptake of food and water. Neither of the investigated groups had apparent clinical symptoms such as diarrhea, fever, or other systemic symptoms throughout the study. Over the 14 days, the body weights of the experimental groups increased more sharply than those of the control beginning 4 days after nanoparticle administration. The intake amount of food and water of experimental groups, however, decreased slightly compared to the control group, beginning about 3 days of nanoparticle after administration (Figure 4A). PAS staining revealed the presence and amount of glycogen in organs, which is one form of energy storage, and is a reaction motivated by insulin.63 Liver, skeletal and cardiac muscle, and kidneys are the main glycogen storage organs. PAS staining in the liver, skeletal muscle, kidney, and cardiac muscle presented increased and much darker staining than in the control (Figure 4B1). The resulting quantitative statistics revealed that the control group had the least PAS staining in each organ, the CS-PGLA nanoparticles exerted enhanced glycogen storage in the liver and muscle, and the PGLA nanoparticles promoted glycogen storage more than the Gen-CS-PGLA nanoparticles in the skeletal muscle, kidney, and cardiac muscle, but less in liver (Figure 4B2). The specific tissue level toxicity study includes inflammatory, hepatoxicity, and nephrotoxicity responses. Our histological assessment found no noticeable tissue damages or toxic effects on the organs. Moreover, the tissue morphology of the kidney, liver, and heart muscle in the experimental group was healthier than that of the control, which indicates that the toxicity was extremely low from these types of nanoparticle (Figure 4C).

Cytotoxicity. The potential biomedical applications were assessed by investigating the cell toxicity of three types of nanoparticles in Caco-2 cells using an MTT assay. As revealed in Figure 5A, each type of nanoparticle was added into Caco-2 cell culture at different concentrations (0 µg/ml, 125 µg/ml, 250 µg/ml, 500 µg/ml, and 1000 µg/ml), and was incubated for 24 h. Higher concentration of nanoparticles did not bring about lower cell viability, instead, PGLA and CS-PGLA seemed to enhance cell viability to a certain degree.


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In vitro quantification of released and absorbed insulin. Glut4 is a glucose transport protein, and its movement to the cell membrane was triggered after the insulin bond to its receptor.64,65 The amount of glut4 protein in the cell membrane may therefore represent the amount of insulin released outside the cell. The amount of glut4 transported on the membrane was shown to have increased in the order of the control, PGLA, CS-PGLA, and Gen-CS-PGLA groups (Figure 5B). At the same time, absorbed insulin is proof that the nanoparticles penetrated into the cell. The CS-PGLA nanoparticles showed the largest amount of absorbed insulin, followed by the Gen-CS-PGLA and PGLA groups. The smallest amount of insulin was absorbed by the control group (Figure 5C).

In vitro uptake of nanoparticles. The assessment of the cellular uptake of the three types of nanoparticles was carried out by incubating Caco-2 cells with insulin loaded nanoparticles for 4 h. Figure 5D shows the absorption of FITC-insulin (green) loaded nanoparticles and Figure 5E shows the small scale fluoresce strength and distribution of Cy5 tagged anti-insulin (red). The cell nucleus was stained with DAPI, showing blue fluoresce. The insulin fluorescence revealed its location around the nucleus, and the Z axis images confirm that the signal was located within the cell, proving that the insulin was successfully delivered by PGLA, CS-PGLA, and Gen-CS-PGLA nanoparticles and absorbed by Caco-2 cells, while free insulin merely entered via the cell membrane. Similar to the former results, CS-PGLA nanoparticles brought the largest amount of insulin into the cell, while the Gen-CSPGLA nanoparticles delivered less, and the PGLA nanoparticles delivered the least amount of insulin.

Discussion An efficient, nontoxic oral insulin delivery system could provide a revolutionary change for T1D patients, with which they could control their blood glucose levels with more ease and comfort than traditional methods.66 This work has two major goals. The first is, the application of a hypoglycemic polymer for the fabrication of a delivery system. Insulin delivery devices play the roles of carriers, protectors, and blood glucose suppressors. Although further study is needed to thoroughly explore the mechanisms behind the combination of the hypoglycemic effects of insulin and medical polymers, this study is the first to link a medical polymer to a nano-sized insulin delivery system. Second, we observed the overall symptoms of diabetes and the long-term efficacy of our insulin delivery systems, which is an innovative and more comprehensive way to evaluate an insulin delivery system. 13 Environment ACS Paragon Plus

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The most obvious and well-understood function of insulin is to mediate blood glucose and the most obvious symptom of diabetes is high blood glucose. Nevertheless, insulin also plays an important role in carbohydrate metabolism, making diabetes more like an energy metabolism disorder.67,68 Therefore, the long-term efficacy of an insulin delivery system, including toxicity and tissue glycogen quantification, and the amelioration of overall diabetic symptoms should be taken into consideration. Muco adhesive properties, epithelial penetration, particle size and charging are important characteristics of an oral delivery system.69 Chitosan and PGLA both have good muco adhesive properties and chitosan has been proven to be an enhancer of epithelial penetration.21,70 Nano-sized particles can be absorbed by M cells in the intestinal epithelium.71,72 Moderate charging is helpful in preventing aggregation and maintaining structure.73 The FT-IR and Raman spectra confirmed the formation of the PGLA, CS-PGLA, and Gen-CS-PGLA nanoparticles. With the modification of W/O/W nanoparticle fabrication method, PGLA was dissolved in the inner water phase instead of the oil phase. PGLA and insulin were entangled forming primary nanoparticles,74 and the oil phase only used to separate the inner water phase, rather than tying many inner water spheres together, too. As a result, the diameter of all three types of nanoparticles were less than 100 nm, which were significantly smaller than previous reports.45,75,76 They were shown to be negatively charged (Figure 2 A-E), which force the primary nanoparticles repelling each other. This force helped to decrease nanoparticle size, in addition, the nanoparticles got even smaller after freezedrying.77 Controlled release, and the protection of the delivered drug are two important properties of any drug delivery system.78 CS-PGLA nanoparticles delayed the insulin release in SGF and significantly delayed insulin release in SIF. CD spectra confirmed that the secondary structure of insulin remained unchanged (Figure 2H, I). The insulin released before and after cell penetration was quantified for the first time in this study. Since insulin would induce the translocation of GLUT4 to the cell membrane,79 the qualification of GLUT4 on the cell membrane may reveal the amount of insulin released outside the cell. Comparing the quantification of GLUT4 on the cell membrane and insulin inside the cell, it possible that chitosan and cross-linked chitosan shell of the nanoparticle may change the chance of cell penetration, which may explain that although the release curves of three types of nanoparticles have little difference, the distribution of insulin in and out of the cell is differed from each other. It is obvious that chitosan may enhance the cell penetration of CS-PGLA and Gen-CS-PGLA nanoparticle, even though these two types of nanoparticle 14 Environment ACS Paragon Plus

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release more insulin outside the cell. However, PGLA nanoparticle release less insulin outside the cell, but it carries less insulin into the cell (Figure 5B). The blood glucose depression efficacy of insulin delivery was evaluated from two aspects, cell level penetration and body level blood glucose control funciton.80 In this work, according to our in vitro and ex vivo exploration, insulin delivered by PGLA, CS-PGLA, and Gen-CSPGLA nanoparticles was indeed absorbed by microvilli and Caco-2 cells (Figure 3D, Figure 5C D E). In vivo test of pharmaceutics shows the rapid blood glucose decrease of PGLA nanoparticle and slow decrease but longer control of blood glucose contributed by CS-PGLA and Gen-CS-PGLA nanoparticle. Three types of nanoparticles have different modes of release with the changes of modifications, the chitosan cover and genipin crosslink both delayed the release of insulin. Insulin bioavailability is an important index to evaluate an insulin oral delivery system.81 The insulin bioavailability of CS-PGLA nanoparticle is the highest one. Most of the research on insulin oral delivery shows a bioavailability of about 5%.82 The three types of nanoparticles in this work have the similar bioavailability with previous reports (Figure 3C). Moreover, long-term observations are required to evaluate the efficacy and toxicity of the insulin delivery system applied in this work, including symptom monitoring and tissue glycogen quantification. The symptoms of type 1 and 2 diabetes include increased food and water intake, but a body weight that remains unchanged or slowly decrease.83,84 The symptoms in our experimental rats were recorded after T1D induction and for the long-term evaluation of delivery systems (Figure 3A, Figure 4A). Since diabetes is an energy metabolism disorder and since insulin has long term effects in mediating carbohydrate metabolism,67,68 the glycogen storage after long term administration of an insulin delivery system is necessary for an accurate evaluation. In accordance with previously published data, CS-PGLA nanoparticles cause the storage of the most glycogen in tissue, which was further confirmed by our results. Thus, our newly designed nanoparticles based on a PGLA were shown to lower blood glucose after one administration and change release mode along with modification, and successfully ameliorated T1D symptoms during long term gavage use, with no significant side effects or toxicity. This combination of a medical material with a nano-carrier is a meaningful attempt in the development of an insulin oral delivery system.

Conclusions


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In conclusion, we reported a new functional polymer platform for blood glucose control. Consisting of blood glucose controlling PGLA, intestinal penetrating chitosan and the natural crosslinker genipin, this platform exerts a relatively high bio-availability. The combination of different polymer functions enhanced the efficacy of the insulin delivery systems from different aspects, which adds to the evidence promoting the use of different polymers for the oral administration of insulin, for which our study has overcome some of the limitations. This design provides a potential delivery strategy for functional polymer development and diabetes treatment.

Notes: The author declare no competing interests and conflicts.

Acknowledgements The expenses of this work were supported by the National Natural Science Foundation of China (31370967, 31170919), the Guangdong Province Universities and Colleges Pearl River Scholar Fund Scheme (2014), China, the Science and Technology Planning Project of Guangdong Province (No.2015A020212033), China.

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Figures and figure captions Figure 1. Schematic mechanism of nanoparticles fabrication. (A) Structure of PGLA, Chitosan and chemical reaction between chitosan and genipin. (B) Schematic representation of synthetic process of PGLA, CS-PGLA, and Gen-CS-PGLA nanoparticles based on W/O/W double emulsion system.

Figure 2. Characteristic of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. (A) FTIR spectra and (B) Raman spectra of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. (C) TEM morphology of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. Scale bar 100 nm. (D)Size and (E) Zeta potential of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. (F) Thermo gravity of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. Black line is pure insulin, dark blue line is PGLA nanoparticles, light blue line is CS-PGLA nanoparticles, and grey line is Gen-CS-PGLA nanoparticles. (G) Loading capacity and loading efficiency of each type of nanoparticle. (H) CD spectra of insulin released from PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. (I) Insulin release of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles in pH=1 and pH=6.8 buffer respectively.

Figure 3. T1D model, in vitro test of absorption and pharmaceutical test. (A) Physical characteristic comparison of induced T1D rat and normal rat. (B) (a)Blood glucose, (b) and (c) amount of food and water took in by rats and (d) their body weight changing during the T1D inducing progress. (C)Blood glucose levels in diabetic rats following oral administration of insulin solution, PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles at an insulin dose of 50 IU/kg, and subcutaneous injection of insulin at an insulin dose of 5 IU/kg (Mean ± SD, n=3). Significant difference from oral insulin solution and from PGLA, CS-PGLA, Gen-CS-PGLA nanoparticles, P < 0.05. (D)Intestinal absorption of saline (a) (b), PGLA nanoparticle (c) (d), CS-PGLA nanoparticle (e) (f), Gen-CS-PGLA nanoparticle (g)(h). Blue fluoresce is DAPI stained nuclei, green fluoresce is FITC-insulin delivered by PGLA, CS-PGLA, Gen-CSPGLA nanoparticles. Scale bar of first line is 50 µm, and of the second line is 15 µm.

Figure 4. Long-term toxicity and efficacy of three type of nanoparticle administration. (A) Symptoms of diabetes changes during the experiment. Dark blue line represents the group gavage PGLA nanoparticles each day, light blue line represents the CS-PGLA group, grey line represents the Gen-CS-PGLA group, and black line represents the control group. (a) 22 Environment ACS Paragon Plus

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Amounts of drinking. (b) Amounts of eating. (c) Body weight. (d) Schematic representation of the changes of T1D rats during the process. (B1) PAS stain of organs from each group. Liver, muscle, kidney, heart from control, PGLA, CS-PGLA and Gen-CS-PGLA group. (B2)Quantitative statistical of glycogen in each organ of each group. (C)H&E stain of organ tissue to show the toxicity of each type of nanoparticle through the tissue morphology. The organs are liver, spleen, kidney, heart, and pancreas.

Figure 5. In vitro test of cell toxicity, glucose absorption and nanoparticle intake. (A) Caco-2 cell viability under incubation of different concentration of PGLA nanoparticles, CSPGLA nanoparticles and Gen-CS-PGLA nanoparticles. (B) The quantity of Glut4 transported to cell membrane. (C) The quantity of insulin absorbed. (D) FITC-Insulin loaded by three nanoparticles absorbed by Caco-2 cells. (E) Cy5 tagged anti-insulin distribution in Caco-2 cell.

Figure 6. Schematic mechanism of three types of nanoparticles based on PGLA backbones. As insulin loaded nanoparticles penetrating through the intestinal epithelium and being transported through blood vessel to the target cell, insulin is gradually released and combine with the receptor on the membrane, triggering series of reaction.


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Figure 1. Schematic mechanism of nanoparticles fabrication. (A) Structure of PGLA, Chitosan and chemical reaction between chitosan and genipin. (B) Schematic representation of synthetic process of PGLA, CSPGLA, and Gen-CS-PGLA nanoparticles based on W/O/W double emulsion system. 136x199mm (300 x 300 DPI)

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Figure 2. Characteristic of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. (A) FT-IR spectra and (B) Raman spectra of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. (C) TEM morphology of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. Scale bar 100 nm. (D)Size and (E) Zeta potential of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. (F) Thermo gravity of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. Black line is pure insulin, dark blue line is PGLA nanoparticles, light blue line is CS-PGLA nanoparticles, and grey line is Gen-CS-PGLA nanoparticles. (G) Loading capacity and loading efficiency of each type of nanoparticle. (H) CD spectra of insulin released from PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles. (I) Insulin release of PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles in pH=1 and pH=6.8 buffer respectively.

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Figure 3. T1D model, in vitro test of absorption and pharmaceutical test. (A) Physical characteristic comparison of induced T1D rat and normal rat. (B) (a)Blood glucose, (b) and (c) amount of food and water took in by rats and (d) their body weight changing during the T1D inducing progress. (C)Blood glucose levels in diabetic rats following oral administration of insulin solution, PGLA, CS-PGLA and Gen-CS-PGLA nanoparticles at an insulin dose of 50 IU/kg, and subcutaneous injection of insulin at an insulin dose of 5 IU/kg (Mean ± SD, n=3). Significant difference from oral insulin solution and from PGLA, CS-PGLA, Gen-CSPGLA nanoparticles, P < 0.05. (D)Intestinal absorption of saline (a) (b), PGLA nanoparticle (c) (d), CS-PGLA nanoparticle (e) (f), Gen-CS-PGLA nanoparticle (g)(h). Blue fluoresce is DAPI stained nuclei, green fluoresce is FITC-insulin delivered by PGLA, CS-PGLA, Gen-CS-PGLA nanoparticles. Scale bar of first line is 50 µm, and of the second line is 15 µm. 187x176mm (300 x 300 DPI)


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Figure 4. Long-term toxicity and efficacy of three type of nanoparticle administration. (A) Symptoms of diabetes changes during the experiment. Dark blue line represents the group gavage PGLA nanoparticles each day, light blue line represents the CS-PGLA group, grey line represents the Gen-CS-PGLA group, and black line represents the control group. (a) Amounts of drinking. (b) Amounts of eating. (c) Body weight. (d) Schematic representation of the changes of T1D rats during the process. (B1) PAS stain of organs from each group. Liver, muscle, kidney, heart from control, PGLA, CS-PGLA and Gen-CS-PGLA group. (B2)Quantitative statistical of glycogen in each organ of each group. (C)H&E stain of organ tissue to show the toxicity of each type of nanoparticle through the tissue morphology. The organs are liver, spleen, kidney, heart, and pancreas.

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Figure 5. In vitro test of cell toxicity, glucose absorption and nanoparticle intake. (A) Caco-2 cell viability under incubation of different concentration of PGLA nanoparticles, CS-PGLA nanoparticles and Gen-CS-PGLA nanoparticles. (B) The quantity of Glut4 transported to cell membrane. (C) The quantity of insulin absorbed. (D) FITC-Insulin loaded by three nanoparticles absorbed by Caco-2 cells. (E) Cy5 tagged anti-insulin distribution in Caco-2 cell.

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Figure 6. Schematic mechanism of three types of nanoparticles based on PGLA backbones. As insulin loaded nanoparticles penetrating through the intestinal epithelium and being transported through blood vessel to the target cell, insulin is gradually released and combine with the receptor on the membrane, triggering series of reaction. 210x297mm (300 x 300 DPI)


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