Poly(2-isobutyl

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pH- and Amylase-Responsive Carboxymethyl Starch/Poly(2-Isobutyl-acrylic acid) Hybrid Microgels as Effective Enteric Carriers for Oral Insulin Delivery Liang Liu, Ying Zhang, Shuangjiang Yu, Zhen Zhang, Chaoliang He, and Xuesi Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00215 • Publication Date (Web): 17 Apr 2018 Downloaded from http://pubs.acs.org on April 18, 2018

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Biomacromolecules

pH- and Amylase-Responsive Carboxymethyl Starch/Poly(2-Isobutyl-acrylic acid) Hybrid Microgels as Effective Enteric Carriers for Oral Insulin Delivery

Liang Liu,a,b Ying Zhang,a,b Shuangjiang Yu,a Zhen Zhang,a,b Chaoliang He,a,b,* Xuesi Chena,b,*

a

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China b

University of Chinese Academy of Sciences, Beijing 100039, P. R. China

* Corresponding authors. E-mail addresses: [email protected] (C. He); [email protected] (X. Chen).

1

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Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract: Oral delivery of insulin has the potential to revolutionize diabetes care since it is regarded as a non-invasive therapeutic approach without the side effects caused by frequent subcutaneous injection. However, the insulin delivery efficiency through oral route was still limited, likely due to the chemical, enzymatic and absorption barriers. In this study, a novel type of pH- and amylase-responsive microgels as an insulin drug carrier for oral administration was developed to improve the drug delivery efficiency. The microgels were prepared via aqueous dispersion copolymerization of acrylate-grafted-carboxymethyl starch (CMS-g-AA) and 2-isobutyl-acrylic acid (iBAA). The resulting hybrid microgels with the PiBAA contents of 13.6 – 45.3 wt% exhibited sharp pH-sensitivity, which was revealed by the changes in particle size of the microgels and the transmittance of the microgel aqueous solution. The accelerated decomposition of the CMS-containing microgels in response to amylase was demonstrated by chromogenic reaction and morphology change. Insulin was loaded into the microgels by swelling-diffusion method, and the insulin release from the insulin-loaded microgels in vitro was found to be triggered by pH change and addition of amylase, which was highly dependent on the microgel component. Cytotoxicity assay was performed to show the good biocompatibility of the microgels. In addition, the tests of cellular uptake by Caco-2 cells and transmembrane transport through the Caco-2 cell monolayers were carried out to confirm the intestinal absorption ability of the insulin-loaded microgels. Finally, the oral administration of insulin-loaded microgels to STZ-induced diabetic rats led to a continuous decline in the fasting blood glucose level within 2 to 4 h, and the hypoglycemic effect maintained over 6 h in vivo. The relative pharmacological availability of the insulin-loaded microgels was enhanced 23 – 38 times compared to free-form insulin solution through oral route. Therefore, the novel starch-based microgels may have potential as an efficient platform for oral insulin delivery. Keywords: microgel; pH-responsive; amylase-responsive; carboxymethyl starch; oral insulin delivery 2


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1. Introduction. Diabetes mellitus, as a metabolic chronic disease characterized by either the inability of pancreatic islets to produce insulin (type 1) or the failure to respond to it (type 2), are currently affecting 415 million patients worldwide, and the number is predicted to increase to 642 million by 2040.1-3 In order to treat hyperglycemia caused by diabetes, the subcutaneous injection of exogenous insulin is the most conventional and efficient way to promote the absorption of glucose and its conversion into glycogen by human body.4-6 However, the subcutaneous approach to peripheral circulation bypasses the portal circulation and may not replicate the normal dynamics of endogenous insulin secretion, which may reduce glucose control efficiency and lead to diabetic complications.4,7 Besides, the long-term frequent injection of insulin may cause low patient compliance and serious side effects, such as local tissue necrosis and infection, nerve damage, hypoglycemia, insulin resistance, weight gain, allergy and hypokalemia.8-10 For a long time, the oral route is considered as the most convenient and comfortable method for administering drugs to patients. Although the oral delivery of insulin has been attempted since its discovery in 1922,11,12 the oral bioavailability of insulin is severely hampered by its inherent instability in the gastrointestinal (GI) tract and its low permeability across biological membranes in the intestine, which has been reported to be less than 1%.13-15 To overcome the gastrointestinal barriers and improve the penetration of insulin through the intestinal epithelial membranes, various oral carriers have been developed in recent years and the clinical trials of several oral insulin formulations have been tested.16-19 However, success in commercialization for oral administration of insulin is still limited.20 Stimuli-responsive microgels have been widely explored for drug delivery systems over the past two decades, attributed to their rapid swelling-deswelling transitions in response to variation of surrounding environments.21-23 Given their small size, fast response and environment-triggered release manner, microgels offer unique opportunities for drug administration in oral route, including 3



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oral insulin delivery.24-26 For instance, a range of microparticles based on poly(ethylene glycol)-grafted-poly(methacrylic acid) (P(MAA-g-EG)) hydrogel particles were developed for oral insulin

delivery

by

Peppas

and

co-workers.

These

carriers

exhibited

pH-dependent

swelling-shrinking transitions in GI tract. Besides, further evaluations demonstrated the good biocompatibility, mucoadhesivity, protease inhibition, absorption enhancement, and marked hypoglycemic effect (~9.5% of bioavailability) of the microparticles.27-30 Sung and colleagues have investigated the feasibility of chitosan nanoparticles (NPs) for oral delivery of insulin.31-33 The NPs were prepared via a simple ionic gelation between chitosan (CS) and poly(γ-glutamic acid) (γ-PGA), with insulin encapsulated in this process. The crosslinked NPs showed high affinity to intestinal mucosa and enhanced insulin absorption through the paracellular pathway, resulting in a bioavailability of 15.1%. Furthermore, many other polymeric carriers based on microgels have been exploited to package insulin effectively for oral delivery. Santos et al. prepared an alginate-dextran sulfate (ADS) microgel with the emulsification/internal gelation technique to protect insulin from gastrointestinal attack and to promote its permeation through intestinal epithelium.34 Nolan et al. developed poly(N-isopropylacrylamide)

(PNIPAm)

microgels

and

a

thin

film

of

poly(N-isopropylacrylamide-co-acrylic acid) (poly(NIPAm-co-AA)) microgels, and investigated the thermally triggered insulin release behaviors of these systems.35,36 Bai et al. prepared microgels by the free-radical emulsion polymerization of acrylate-grafted-hydroxypropyl cellulose and poly(L-glutamic acid-2-hydroxylethyl methacrylate) for oral delivery of insulin.37 The thermo- and pH-responsiveness of the microgels were investigated in vitro. The loading and release of insulin were studied in the simulated environment of GI tract. Kim and co-workers developed the insulin-loaded beads for oral administration based on the terpolymers of N-isopropylacrylamide, butyl methacrylate and acrylic acid. The release manner of insulin from the beads was found to depend on the molecular weight (MW) of the terpolyers. By changing the MW, the polymeric beads 4


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could be adapted to release insulin in duodenum, lower small intestine and colon, respectively.38 Most of the oral microgel devices mentioned above focused on the shrinking-to-swelling transitions triggered by the variation of pH from strongly acidic condition of the stomach to neutral condition of the small intestine. Thus, insulin encapsulated inside the microgels may be protected in the gastric environment but quickly released at desired sites in the intestinal lumen, where insulin is expected to be absorbed by the intestinal epithelial membrane into systemic circulation. Nevertheless, the interactions between microgels and intestinal cells, the absorption process of insulin by small intestine, and the in vivo hypoglycemic effects still remained unclear. In this work, a series of pH- and amylase-responsive microgel carriers were developed for oral administration of insulin, focusing on the pH change in GI tract and the amylolytic environment in small intestine. The microgel carriers, denoted as CMS/PiBAA hybrid microgels, were prepared via aqueous

dispersion

copolymerization

acrylate-grafted-carboxymethyl

starch

of

2-isobutyl-acrylic

(CMS-g-AA).

acid

(iBAA)

and

Poly(2-isobutyl-acrylic acid) (PiBAA)

undergoes abrupt ionization-deionization transition at pH 6.0 around the pKa of iBAA, which may be an ideal material for protecting insulin in gastric acid environment but selectively releasing the protein in neutral small intestine (Scheme 1). Moreover, carboxymethyl starch (CMS) possesses advantages in the oral delivery systems including nontoxicity, biocompatibility, bioadhesion to mucosa membranes with hydrogen bond, and rapid enzymatic degradation property in response to several enzymes in the small intestine, such as α-amylase.39-41 Hence, the CMS/PiBAA hybrid microgels are designed to act as a platform for the intestine-targeting oral delivery system, which are expected to timely decompose in the neutral small intestine in the presence of α-amylase. Compared to the microgels with single pH-sensitivity, the pH- and amylase-responsive microgels are expected to exhibit accelerated insulin release rates, which were triggered by the pH-variation and enzymatic degradation in intestinal lumen (Scheme 1). Thus, more rapid hypoglycemic effect might be achieved for practical application. To investigate the cellular internalization of the microgel carriers 5



and the intestinal absorption of insulin, the endocytic assay and transmembrane transport of insulin-loaded microgels were carried out on Caco-2 cells and cell monolayers, mimicking the enterocytes and intestinal epithelium. The pharmacodynamics assay on Type-1 diabetic rats was performed to evaluate the oral availability of the insulin-loaded microgels. Overall, the feasibility of using the CMS/PiBAA hybrid microgels as a carrier for oral insulin delivery was studied in vitro and in vivo.

6


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Scheme 1. Proposed schematic illustration for the behaviors of the insulin-loaded microgels during the delivery process in the GI tract.

2. Materials and methods. 2.1. Materials. Carboxymethyl starch (CMS, Medicinal Grade), ammonium persulfate (APS, 99.99% metals basis), 4-dimethylaminopyridine (DMAP, 99%), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl, 98%) were purchased from Aladdin, Shanghai, China. Acrylic acid (AA) was obtained from Shanghai Chemical Reagent Co. (China) and purified by vacuum distillation. Diethyl isobutylmalonate (98%), diethylamine hydrochloride (99%), and paraformaldehyde (96%) were acquired from Adamas. Co., Shanghai, China. Porcine insulin (29.5 IU/mg) was provided by Xuzhou Wanbang Bio-Chemical Co., Ltd. (Jiangsu, China). Pepsin (250 U/mg) from porcine gastric mucosa, trypsin (1500 U/mg) from porcine pancreas, α-amylase (50 U/mg) from porcine pancreas, carboxymethyl cellulose (CMC, 0.7 carboxymethyl groups per anhydroglucose unit, 90,000 Da), rhodamine B (RhoB), and fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The porcine Insulin ELISA Kits were purchased from Mercodia, Sweden. 2-isobutyl-acrylic acid (iBAA) was synthesized through a procedure similar to the previous literatures (supporting information: Section S1.1).42,43 The acrylate-grafted carboxymethyl starch (CMS-g-AA) was synthesized by a coupling reaction according to our previous work (supporting information: Section S1.2).44 The artificial gastric fluid (AGF, pH 1.2) and the artificial intestinal fluid (AIF, pH 6.8) were prepared according to our previous method.44,45 To prepare AGF, 3.5 mL of hydrochloric acid (37 wt%) and 1.0 g of sodium chloride were dissolved in 500 mL of deionized water. To obtain AIF, potassium dihydrogen phosphate (3.4 g) was dissolved in 250 mL of deionized water, then the pH value was adjusted to 6.8, followed by diluting the solution to 500 mL. 7



2.2. Preparation of CMS/PiBAA hybrid microgel. The CMS/PiBAA hybrid microgels were prepared via the following procedures. Typically, the mixtures of CMS-g-AA and iBAA were dissolved and stirred in 500 mL of deionized water. APS was added to initiate the free radical copolymerization at the temperature of 70 oC. The reaction was allowed to proceed under nitrogen protection for 12 h. The feeding amounts of the monomer and other reactants are listed in Table 1. Finally, the microgel products were collected by centrifugation at 15,000 rpm, neutralized with dilute NaOH solution, and purified by dialysis against deionized water for 3 days, followed by lyophilization. As shown in Table 1, with varying the iBAA content, four different hybrid microgels were obtained, which were tagged as Microgel-1, Microgel-2, Microgel-3 and Microgel-4.

2.3. Preparation of rhodamine B labeled microgel. The CMS/PiBAA hybrid microgels were labeled by rhodamine B (RhoB), which is widely used as a natural fluorescent dye with excitation wavelength of 552 nm and emission wavelength of 610 nm at physiological condition.46 Typically, RhoB (0.048 g) was dissolved in deionized water with EDC·HCl (0.023 g) and DMAP (0.005 g) for activation of the carboxyl groups. After 24 h, 1 mL of the microgel suspension in deionized water (2 mg/mL) was added into the mixture, followed by stirring for another 48 h. Finally, the RhoB-labeled microgel (named as RhoB-MG) was collected by centrifugation at 15,000 rpm, and purified through dialysis against deionized water for 3 days to get rid of the dissociative RhoB, followed by lyophilization. The amount of RhoB labeled on the microgel was determined by spectrofluorometer (Photon Tech. Int’l. Inc., ASOC-10), as listed in

Table S1 (supporting information). RhoB labeled Microgel-1, Microgel-2, Microgel-3 and Microgel-4 were tagged as RhoB-MG-1, RhoB-MG-2, RhoB-MG-3 and RhoB-MG-4, respectively.

2.4. Characterization. 1

H and 13C NMR spectra were recorded on a Bruker 400-MHz spectrometer. CDCl3 was used as the

solvent for 2-isobutyl-malonate and iBAA, while D2O for CMS and CMS/PiBAA microgel samples. 8


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FT-IR spectra were recorded on a Bruker Vertex 70 Fourier Transform Infrared spectrometer with the KBr disk method. Elemental analysis was conducted on a Vario-EL-Cube elementar (Elementar Co., Germany) to test the substitution degree of acrylate groups in CMS-g-AA and the components of the resulting CMS/PiBAA microgels as listed in Table 1.44,45 Meanwhile, the conductometric titrations were performed on a REX-DDSJ-308F conductivity titrator (INESA Scientific Instrument Co., Ltd, Shanghai, China) to further determine the compositions of the microgels through a procedure similar to the previous literatures (supporting information: Section S1.3).47,48

2.5. Particle size and zeta-potential of CMS/PiBAA microgel. The particle size and zeta-potential of the microgel samples in aqueous solution were measured by a Zeta Potential/BI-90Plus Particle Size Analyzer (Brookhaven, USA). All samples were prepared in aqueous solution at the concentration of 0.1 mg/mL. The measurements were carried out at 37 oC by adjusting the pH condition for each sample from 1.0 to 8.0 with dilute HCl or NaOH solution, and the ionic strength was controlled to be 0.15 M by adding sodium chloride (NaCl). By alternately changing the pH value of the microgel suspensions to pH 1.2 (simulating the pH environment of the stomach) or to pH 6.8 (simulating the pH environment of the small intestine), the pH-cycling experiments were conducted to evaluate the pH-dependent swelling-shrinking-swelling transitions of the microgels. The particle size and zeta-potential were both monitored in this process. The microgels were held at the given pH for 30 min to reach the equilibrium prior to the measurements. The dispersion stability of the microgel suspensions was investigated by testing the particle size of the microgels in the artificial gastric fluid (AGF, pH 1.2) or in the artificial intestinal fluid (AIF, pH 6.8) at distinct time intervals within 72 h.

2.6. Morphology of CMS/PiBAA microgel. The morphology of the CMS/PiBAA microgels was investigated by JEM-1200EX TEM System (NEC, Tokyo, Japan). A drop of microgel solution (dispersed in AGF or in AIF, with or without 10 9



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U/mL of α-amylase) was deposited onto a holey carbon TEM grid and air-dried. Then the measurements were performed to observe the morphology under different conditions. The degradation of microgel under α-amylase was terminated by adding trypsin solution at a given time point.49

2.7. Enzymatic degradation of CMS/PiBAA microgel. The enzymatic degradation rate of the CMS/PiBAA microgel was investigated by observing the chromogenic reaction degree between starch and iodine. The CMS component in the CMS/PiBAA microgel might be rapidly degraded by α-amylase, thus the microgel solution gradually turned from dark blue to colorless under the indicator of iodine. Typically, the carboxymethyl cellulose (CMC), the carboxymethyl starch (CMS) and the microgel sample were dissolved in AIF at the concentration of 1.0 mg/mL, 1.0 mg/mL and 1.4 mg/mL, respectively. Then, 10 µL of fresh iodine indicator was added into each of the sample solutions (2 mL). Then, α-amylase (10 U/mL) was added to each solution. The degradation was monitored every half an hour at 37 oC away from light.

2.8. In vitro drug loading and release study. Insulin was loaded into the CMS/PiBAA microgels with a swelling-diffusion method.37 Typically, insulin solution was prepared at the concentration of 0.2 mg/mL (pH = 7.4). Then, 40 mg of dried microgel sample was immersed into 100 mL of insulin solution and stirred for 24 hours to reach the swelling equilibrium at 37 oC. The insulin-loaded microgels were collected by centrifugation at 15,000 rpm, and rinsed to remove the unloaded insulin with AGF, followed by lyophilization. The combined supernatant was diluted to detect the concentration of remained dissociative insulin on a UV/Vis spectrophotometer (Shimadzu UV-2401PC) at the wavelength of 280 nm. The drug loading content (DLC) and drug loading efficiency (DLE) were calculated using the following equations.31 DLCሺ%ሻ=

WIL WMG +WIL

DLEሺ%ሻ=

×100

10


WIL WI0

×100

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where WIL is the weight of insulin encapsulated inside the microgel, WI0 is the total weight of the insulin before loading, and WMG is the weight of the dry microgel specimen. The tests were performed in triplicate. To investigate the drug release behavior, dried powder of the insulin-loaded microgel was dispersed into 10 mL of AGF or AIF (with or without α-amylase, 10 U/mL), poured into a precise dialysis bag (MWCO 10 kDa) and placed in a beaker containing 90 mL of fresh release medium (AGF or AIF), under a shaking rate of 100 rpm at 37 oC to mimic the peristalsis of GI tract. At distinct time points, 0.5 mL of external medium was taken out and replaced by 0.5 mL of fresh medium. The concentration of the released insulin was determined by a UV/Vis spectrophotometer, and the cumulative release curve in vitro was calculated. All the release points for each sample were carried out in triplicate.

2.9. Internalization of CMS/PiBAA microgel by Caco-2 cells. In vitro cell culture studies were carried out on Caco-2 cells and cell monolayers.27,29,31 Caco-2 cells (human colon carcinoma, clone 1) were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Caco-2 cells were cultivated on cell culture flasks using MEM cell culture medium (Grand Island, NY, USA) with 10% fetal bovine serum. To evaluate the distinction in endocytic capacity of different microgel carriers, the cellular uptake study was performed with the RhoB-labeled microgel samples (RhoB-MG, as prepared in Section

2.3). Typically, Caco-2 cells were seeded on 12-well plates at 2.0×105 cells/well and incubated for 24 h. Then, the culture medium was replaced by the uptake medium, into which the RhoB-MG samples were dispersed. With approximately equal amount of rhodamine B dye tagged on the four groups of microgels (Table S1, supporting information), the concentration of RhoB-MG sample in the uptake medium for each group was set as 1.0 mg/mL. After 60 min of incubation, the amount of microgel particles inside Caco-2 cells was determined by testing the intracellular fluorescent intensity of rhodamine B with a Guava EasyCyte flow cytometer (Guava Technologies). Herein, the 11



free-form rhodamine B dye was used as the negative control for the cellular uptake assay at the concentration of 10 µg/mL, which was determined according to the weight content (approximately 1.0 wt%) of rhodamine B dye labeling on the microgel sample for each group. Insulin was covalently labeled with FITC.46 And the resulting FITC-labeled insulin (FITC-Ins) was loaded into the microgel samples. The drug loading contents (DLC) and drug loading efficiencies (DLE) of FITC-Ins for the samples were all shown in Table S2 (supporting information). The FITC-Ins loaded microgel samples were employed to conduct the cellular uptake assay described above in Caco-2 cells, to investigate the endocytosis of insulin by enterocytes after encapsulation into the microgel carriers. In this process, the FITC-Ins loaded microgel sample was dispersed into the uptake medium of each well (containing 50 µg/mL of FITC-Ins), and Caco-2 cells were placed in the uptake medium for 60 min. The free-form FITC-Ins (50 µg/mL in the uptake medium) was used as the negative control. The endocytic capacity of FITC-Ins for each group was reflected by the fluorescent intensity of FITC in Caco-2 cells. Also, Caco-2 cells were incubated for 60 min with the mixture of the free-form FITC-Ins (50 µg/mL in the uptake medium) and the empty microgel sample. The dosage of empty microgel sample in the mixture was equal to that for the FITC-Ins loaded microgel sample in this cellular uptake assay, and the endocytic capacity of FITC-Ins was also determined by the flow cytometer. Furthermore, the impacts of enzymatic degradation on the microgel carriers under the effect of amylase were taken into consideration in the cellular uptake assay via the following procedure. Typically, the culture medium was replaced by the uptake medium, in which the FITC-Ins loaded microgels were dispersed. And each group was ensured to contain 50 µg/mL of FITC-Ins in the uptake medium. Also, α-amylase (10 U/mL) was added into the uptake medium for each group to mimic the amylolytic environment in small intestine. The medium both containing free-form FITC-Ins (50 µg/mL) and α-amylase (10 U/mL) was used as the negative control. After incubation

12


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for different time intervals (15 min, 30 min, 60 min or 120 min), the cells were collected to determine the endocytic fluorescent intensity of FITC-Ins with flow cytometer. To visualize the cellular uptake of FITC-Ins that was loaded in the microgels, and the empty RhoB-MG samples, Caco-2 cells were seeded on coverslips in 6-well plates at 2.0×105 cells/well and incubated for 24 h. After that, the culture medium was replaced by the uptake medium, into which the FITC-Ins loaded microgel samples (containing 50 µg/mL of FITC-Ins for each group) or the empty RhoB-MG samples (1.0 mg/mL) were added. After incubation in the uptake medium for 60 min, the Caco-2 cells on cover-slips were washed thoroughly with PBS to remove the extracellular residue, and fixed with 3.7% paraformaldehyde for 15 min at room temperature. The cell nucleus were stained by 4'-6-diamidino-2-phenylindole (DAPI, 1.0 mg/mL, 1 µL per well) for 15 min, and the cell membranes were stained with 5 µL/well Alexa Fluor 488 phalloidin for 20 min at 37 oC. Finally, the cover-slips were taken out and placed on the slides, enclosed with glycerol. The samples were observed by CLSM (ZEISS-LSM780, Germany).

2.10. Transmembrane transport. The transmembrane transport was carried out to investigate the permeability of the mirogels loaded with FITC-Ins through Caco-2 cell monolayers. Briefly, Caco-2 cells were cultured on the polycarbonate filter membrane at a density of 3.0 × 105 cells/well in Costar Transwell 24 well/plates (membrane pore diameter of 3 µm, Corning Costar Corp., NY), and were used for transport experiments about 21 days after seeding. The transepithelial electric resistance (TEER) values were measured with an Epithelial Volt-Ohm Meter (Millicell ERS-2 MILLIPORE Corp., USA) to verify the integrity of the cell monolayers. When the TEER values were in the range from 180 to 200 Ω·cm2, the grown cell monolayers were incubated with different FITC-Ins loaded microgels in the upper compartments. The concentration of FITC-Ins was set as 50 µg/mL in each well. In the transport process, the culture medium of the lower compartments was detected by spectrofluorometer to determine the cumulative amount of FITC-Ins that permeated through Caco-2 cell monolayers 13



from the upper compartments. Additionally, the transmembrane transport of the empty RhoB-labeled microgel (RhoB-MG) was investigated according to the same procedure. The grown cell monolayers were incubated with different RhoB-MG samples (1.0 mg/mL) in the upper compartments, and the medium of the lower compartments was detected for the fluorescent intensity of rhodamine B to determine the cumulative amount of the microgels across Caco-2 cell monolayers.

2.11. In vivo pharmacodynamics assay. Type 1 diabetic rats (Wistar rat, male, 180 – 220 g) were induced by streptozotocin (STZ, dissolved in citrate buffer solution, pH 4.5) via a single intraperitoneal injection at the dose of 55 mg/kg body weight, and with the fasting blood glucose level of over 16.7 mmol/L after injection. The diabetic rats were fasted overnight and remained fasted during the period of experiment, but were allowed to drink water.44,45 The rats were randomly divided into seven groups (six rats per group, n = 6). The dehydrated insulin-loaded microgel powder or blank microgel powder was dispersed in AGF, followed by immediately intragastric administration with an oral gavage needle. For pharmacodynamics assay, the rats were orally administered according to the following formulations: (a) the insulin-loaded Microgel-1 in the insulin dosage of 60 IU/kg, (b) the insulin-loaded Microgel-2 in the insulin dosage of 60 IU/kg, (c) the insulin-loaded Microgel-3 in the insulin dosage of 60 IU/kg, (d) the insulin-loaded Microgel-4 in the insulin dosage of 60 IU/kg, (e) the free-form insulin with 60 IU/kg dose, and (f) the empty Microgel-4 at the dose of 20 mg/kg. Besides, the group treated with subcutaneous (S.C.) injection of insulin solution (5 IU/kg) was regarded as the positive control with 100% pharmacological availability of insulin. The bioactivities (IU/mg) for the native porcine insulin and the insulin-loaded microgels were determined with the porcine Insulin ELISA Kits, and were provided in Table S2 (supporting information). During the experimental process, blood samples were collected from the tail veins of rats at distinct time intervals after treatment.32,46 The blood glucose levels were determined with a glucocard G meter (GT-1810, ARKRAY Inc., Japan). The 14


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relative pharmacological availability (rPA, %) in oral route was derived from the activity curves with the equation below.44,45 rPAሺ%ሻ=

[AAC]Oral ×DoseS.C. ×100 [AAC]S.C. ×DoseOral

where [AAC]oral and [AAC]s.c. are the area above curves, that are relative to oral administration and subcutaneous injection of insulin. Doseoral and Doses.c. are the insulin doses administered in oral and S.C. route.

2.12. Animal procedure: The experiments on animals were carried out according to the guide for the care and use of laboratory animals, provided by Jilin University, Changchun, China, and the procedure was approved by the local Animal Ethics Committee.

2.13. Statistical analysis. The statistical differences of the control and experimental groups were analyzed by using the One-Way ANOVA test. In all cases, when p value was less than 0.05 (p < 0.05), the difference was considered to be statistically significant. All experiments were performed at least three times, and the data are presented as mean ± standard deviation.

3. Results and discussion. 3.1. Synthesis and characterization of CMS/PiBAA hybrid microgel. As shown in Scheme 2A, the monomer of 2-isobutyl-acrylic acid (iBAA) was successfully synthesized by two steps of reactions (supporting information: Section S2.1, Fig. S1). Meanwhile, CMS-g-AA, which was used as both the co-monomer and the macromolecular crosslinker, was prepared by the esterification between the hydroxyl groups of carboxymethyl starch (CMS) and the carboxyl groups of acrylic acid (AA), with EDC·HCl and DMAP as the coupling agent and catalyst, respectively (Scheme 2B) (supporting information: Section S2.2, Fig. S2).

15



Scheme 2. Synthetic route of 2-isobutyl-acrylic acid (iBAA), acrylate-grafted-carboxymethyl starch (CMS-g-AA), and the CMS/PiBAA hybrid microgel.

The CMS/PiBAA hybrid microgels with different CMS/PiBAA proportions were prepared via the aqueous dispersion co-polymerization of acrylate-grafted-carboxymethyl starch (CMS-g-AA) and 2-isobutyl-acrylic acid (iBAA), using ammonium persulfate (APS) as the free radical initiator at 70 o

C (Scheme 2C). As shown in Table 1, by increasing the feed ratios (wt/wt) of iBAA/CMS-g-AA

from 0.15:1 to 0.90:1, four hybrid microgels with the PiBAA contents of 13.6 – 45.3 wt% were obtained, and were denoted as Microgel-1, Microgel-2, Microgel-3 and Microgel-4, respectively. As the co-polymerization proceeded, the formation of PiBAA segments afforded adequate hydrophobicity to drive the precipitation of the polymer chains and nucleation of the hybrid polymeric networks. Meanwhile, the incorporation of hydrophilic CMS chains sterically stabilized the particles, preventing the formation of macroscopic aggregates. Thus, due to the above mechanism, the conventional surfactants could be avoided in the preparation of the CMS/PiBAA hybrid microgels. After purification, the acquired microgel suspensions were adjusted to neutral, leading to 16


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the ionization of the carboxyl groups. The freeze-dried microgels were characterized by 1H NMR in deuterium water (D2O) (Fig. 1A). The peaks at approximately 3.65 ppm in the 1H NMR spectra were ascribed to the protons on the carbons of the anhydroglucose units in CMS or the microgels. In comparison with CMS, a new peak at 0.72 ppm assigned to the methyl protons (-CH3) of isobutyl groups of PiBAA was observed in the 1H NMR spectra of the microgels. Moreover, the peak intensity of the methyl protons of iBAA enhanced in the order of Microgel-1, Microgel-2, Microgel-3 and Microgel-4, indicating the increase of PiBAA content in the microgel networks. Furthermore, the FT-IR spectra of CMS-g-AA, Microgel-1, Microgel-2, Microgel-3 and Microgel-4 were measured, as shown in Fig. 1B. Compared with CMS-g-AA, the IR spectra of the microgel samples displayed a new absorption peak at 1727 cm-1 (νC=O”), which might be assigned to the carbonyl group of iBAA. Also, the absorption peaks at 1603 cm-1 and 1653 cm-1 appeared for both CMS-g-AA and the microgels, attributing to the carbonyl of the carboxymethyl from CMS and the acrylate side groups. These results clearly suggested the successful preparation of the hybrid microgels composed of PiBAA and CMS. As listed in Table 1, elemental analysis and conductometric titration analysis were performed to estimate the compositions of the microgels.47,48 According to the titration analysis, the PiBAA contents and conversions of iBAA were in the ranges of 13.6 wt% – 45.3 wt% and 85.9% – 67.2%, respectively, with increasing the iBAA/CMS-g-AA feed ratio (Table 1).

Table 1. Feed ratios and compositions of microgel samples

Sample codes

Feeding amounts

PiBAA (wt%)[a]

PiBAA (wt%)[b]

Yield (%)[c]

Conversion of iBAA (%)[d]

50

10 ± 2

13.6 ± 1.3

82

85.9 ± 8.1

0.30

50

18 ± 1

24.1 ± 2.1

78

81.2 ± 6.9

0.60

50

28 ± 2

36.9 ± 3.7

79

78.1 ± 7.8

CMS-g-AA [g]

iBAA [g]

APS [mg]

Microgel-1

1.0

0.15

Microgel-2

1.0

Microgel-3

1.0

17



Microgel-4 a)

1.0

0.90

50

38 ± 2

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45.3 ± 3.1

70

67.2 ± 4.6

Weight content of PiBAA in dried microgels, calculated from elemental analysis results.

b)

Calculated from conductometric titration results. c)Calculated from the following equation: Yield

(%) = WM/(WC + Wi), where WM is the weight of freeze-dried microgel samples, WC is the feeding weight of CMS-g-AA and Wi is the feeding weight of iBAA. d)Conversion of the monomer iBAA in the copolymerization with CMS-g-AA, calculated from conductometric titration results.

Figure 1. (A) 1H NMR spectra of CMS, Microgel-1, Microgel-2, Microgel-3 and Microgel-4 in D2O, respectively. (B) FT-IR spectra of CMS-g-AA (a), Microgel-1 (b), Microgel-2 (c), Microgel-3 (d) and Microgel-4 (e), respectively.

3.2. pH-sensitivity and swelling behaviors of the microgels. Due to the ionization-deionization transition of the carboxyl groups of the PiBAA and CMS segments in response to the pH change, the CMS/PiBAA microgels exhibited pH-dependent swelling-deswelling behaviors, likely leading to the change in particle size and zeta-potential. Therefore, the particle size and zeta-potential measurements were performed for the microgel suspensions in different pH conditions from 1.0 to 8.0. As shown in Fig. 2A, it was found that, with varying the pH values, the microgel with a higher PiBAA content displayed a more marked change 18


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in particle size, implying a more drastic swelling-to-deswelling transition. For instance, with increasing pH from 1.0 to 8.0, the microgel with the lowest PiBAA content of 13.6 wt% (Microgel-1) showed an increase in particle size from 480.1 nm to 642.8 nm, whereas the sample with the highest PiBAA content of 45.3 wt% (Microgel-4) exhibited a drastic augment of the particle size from 415.6 nm to 933.3 nm. This phenomenon could be attributed to the following two aspects: On one hand, a lower CMS content led to a lower crosslinking density and therefore a higher swelling degree of the microgel networks when the carboxyl groups were ionized at a neutral pH value. Additionally, the hydrophobic isobutyl side group of iBAA resulted in an enhanced pKa value of PiBAA segments compared to polyacrylic acid (PAA) and polymethacrylic acid (PMAA) (pKa < 5.0), rendering an abrupt protonation-to-deprotonation transition when the pH increased over 6.0.42,43,45 Furthermore, in Fig. 2B, with pH increased from 1.0 to 8.0, the zeta-potential of the microgels presented obvious decrease, indicating that the polymeric microgels transformed from uncharged to negatively charged networks, along with the gradual ionization of the carboxyl groups. Moreover, the pH-cycling tests were performed to show the redispersion property of the microgels at acidic stomach pH and neutral intestinal pH, respectively. By alternately switching the pH value to 1.2 or 6.8, the microgel suspensions exhibited reversible swelling-deswelling transitions (Fig. 2C), as well as repeated ionization-deionization transitions (Fig. 2D). The results suggested well redispersibility of the microgels in the intestinal pH.

19



Figure 2. (A) Particle size and (B) Zeta-potential profiles of the microgel suspensions in different pH conditions (I = 0.15 M). (C) Particle size and (D) Zeta-potential for the microgel suspensions in the pH-cycling tests between pH 1.2 (simulating the pH environment of the stomach) and pH 6.8 (simulating the pH environment of the small intestine).

3.3. pH-dependent turbidity of the microgels. To investigate the turbidity, UV-Vis transmittance of the microgel suspensions at 550 nm was tested with varying pH (supporting information: Section S1.4). The detection was proceeded for 2 h with each sample at the given pH by continuously recording the UV-Vis transmittance (550 nm), as shown in Fig. S3 (supporting information). With the pH decrease from neutral medium to acidic medium, the microgels underwent swelling-to-shrinking transitions due to the protonation of carboxyl groups, leading to the changes of the refractive index contrast in the microgel suspensions. Therefore, the transmittance of the solutions displayed a marked decline. It was noteworthy that the 20


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solutions showed good transparency at pH 7.0 and 8.0, while the sudden decline in transmittance was observed at pH 6.0 (Fig. S3 and S4, supporting information). This indicated that the PiBAA segments displayed an enhanced pKa value at approximately 6.0. Besides, it was found that the solutions of Microgel-3 and Microgel-4 displayed greater transmittance change in response to pH decrease as compared with Microgel-1 and Microgel-2 solutions. This phenomenon might be attributed to the higher contents of PiBAA segments in Microgel-3 and Microgel-4. 3.4. Dispersion stability of the microgels. The dispersion stability of the microgel suspensions was investigated by testing the particle size of the microgels in the artificial gastric fluid (AGF, pH 1.2) or in the artificial intestinal fluid (AIF, pH 6.8) at distinct time intervals within 72 h. As shown in Fig. 3A, the microgels were well re-dispersed in AGF or AIF, and could keep stable in the subsequent 72 h. After encapsulation of insulin, the redispersibility of the insulin-loaded microgels was considered to be unaffected (Fig. 3B). Also, the particle size measurements for the insulin-loaded microgels after redispersing for 8 h in AGF (pH = 1.2), AIF (pH = 6.8) and MEM cell culture medium (pH = 7.4) were carried out in Fig. S5 (supporting information).

Figure 3. (A) The particle size vs. time profiles of the microgels after re-dispersion in AGF (hollow symbol) or in AIF (solid symbol) within 72 h. (B) The images of the insulin-loaded Microgel-3 after 21



freeze-drying to powder (left), re-dispersing in AGF (center, pH = 1.2, 1.0 mg/mL) and in AIF (right, pH = 6.8, 1.0 mg/mL).

3.5. Enzymatic degradation and morphology of the microgels. To study the enzymatic degradation behavior of the CMS/PiBAA microgels triggered by amylase, the chromogenic reaction between starch and iodine was used to reflect the degree of disintegration of the CMS segments according to the color depth. As shown in Fig 4A, the α-amylase-triggered degradation of Microgel-3 with the CMS content of 63.1 wt% was revealed, with carboxymethyl cellulose (CMC) and carboxymethyl starch (CMS) as the negative and positive control groups, respectively. Before addition of iodine, the CMC, CMS and Microgel-3 solutions in AIF were colorless. After addition of α-amylase and iodine as the indicator, both the CMS and Microgel-3 solutions turned into dark blue immediately, while the CMC solution only showed the yellow color from iodine. After incubation with α-amylase, the CMS solution turned into light blue at 90 min and further changed into colorless at 120 min, indicating the nearly entire decomposition of the CMS skeleton. It was observed that the color of the microgel solution turned into light blue after incubation with α-amylase for 180 min. The result suggested the degradation of the CMS segments in the microgels under the effect of α-amylase. However, the reduced enzymatic degradation rate of the microgel suspensions compared to the dissociative CMS solution might be related to the lower enzyme accessibility of the CMS segments within the microgel networks. The morphologies of the microgels before and after enzymatic degradation were investigated by TEM (Fig 4B). Similar to the measurement of particle size (Fig. 2), the microgels exhibited a spherical morphology with higher size in neutral AIF (b-2). In contrast, the microgels shrunk markedly in acidic AGF (b-1). Furthermore, after incubation with α-amylase in AIF, the microgels showed an obvious change in morphology from sphere to irregular shapes. The amylolytic degradation of CMS segments led to the reduction of the crosslinking density for the microgel 22


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networks. Thus, as shown in Fig. 4B and Fig. S6 (supporting information), the degraded microgels underwent pronounced swelling in the initial stage, but gradually collapsed into smaller fragments. Therefore, the chromogenic reaction and morphology change clearly indicated that the degradation of the microgels occurred in the presence of α-amylase.

Figure 4. (A) Diastasic degradation behavior of Microgel-3 suspension, with the solutions of free-form CMS and CMC as the positive and negative controls, respectively. Through adding iodine as the indicator, the chromogenic reaction was used to monitor the degradation of starch by α-amylase. (B) Morphologies of Microgel-3 suspensions in AGF (b-1) or in AIF (b-2, without α-amylase), as well as morphologies of Microgel-3 suspensions after incubation in AIF with α-amylase for 30 min (b-3), 60 min (b-4), 120 min (b-5) and 180 min (b-6), respectively. The scale bars for all the images represent 1 µm.

3.6. In vitro drug loading and release. Insulin was loaded into the microgels by a swelling-diffusion method, and the insulin-loaded microgels were allowed to shrink by rinsing with acidic solution. The drug loading contents (DLC) and drug loading efficiencies (DLE) of insulin for the microgels were determined, as presented in Fig. S7 and Table S2 (supporting information). The DLCs and DLEs for the four types of microgels were 2.8 – 6.8% and 5.9 – 15.0%, respectively. It was found that the microgels with higher PiBAA 23



content showed a lower DLC and DLE. This may be due to the hindering effect of the abundant isobutyl groups of PiBAA segments, which restricted the penetration of hydrophilic insulin molecules through the meshes of the microgels. To investigate the drug release behaviors in different media, the insulin-loaded microgels were dispersed in AGF, AIF with α-amylase, and AIF without α-amylase, respectively. As shown in Fig. 5, the release rates for the four types of microgels were all restricted in AGF (pH 1.2) due to the shrink of the microgel networks in the acidic condition. With increasing the content of PiBAA segments in the microgels (from Microgel-1 to Microgel-4), the cumulative release of insulin at the given time was further suppressed, implying a better protection of insulin from the acidic stomach environment. It is noteworthy that the release of insulin was markedly accelerated after incubation in AIF (pH 6.8), and the release rates were further enhanced in the presence of α-amylase. Without the presence of α-amylase, insulin gradually diffused out from the swollen microgel networks by a concentration gradient, and the diffusion rate was reduced with increasing the PiBAA content. Notably, with the presence of α-amylase, over 75% of insulin was released from all the four types of insulin-loaded microgels within 2 h. This may be beneficial for a relatively rapid insulin release at the desired sites with the diastasic environment in intestinal lumen.

Figure 5. Cumulative release profiles of the insulin-loaded microgels in AGF (A) or in AIF (B) with α-amylase (solid symbol) and without α-amylase (hollow symbol) (n = 3).

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3.7. Enterocyte absorption of the microgels. It is known that the dimension of the intercellular spaces between the intestinal epithelial cells is in the order of 10 Å, thus the solutes with a molecular radius exceeding 15 Å (approximately 3.5 kDa) can hardly be transported via the paracellular pathway.50 Insulin, as a hydrophilic protein (approximately 5.7 kDa), was severely limited to be absorbed by the small intestine through the paracellular transport.32 Besides, the hydrophilicity of insulin molecules in intestinal lumen also inhibited the transcytosis by enterocytes, leading to a poor transcellular transport performance.17 Hence, due to the intestinal absorption barriers, oral administration of free-form insulin was considered to be invalid in diabetic treatment.17,32 To improve the permeation of insulin through intestinal epithelium, the microgels were used to encapsulate insulin and served as the absorption enhancer for the enterocytes. Based on cytotoxicity tests (supporting information: Section S1.5), the CMS/PiBAA hybrid microgels exhibited good cytocompatibility against Caco-2 cells as a model of enterocytes in the small intestine (supporting information: Section S2.3, Fig. S8). Then, the cellular internalization of blank RhoB-labeled microgel (RhoB-MG) by Caco-2 cells was detected by flow cytometer based on the intracellular fluorescent intensity of rhodamine B (RhoB). As displayed in Fig. 6A, the intracellular fluorescent intensity of RhoB in Caco-2 cells after incubation with the RhoB-MG samples was markedly higher than that in the cells incubated with free-form RhoB dye. Also, it was notable that the RhoB-labeled microgel with the higher CMS content showed the more pronounced intracellular fluorescent intensity of RhoB. These results indicated that the CMS/PiBAA microgels might serve as a promising absorption promoter for enterocytes by encapsulating insulin inside. Furthermore, the cellular uptake assay of microgels loaded with FITC-labeled insulin (FITC-Ins) was carried out on Caco-2 cells (Fig. 6B). Similarly, the intracellular fluorescent intensity of FITC in the Caco-2 cells after incubation with the FITC-Ins loaded microgels was significantly higher than that in the cells incubated with free-form FITC-Ins. Meanwhile, the FITC-Ins loaded microgels with the highest 25



CMS content (FITC-Ins loaded Microgel-1) showed the highest intracellular fluorescent intensity of FITC, suggesting that the intracellular uptake of FITC-Ins was affected by the microgel carriers depending on the CMS content. Besides, Caco-2 cells were also incubated with the blends of free-form FITC-Ins and different empty microgel carriers, and the cellular uptake of FITC-Ins was determined in the same procedure (Fig. 6C). It was noteworthy that no obvious enhancement of FITC-Ins endocytosis in Caco-2 cells was detected by blending free-form FITC-Ins with empty microgels, compared to the marked increase of insulin uptake by the cells after encapsulating FITC-Ins in the microgels. Similarly, it was found that the presence of dissociative CMS or D-glucose at different concentrations did not apparently promote the cellular uptake of free-form FITC-Ins (supporting information: Section S1.7, Fig. S9). Considering the rapid release of insulin triggered by the disintegration of the microgel networks in the amylolytic environment, the microgel-assisted endocytosis was further evaluated in the presence of α-amylase. As shown in Fig. S10 (supporting information), the release behaviors of FITC-Ins loaded microgels were evaluated in MEM cell culture medium containing α-amylase at 37 oC. Also, the impacts of the degradation of the microgel carriers in the presence of amylase were considered in the tests of cellular uptake. As shown in Fig. 6D – 6H, gradually enhanced intracellular fluorescent intensity of FITC was observed in the cells incubated with FITC-Ins loaded microgels with the increase of incubation time from 15 – 120 min. Thus, despite insulin was unavoidably released in a rapid rate, the remained intact or partially degraded microgel carriers might still maintain the ability to promote the transportation of the encapsulated insulin. Moreover, it has been reported that the endocytosis of nanoparticulates in the gut may occur rapidly, being finished within 1 h.51 Therefore, in the small intestine, the proposed microgel-assisted endocytosis was predicted to be effective for improving the absorption of insulin by enterocytes.

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Figure 6. (A) Intracellular fluorescent intensity of rhodamine B (RhoB) in Caco-2 cells after incubation of Caco-2 cells with the empty RhoB-labeled microgel for 60 min, and with the free-form rhodamine B dye as a control. (B) Intracellular fluorescent intensity of FITC after incubation of Caco-2 cells with the FITC-Ins-loaded microgels for 60 min, and with the free-form FITC-Ins as a control. (C) Intracellular fluorescent intensity of FITC after incubation for 60 min of Caco-2 cells with the blend of FITC-Ins and empty microgels, and with the free-form FITC-Ins as a control. Intracellular fluorescent intensity of FITC at different time intervals, after incubation of Caco-2 cells with free-form FITC-Ins (D), FITC-Ins-loaded Microgel-1 (E), FITC-Ins-loaded Microgel-2 (F), FITC-Ins-loaded Microgel-3 (G), and FITC-Ins-loaded Microgel-4 (H), in presence of α-amylase for each group.

The percentage of Caco-2 cells that endocytosed insulin only, microgels only, or both insulin and microgels was determined by simultaneous detection of the fluorescent intensities of FITC and rhodamine B, through incubation of the cells with empty RhoB-MG or RhoB-MG loaded with 27



FITC-Ins (FITC-Ins loaded RhoB-MG). The percentages (%) of the fluorescigenic cells were shown in Fig. 7, and the histogram was provided in Fig. S11 (supporting information). When the cells incubated with empty RhoB-MG samples, the percentage (%) of the cells showing red fluorescence (rhodamine B) continuously reduced with increasing PiBAA content in the microgels, indicating the decrease in endocytosis of the microgels by Caco-2 cells. This result was consistent with the data shown in Fig. 6A. As the cells incubated with FITC-Ins loaded RhoB-MG, the fluorescence for both FITC and rhodamine B were detected in most of the cells, implying the internalization of both insulin and microgel carriers by Caco-2 cells. From these results, it was further confirmed that the microgel-assisted endocytosis occurred by incubating the obtained insulin-loaded microgels with enterocytes.

Figure 7. Intracellular fluorescence intensity of FITC (X-axis) or RhoB (Y-axis) in Caco-2 cells after incubation with the FITC-Ins loaded RhoB-MG samples. The group treated with PBS was set as the negative control.

Moreover, the cellular uptake of empty RhoB-labeled microgel (RhoB-MG), and the endocytosis of microgels loaded with FITC-Ins were both visualized by confocal laser scanning microscopy (CLSM), as shown in Fig. 8 and Fig. S12 (supporting information). The red fluorescence of

28


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rhodamine B from the RhoB-labeled microgel was clearly observed in Caco-2 cells (Fig. 8). The microgel with the highest CMS content (Microgel-1) showed the highest fluorescent intensity, which was in accordance with the data of flow cytometry. Besides, the successful internalization of insulin into Caco-2 cells was also confirmed by observation of the fluorescence of FITC in the cells after incubation with the microgels loaded with FITC-Ins (Fig. S12, supporting information).

Figure 8. Visualization by CLSM for the endocytosis of empty RhoB-labeled microgel (RhoB-MG) in Caco-2 cells. From left to right, the groups of RhoB-MG-1, RhoB-MG-2, RhoB-MG-3 and RhoB-MG-4 were arrayed in succession (scale bar: 50 µm).

Overall, the results of flow cytometry and CLSM clearly indicated that the insulin-loaded microgels could be internalized by Caco-2 cells, which may promote the transcellular transport of insulin in the small intestine during the oral delivery route. The mechanism for the microgel-assisted endocytosis was remained unclear. Several previous studies have demonstrated the feasibility for the nanoparticle-assisted transport of insulin across the enterocytes via the receptor-mediated transcytosis.51-54 In general, this transportation comprises three steps.50 First step includes the contact and adhesion of the nanoparticles to the cell apical membranes. In this process, either a receptor-specific ligand for the surface-attached receptors or a receptor for the surface-attached 29



ligands is required in the carrier system. Followed, the invagination of enterocytes occurs, leading to the formation of a vesicle. Thirdly, the nanoparticles are internalized through the coated vesicles into endosomal compartments, and are transported through the cells to release insulin at the basolateral pole. In our study, it was predicted that the polysaccharide component (CMS) could provide the CMS/PiBAA hybrid microgels with affinity to the intestinal epithelia via the interaction with the glycocalyx and lectins on the cell membranes.55,56 Hence, the microgel with the higher CMS content in networks displayed enhanced endocytosis of the insulin-loaded carrier. To investigate the penetrability of insulin across the intestinal epithelium, the transmembrane transport assay for the microgels loaded with FITC-Ins was carried out on Caco-2 cell monolayer, simulating the epithelial barrier in small intestine. After a distinct period of incubation time, the culture medium of the lower compartment was measured for the amount of FITC-Ins that permeated through the Caco-2 cell monolayer from the upper compartment. As shown in Fig. 9A, it could be inferred that the cumulative insulin in the lower compartment increased as the incubation time increased from 1 h to 8 h. In comparison with free-form FITC-Ins, the significantly enhanced FITC-Ins was detected in the lower compartment after being encapsulated into the microgels, indicating that the insulin loaded in the microgels was effectively transported through the cell monolayer. Besides, it was found that the microgels with higher CMS content displayed higher rate of transmembrane transport of insulin. This may be related to the higher cellular uptake of the microgels with higher CMS content (Fig. 6 – 8). Moreover, the transmembrane transport assay for the empty RhoB-labeled microgel (RhoB-MG) was performed in Fig. 9B to evaluate the transport of the four types of microgel carriers across the cell monolayer. Also, the RhoB-MG samples that permeated through the cell monolayer were visualized in Fig. S13 (supporting information), which could be reflected by the red color of rhodamine B dye. After incubation for 4 h, over 60% of the microgels were detected in the lower compartment, indicating possible transcytosis of the microgel

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carriers.50 Therefore, the CMS/PiBAA hybrid microgels may be a promising carrier for improving the penetration of insulin through the intestinal epithelium.

Figure 9. (A) Cumulative transportation of insulin (of initial %) at distinct time intervals. The groups for FITC-Ins loaded Microgel-1 (red), FITC-Ins loaded Microgel-2 (blue), FITC-Ins loaded Microgel-3 (green), FITC-Ins loaded Microgel-4 (pink) and free-form FITC-Ins (grey) were shown (n = 3) (***p < 0.001, **p < 0.01, *p < 0.05). (B) Cumulative transportation of RhoB-labeled microgel (of initial %) at distinct time intervals. The groups for RhoB-MG-1 (red), RhoB-MG-2 (blue), RhoB-MG-3 (green) and RhoB-MG-4 (pink) were shown (n = 4) (***p < 0.001, **p < 0.01, *p < 0.05).

3.8. In vivo hypoglycemic effect. The in vivo hypoglycemic effect of the insulin-loaded microgels was evaluated by the oral administration into streptozotocin-induced Type 1 diabetic (T1D) rat model. While regarding the blood glucose level before drug administration (at 0 h) as 100% for each group, the changes in blood glucose level (of initial %) of the diabetic rats as a function of time were displayed after the oral administration of various formulations or subcutaneous (S.C.) injection of free-form insulin solution (Fig. 10). It was found that no apparent hypoglycemic effect was observed after oral administration of blank microgel sample (20 mg/kg) or free-form insulin solution (60 IU/kg), indicating that the oral intake of empty microgel carriers or free-form insulin exhibited nearly no oral pharmacological 31



availability. In contrary, the subcutaneous (S.C.) injection of free-form insulin solution (5 IU/kg) resulted in marked reduction of the blood glucose level within 2 h, but the blood glucose level rapidly returned to high level subsequently. It was noteworthy that the oral intake of insulin-loaded microgels (60 IU/kg) also led to significant reduction in blood glucose level. Despite the reduction rates in blood glucose level was slower than that by S.C. injection of insulin solution, the pronounced decline in blood glucose level was achieved 2 h after oral administration of the insulin-loaded microgels, and the hypoglycemic effect was maintained over 6 h. The relative pharmacological availabilities (rPA, %) of the different insulin-loaded microgels were calculated by regarding the rPA of S.C. injection as 100%, and were listed in Table 2. It was shown that the oral administration of the insulin-loaded Microgel-3 exhibited the highest rPA of 5.7%, which was approximately 38 times higher than that of the oral delivery of free-form insulin (rPA = 0.15%). The differences in the oral pharmacological availabilities of the four types of insulin-loaded microgels were attributed to the combination effects of the drug loading efficiency, the protection of insulin from proteolysis in the stomach, the accelerated release under intestinal amylase, and the absorption of insulin in the small intestine. In this study, the encapsulation and release of insulin-loaded microgels in the gastrointestinal tract, and the penetration of insulin-loaded microgels across the intestinal epithelium were considered as the two important factors affecting oral insulin availability. Based on the in vitro release study in Fig. 5A, it was observed that the premature releases of insulin in the acidic stomach-mimicking environment from Microgel-1 and Microgel-2 were obviously higher than those from Microgel-3 and Microgel-4. The suppressed premature release of insulin by Microgel-3 and Microgel-4 with higher PiBAA contents may be beneficial for the protection of insulin from the proteolysis. Moreover, according to the cellular uptake assay and the transmembrane transport study (Fig. 6 – 9), insulin-loaded Microgel-3 exhibited enhanced internalization by the enterocytes and increased transportation across Caco-2 cell monolayers, compared to insulin-loaded Microgel-4. Therefore, the microgels with appropriate CMS and PiBAA components, such as Microgel-3 in this 32


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study, may possess the ability to both protect insulin in acidic stomach environment and showed moderate enhancement of insulin transportation. Accordingly, in the in vivo pharmacodynamics tests (Fig. 10), the insulin-loaded Microgel-3 exhibited the highest hypoglycemic effect. Overall, the results of the in vivo oral insulin delivery based on the CMS/PiBAA hybrid microgel carriers further confirmed the capacity of the microgels in marked enhancement of the oral insulin availability. Notably, the relative pharmacological availability of the insulin-loaded CMS/PiBAA microgels (3.4 – 5.7%) is comparable to that of the recently reported devices for oral insulin delivery, including PMAA-chitosan-PEG hydrogel encapsulated with methyl-β-cyclodextrin complexed insulin (1.8%),57 poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan IPN hydrogel (4.1%),58 poly(NIPAm-co-β-propyl acrylic acid) hydrogel (4.8%),45 P(MAA-g-EG) microparticles (9.5%),30 along with chitosan and poly(γ-glutamic acid) nanoparticles (15.1%).32

Figure 10. Blood glucose level (of initial %) changes versus time curves of the diabetic rats after oral administration of insulin-loaded Microgel-1 (green), insulin-loaded Microgel-2 (blue), insulin-loaded Microgel-3 (red), insulin-loaded Microgel-4 (yellow), and free-form insulin solution (pink), all at the insulin dosage of 60 IU/kg. The group by subcutaneous injection (S.C., black) of insulin at 5 IU/kg was set as a positive control, while the group orally administrated with empty Microgel-4 (grey) at 20 mg/kg served as a negative control. (n = 6) The significances of differences 33



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were compared between the group orally administrated with insulin-loaded Microgel-3 and the group orally administrated with free-form insulin solution both at the insulin dosage of 60 IU/kg. Thus, in the blood glucose level vs. time curve for the group of oral insulin-loaded Microgel-3 (red), the p values for different time intervals were 3h**, 4h* and 6h*, where **p < 0.01, *p < 0.05.

Table 2. Pharmacological effects of different insulin formulations, following S.C. injection and oral administration to diabetic rats

a)

Formulation

[AAC]0–12 h (% glucose reduction, h)[a]

rPA (%)[b]

rPA Ratio[c]

Insulin solution (S.C., 5 IU/kg)

397.7 ± 47.5

100

-

Insulin solution (oral, 60 IU/kg)

7.0 ± 61.8

0.15 ± 1.3

1

Insulin loaded Microgel-1 (oral, 60 IU/kg)

163.1 ± 75.7

3.4 ± 2.0

23


234.6 ± 52.8

4.9 ± 1.7

33


273.5 ± 109.3

5.7 ± 3.0

38


224.0 ± 115.9

4.7 ± 3.0

31

[AAC]0–12 h was the area above the blood glucose level vs. time curve within 12 hours.

b)

rPA

represented for the relative pharmacological availability for each group. c)rPA Ratio represented the ratios between rPA for the insulin-loaded microgel fomulations and that for the free-form insulin solution via oral administration.

4. Conclusion. In this study, a novel type of CMS/PiBAA hybrid microgels that responded to the pH change in GI tract and α-amylase in the small intestine was developed. With increasing pH from acidic environment to neutral condition, the hybrid microgels exhibited the abrupt increases in the particle 34


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Biomacromolecules

size of the microgels and the light transmittance of the microgel solution. In the presence of α-amylase, the hybrid microgels decomposed gradually. Insulin was loaded in the microgels with the DLC of 2.8 – 6.8%. The release of insulin was found to be suppressed in acidic AGF, while was markedly accelerated in AIF, especially in the presence of α-amylase. The insulin-loaded microgels displayed the enhanced cellular uptake by Caco-2 cells and promoted the transmembrane transport of insulin through the Caco-2 cell monolayer in vitro. After oral administration of the insulin-loaded microgels to STZ-induced diabetic rats, a continuous reduction in the fasting blood glucose level of the rats was observed within 2 to 4 h, and the hypoglycemic effect was prolonged over 6 h. The relative pharmacological availability of insulin was enhanced markedly in vivo. Therefore, the hybrid microgels may serve as an effective platform for oral delivery of insulin and other proteins.

ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publications website. Additional experimental details for synthesis and characterization of iBAA and CMS-g-AA, characterization data of microgels, drug release in vitro, as well as tests for cytocompatibility and intracellular uptake in vitro.

AUTHOR INFORMATION Corresponding Authors *Email: [email protected]; [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS 35



The financial supports from the National Natural Science Foundation of China (projects 21574127, 51622307, 51390484, 51520105004), and the Youth Innovation Promotion Association CAS are gratefully thanked.

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TOC:

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Poly(2-isobutyl

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