Development of Functional or Medical Foods for Oral Administration of

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Bioactive Constituents, Metabolites, and Functions

Development of functional or medical foods for oral administration of insulin for diabetes treatment: Gastroprotective edible microgels Quancai Sun, zipei zhang, Ruojie Zhang, Ruichang Gao, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00233 • Publication Date (Web): 27 Apr 2018 Downloaded from http://pubs.acs.org on April 28, 2018

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Journal of Agricultural and Food Chemistry

Development of functional or medical foods for oral administration of insulin for

diabetes treatment: Gastroprotective edible microgels

Quancai Suna,1, Zipei Zhangb,1, Ruojie Zhangb, Ruichang Gao a*, and David Julian McClementsb*

a

School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212001,

China b

Department of Food Science, University of Massachusetts Amherst, Amherst, MA

01003, USA

1

These authors contributed equally to the present work.

*

Corresponding authors: Ruichang Gao, School of Food and Biological Engineering.

Tel.: +8651188780201; e-mail: [email protected].

D.J. McClements, Department of Food Science, University of Massachusetts,

Amherst. Tel.: +1 413-545-2275; fax: +1 413-545-1262; e-mail:

[email protected]

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Abstract

Insulin and an antacid (Mg(OH)2) were co-encapsulated inside calcium alginate

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microgels (diameter = 280 µm) using a vibrating nozzle injector.

Confocal

4

microscopy indicated that insulin was successfully encapsulated inside the microgels

5

and remained inside them after they were exposed to simulated gastric conditions.

6

Localized fluorescence intensity measurements indicated that the internal pH of the

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antacid-loaded microgels was around pH 7.4 after incubation in acidic gastric fluids,

8

but below the limit of detection (pH < 4) in the antacid-free microgels. After

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incubation in small intestine conditions, around 30% of the insulin was released from

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the antacid-loaded microgels over a 2-hour period. Encapsulation of insulin within the

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antacid-loaded microgels increased its biological activity after exposure to simulated

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gastric conditions.

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phosphorylation at both Thr308 and Ser473 in L6 myotubes when compared to free

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insulin.

In particular, the encapsulated insulin significantly increased Akt

15

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Keywords: alginate; microgels; insulin; antacid agent; diabet

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Introduction

A major challenge in the design of functional and medical foods is to keep

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bioactive ingredients stable during food processing, storage, and preparation, as well

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as they travel through the gastrointestinal (GI) system after ingestion.1

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the bioactive ingredients must be delivered in a form that is acceptable to consumers,

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otherwise they will not be eaten.

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bioactive ingredients encapsulated within nanoparticles or microparticles, are

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particularly powerful tools in the design and development of these kinds of functional

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and medical foods.2

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In addition,

Colloidal delivery systems, which consist of

Diabetes is a global health concern, which reduces the quality of life of numerous

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people, and is a major economic burden on society due to increased health costs and

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lost economic activity.3-4

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advanced Type 2 diabetes, have a chronic health condition in which their pancreases

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cannot produce sufficient insulin to properly regulate their blood sugar levels.5-6

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Insulin is a peptide-based hormone that normally regulates the uptake of glucose into

32

cells where it can be used as an energy source, and so individuals with diabetes need

Individuals with Type 1 diabetes, as well as those with

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to receive regular doses of insulin to remain healthy.7

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subcutaneous injections are the most common means of administering insulin, which

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is associated with local discomfort and the possibility of infection.5, 8-9

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insulin through the oral route would be much more preferable, as it would be more

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convenient and less painful than injections.10-11

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At present, regular

Delivery of

However, there are a number of challenges that currently hold back the

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development of successful oral insulin delivery systems.

First, insulin is highly

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susceptible to degradation in the stomach due to the high proteolytic activity and

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acidity of the gastric fluids.12-13

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hydrophilic peptide, that is actually absorbed by the epithelium cells is relatively low.

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14

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gastric stability of insulin.

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stability of insulin within the stomach is to encapsulate it inside colloidal particles,

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such as nanoparticles or microparticles.11, 15-17

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be assembled from synthetic polymers, natural polymers, lipids, surfactants, or

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phospholipids suitable for oral ingestion, using various types of particle preparation

Second, the fraction of ingested insulin, which is a

In the current study, we focus on the first of these challenges – improving the

One of the most promising strategies for increasing the

These colloidal delivery systems may

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methods.

In this study, we examined the possibility of encapsulating insulin within

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biopolymer microgels.

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biopolymer microgels can modulate the gastrointestinal fate of encapsulated

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nutraceuticals and probiotics, including microgels fabricated from food-grade proteins

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and/or polysaccharides 18-21.

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preparation methods, including simple injection at ambient temperature, which is

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important to minimize any loss of insulin activity during the loading procedure.22

Previous research has shown that various kinds of

These microgels can be assembled using mild

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In this study, we focused on the utilization of biopolymer microgels formed by

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injecting an alginate solution into a calcium solution that induces particle formation

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and cross-linking 23.

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alginate microgels for encapsulating, protecting, and delivering insulin.

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been encapsulated within calcium alginate microgels fabricated using an

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emulsification/gelation method, but this system was unable to protect the insulin from

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degradation under simulated gastrointestinal conditions. 10 Presumably, this lack of

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protection was because these simple microgels had relatively large pores and were

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therefore unable to protect insulin from attack by the acids and pepsin in the gastric

A number of researchers have examined the efficacy of calcium

Insulin has

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juices.

Structural design principles have therefore been employed to improve the

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encapsulation and protection properties of alginate microgels.

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alginate microgels have been coated with cationic biopolymers such as poly-L-lysine

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24

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has been used in combination with other biopolymers to form a mixed hydrogel

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matrix inside the microgels, such as cellulose derivatives, pectin, and chitosan.27-29

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These studies showed that the insulin could be successfully entrapped and retained

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within the microgels, but that it was still susceptible to degradation under simulated

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gastrointestinal conditions.

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biopolymer microgel cores or coatings are still relatively large compared to the size of

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small ions (H+) or digestive enzymes (pepsin) in the gastric fluids.

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these molecular species easily diffuse into the interior of the microgels and promote

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insulin degradation.

or chitosan 25-26 to form a protective coating around them.

For instance, anionic

Alternatively, alginate

Again, the reason for this is because the pores in

Consequently,

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In the current study, a different approach was adopted to create alginate microgels

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that could protect insulin from acid and pepsin-induced deactivation when exposed to

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gastric conditions.

In this approach, insulin was co-encapsulated with magnesium

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hydroxide inside the microgels. Mg(OH)2 is a widely used food-grade antacid, which

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is soluble in water at acidic pH, but insoluble at neutral and basic pH.30 Consequently,

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the pH inside antacid-loaded microgels stays close to neutral for an extended time

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when dispersed in simulated gastric fluids, because some of the antacid dissolves and

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releases hydroxyl ions (OH-) when hydrogen ions (H+) diffuse into them.23, 31

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been reported that pepsin loses most of its enzyme activity at neutral pH.32

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Consequently, any gastric pepsin that diffuses into the neutral interior of

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antacid-loaded microgels may lose its activity, and therefore be unable to hydrolyze

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the encapsulated insulin.

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biopolymer microgels could protect insulin from both acid- and protease-degradation

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in gastric fluids.

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determine the effectiveness of the antacid-loaded microgels for the encapsulation,

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retention, protection, and release of insulin under simulated gastrointestinal

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conditions.

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Materials and Methods

It has

We therefore postulated that these antacid-loaded

For this reason, we carried out a series of experiments to

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Materials

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The pH-sensitive fluorescence probe, fluorescein tetramethylrhodamine dextran

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(FRD, average Mr 70,000), was purchased from Molecular Probes (Eugene, OR). L6

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cells were from American Type Culture Collection (Manassas, VA). α-minimum

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Eagle’s medium (α-MEM) was from Invitrogen (Carlsbad, CA). RIPA buffer,

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phosphatase inhibitor and protease inhibitor cocktail were from Thermo Scientific,

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Rockford, IL). Protein (or insulin) concentrations were determined with a protein DC

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assay kit (Bio-Rad Co., Hercules, CA). Immobilin P membrane was from Millipore

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(Bedford, MA). Primary rabbit antibodies including protein kinase B (AKT),

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phospho-AKT Thr308, phosphor-Akt Ser473, β-actin and horseradish peroxidase

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conjugated goat anti-rabbit IgG were obtained from Cell Signaling Technology

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(Danvers, MA). Enhanced chemi-luminescence detection kit (Bio-Rad Co., Hercules,

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CA). The following chemicals were purchased from the Sigma Chemical Company

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(St. Louis, MO): fetal bovine serum; Penicillin; Streptomycin; alginic acid (sodium

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salt); pepsin from porcine gastric mucosa; fluorescein isothiocynate (FITC) isomer I;

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calcium chloride dehydrate; and magnesium hydrate. Sodium phosphate and calcium

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chloride were purchased from Fisher Chemical Company (Pittsburgh, PA). All

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chemicals used were analytical grade. Double distilled water was used to make all

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solutions.

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Microgels preparation

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The microgels were prepared using an injection-gelation method as described

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previously,23, 31 with some slight modifications. Aqueous alginate solutions were

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prepared by dispersing powdered sodium alginate (1%, w/w) into phosphate buffer

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solution (5 mM, pH 7.0), and then stirring at 60 °C for 60 min. The temperature was

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then reduced to 35 °C with continuous stirring until the alginate was fully dissolved.

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The alginate solution was then mixed with powdered 1 mg/mL insulin and either 0%

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(antacid-free) or 0.15% (antacid-loaded) Mg(OH)2, and then stirred until the system

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was homogeneous.

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through a 120 µm vibrating nozzle into a 5% w/w calcium chloride solution using a

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dedicated encapsulation device (Encapsulator B-390, BUCHI, Switzerland). The

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operating conditions used for the encapsulation device were as follows: frequency =

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800 Hz; electrode potential = 650 V; and operating pressure = 300 mbar. The

Microgels were then prepared by injecting these mixtures

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biopolymer microgels formed were held in the Ca2+ solution for 20 min at ambient

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temperature to promote hardening before being removed. They were then filtered and

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washed with buffer solution to remove any residual hardening solution.

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Simulated gastrointestinal conditions

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The impact of incubation of the microgels in simulated gastric fluid (SGF) on

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their internal pH and on insulin activity was determined using a method described

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previously, 23 with some slight modifications. Specifically, microgels (with or without

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Mg(OH)2) were added at a ratio of 1:4 (w/w) to SGF (containing 0.0032 g/mL pepsin)

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that had been preheated to 37 °C and adjusted to pH 2.5.

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then incubated within a shaking device for 2 h at 37 °C to mimic stomach conditions.

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The resulting mixture was

After exposure to stomach phase, the microgels were then exposed to simulated

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small intestinal conditions by mixing them with phosphate buffer solution (pH 7.0,

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37 °C) at a fixed mass ratio of 1:4, and then the mixture was adjusted to pH 7.0. For

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the release experiments, the microgels were incubated in this simulated small

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intestinal phase for 2 h at 37 °C.

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Confocal Laser Scanning Microscopy

The microstructural analysis of the biopolymer microgels was carried out using a

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confocal scanning laser fluorescence microscope with a 20 × objective lens (Nikon

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D-Eclipse C1 80i, Nikon, Melville, NY, U.S.). The insulin was dyed using FITC

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solution and then stored at 5 °C overnight. The excitation and emission wavelength

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used for detection of the FITC-labeled insulin were 488 nm and 515 nm, respectively.

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The pH inside the biopolymer microgels was measured using a fluorescence

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intensity method described previously.33

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(10 mg/mL FRD) was dissolved in phosphate buffer (5 mM, pH 7.0), and then mixed

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with alginate solution (1:200 v/v) with or without 0.15% Mg(OH)2. Microgels were

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then fabricated using the encapsulation device described in Section 2.2. Images of the

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microgels were acquired using the confocal laser scanning fluorescence microscope

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with a 20×objective lens. Images of the pH-sensitive dye were obtained using

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excitation wavelengths of 543 and 488 nm, and detection wavelengths/bandwidths of

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650 nm/LP and 590 nm/50 nm, respectively.34-35 All samples were imaged using an

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exposure time of 0.5 s and a 12.5% excitation power level for both channels.

A known amount of the pH-sensitive dye

Full

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images of each sample were typically acquired in less than 2 min and then stored on

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the computer using the microscope’s software (NIS-Elements, Nikon, Melville, NY,

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USA). The images were then analyzed using Image J software (1.50I, imagej.nih.gov).

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The ratio of pixel intensities of the two images obtained at two different wavelengths

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(488 and 543 nm) were calculated and correlated with the pH obtained using a

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standard curve, as described previously.33

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from at least eight measurements.

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Particle size characterization

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The average intensity ratio was calculated

Particle size distributions were carried out using a static light scattering

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instrument (Mastersizer 2000, Malvern Instruments Ltd., Malvern, Worcestershire,

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UK). Phosphate buffer (5 mM, pH 7.0) was used for the dilution of the initial samples

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and acidified distilled water (pH 2.5) was used to dilute the gastric samples to avoid

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multiple scattering effects.

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Insulin activity assay with L6 myotubes and immunoblotting

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The activity of insulin was assayed by determining its ability to stimulate Akt

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phosphorylation in L6 myotubes. L6 cells were maintained in α-MEM with 10% fetal

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bovine serum, 100 µg/ml streptomycin, and 100 U/ml penicillin at 37 °C in a

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humidified 5% CO2 atmosphere. Cells were differentiated into myotubes for 6 days as

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has been reported previously.36

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mixing them with a calcium chelating agent (EDTA).

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dissolved in 2 mL of 10% EDTA solution and then immediately diluted with

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α-minimum Eagle’s medium by 100 times before treating myotubes for 10 minutes.

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Cells were lysed with RIPA buffer supplemented with phosphatase inhibitor and

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protease inhibitor cocktail. Protein concentrations were measured using a protein DC

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assay kit. Cell lysates containing 50 µg of protein were separated with 10 %

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SDS-polyacrylamide gel and transferred to Immobilin P membrane and incubated

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with primary antibodies overnight at 4 °C and then secondary antibodies for 1 hour.

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Detections were performed with an enhanced chemi-luminescence detection kit.

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The images acquired were quantified using the Image J software described earlier.

Prior to analysis the microgels were dissociated by

Filtered microgels were

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Statistical analysis

All experiments were performed on at least three freshly prepared samples, and

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the results reported as averages and standard deviations calculated using Excel

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(Microsoft, Redmond, VA, USA).

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Results and Discussion

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Preparation and characterization of insulin-loaded microgels

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The ability of the biopolymer microgels to encapsulate insulin and then retain it

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within the stomach phase was determined using confocal fluorescence microscopy

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(Fig. 1).

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composition of the microgels, which were based on our previous studies with similar

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systems 23, 31.

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be suitable for encapsulating a high percentage (around 82%) of the insulin inside the

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antacid-loaded microgels.

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insulin (stained green) was distributed evenly throughout the interior of the microgels

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indicating that it had been successfully encapsulated.

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the microgels was still stained green, indicating that the insulin was retained within

Preliminary experiments were carried out to establish the optimum

A composition of 1% alginate and 5% calcium chloride was found to

Prior to exposure to simulated gastric conditions, the

After exposure, the interior of

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the microgels.

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intensity of the insulin after exposure to the stomach phase.

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be attributed to the fact that the intensity of the fluorescence probe used to stain the

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insulin (FITC) decreases under acidic conditions.33 It should be noted that the

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fluorescence measurements only indicate the location of the insulin within the system,

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rather than its biological activity, and so this was measured using another method

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(described later).

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However, there was a significant decrease in the fluorescence

This effect can mainly

The confocal microscopy images indicated that the microgels had a roughly

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spherical shape, and remained intact after exposure to the simulated gastric

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conditions, which is in agreement with earlier studies.23, 31 The light scattering

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measurements indicated that the microgels had a monomodal particle size distribution

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both before and after exposure (Fig. 2).

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slight shrinkage of the microgels after they were incubated in the gastric fluids, with

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the mean particle diameter decreasing from around 280 µm before exposure to 265

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µm after exposure.

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attributed to a decrease in the electrostatic repulsion between the alginate chains when

In addition, they indicated that there was a

Microgel shrinkage under highly acidic conditions can be

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the carboxyl groups become more protonated: −COO- + H+ ↔ −COOH, pKa = 3.5.23

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This phenomenon is advantageous for the retention of the insulin within the microgels

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in the stomach, since a cross-linked biopolymer network with smaller pores will trap

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the insulin molecules more effectively.

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Mapping the pH inside the microgels

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As mentioned earlier, one of the main hurdles to the oral administration of insulin

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is its tendency to degrade in the stomach due to the high acidity and protease activity

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of the gastric fluids.13, 37 Protection against these effects is therefore an important

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strategy for the development of effective oral delivery systems for insulin.38-39 We

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hypothesized that antacid-loaded microgels would protect insulin from both acid- and

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protease-degradation by maintaining a neutral pH inside them.

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degrade under acidic conditions,37 and therefore keeping the local environment

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neutral should protect it from this type of degradation.

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activity of pepsin has been reported to be around pH 2 to 3,40 and it is known to be

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deactivated around pH 7.32 Consequently, maintaining a neutral environment inside

Insulin is known to

Moreover, the optimum

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the microgels may also protect the insulin from protease-induced degradation by

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reducing the local pepsin activity.

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In this study, an insoluble antacid (Mg(OH)2) was co-encapsulated with the

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insulin to control the internal pH of the microgels under gastric conditions. The local

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pH inside the microgels was measured using a pH-sensitive fluorescence probe (FRD)

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based on a fluorescence intensity ratio method described earlier.33

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has both pH-dependent (FITC) and pH-independent (TMR) fluorescence groups, and

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so the pH can be determined by measuring the FITC-to-TMR fluorescence intensity

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ratio.

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(dextran) attached to inhibit its release from the biopolymers microgels.

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The FRD probe

In addition, the FRD probe selected for this study had a polymer chain

Confocal fluorescence microscopy images of the FRD probe within the microgels

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was acquired before and after exposure to simulated gastric fluids (Fig. 3).

For the

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antacid-loaded microgels, the fluorescence intensity of the FITC channel

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(pH-dependent) was relatively strong both before and after incubation in the stomach

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phase.

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FITC channel changed from relatively strong before incubation to very weak after

Conversely, for the antacid-free microgels, the fluorescence intensity of the

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incubation.

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the antacid-loaded microgels after exposure to the simulated gastric fluids, but below

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the limit of detection (pH < 4) for the antacid-free microgels.

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that the co-encapsulated magnesium hydroxide was able to maintain a neutral pH

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inside the microgels, which is in agreement with our previous studies with lipase- and

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lactase-loaded alginate microgels.23, 31

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of the Mg(OH)2 particles to partially dissolve when hydrogen ions diffuse into the

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microgels, thereby releasing OH- ions that neutralize the H+ ions and maintain a

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neutral pH.

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Influence of encapsulation on insulin activity

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The average internal pH value was determined to be around pH 7.4 for

These results indicate

This effect has been attributed to the ability

In this series of experiments, a biological model was used to test the activity of

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insulin after it had been exposed to simulated gastric conditions.

In its active form,

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insulin is known to stimulate Akt phosphorylation in L6 myotubes,36 and so this assay

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was used to determine the activity of free and encapsulated insulin.

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encapsulated in both antacid-free and antacid-loaded microgels to ascertain the impact

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of the antacid on its stability under gastric conditions.

Insulin was

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The Akt phosphorylation at both Thr308 and Ser473 was significantly higher for

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insulin encapsulated in the antacid-loaded microgels, than for free insulin or for

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insulin encapsulated in antacid-free microgels (Figs. 4 and 5). This result suggests

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that the microgels containing the antacid protected the insulin under gastric conditions,

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which can be attributed to its ability to maintain a neutral pH inside the microgels,

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thereby inhibiting both acid- and pepsin-induced degradation.

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A number of previous researchers have focused on the impact of encapsulation of

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insulin on its activity under gastric conditions.

Studies have shown that insulin can

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be encapsulated in biopolymer nanoparticles fabricated from alginate and dextran

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sulfate, and that its biological activity was retained after encapsulation.41 However,

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the activity of the insulin in this study was only tested by injecting insulin-loaded

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nanoparticles subcutaneously into diabetic rats.

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insulin would still remain active if insulin-loaded nanoparticles were orally

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administered to the rats.

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poly(lactic-co-glycolic acid) nanoparticles, which were shown to protect it from

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gastric degradation, and to maintain its activity after oral administration to diabetic

Hence, it is unclear whether the

Insulin has also been successfully loaded into

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rats.42

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encapsulating insulin, protecting it from degradation under gastrointestinal conditions,

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and maintaining its activity after oral administration to diabetic rats.43

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Insulin-loaded resistant starch-coated microparticles were also reported to decrease

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plasma glucose levels after oral administration.44

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on short-chain glucan and proanthocyanidins have been developed and proven to be

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effective in reducing the blood glucose level in diabetic rats.45

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of polymer-based microgels seem to have promise as oral delivery systems for insulin

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.46

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commercial viability of the ingredients and fabrication processes used to prepare

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them, and their functional attributes.

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Release of insulin in a simulated small intestine phase

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Alginate-chitosan microgels have also been shown to be effective at

Recently,nanocomposites based

Thus, various types

Each system has its own advantages and disadvantages in terms of the

Insulin that survives intact after passage through the stomach has to be absorbed

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by the epithelium cells after reaching the small intestine so that it can enter the

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systemic circulation.

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intestinal enterocytes through various internalization mechanisms.47-48

It has been reported that free insulin can be absorbed by

It is therefore

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important to understand the release properties of the insulin from the microgels in the

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small intestine.

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In this section, we determined the release profile of insulin from the

302

antacid-loaded microgels during incubation in simulated small intestinal fluids for

303

two hours (Fig. 6). Around 21% of the insulin was released fairly rapidly during the

304

first 30 minutes and then another 10% was released more gradually over the next 90

305

minutes.

306

at longer incubation times, which may be useful for sustained release applications.

307

The release of insulin from the biopolymer microgels in the small intestine phase can

308

be attributed to changes in the electrical properties of the alginate and insulin

309

molecules, as well as alternations in the structure of the alginate microgels. The

310

insulin and alginate molecules are both negatively charged in the small intestine phase

311

(pH 7) and so they will electrostatically repel each other, which promotes insulin

312

release.49

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neutral conditions because of the increase in electrostatic repulsion between the

314

anionic alginate chains.50 This leads to an increase in hydrogel pore size, which can

Presumably, the remainder of the insulin would also be released gradually

Moreover, alginate microgels swell when they are moved from acidic to

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increase the release of any encapsulated substances.51

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that insulin could be successfully released from the microgels in the small intestine

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phase, which would facilitate their subsequent absorption.

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possible that the insulin would still be degraded in the small intestinal fluids prior to

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adsorption due to the presence of digestive enzymes, such as proteases.

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it will be important to determine the fraction of insulin that is actually absorbed and

321

enters the systemic circulation where it can have its beneficial effects. In future

322

studies, it will therefore be important to test the efficacy of these antacid-loaded

323

microgels using animal feeding studies, e.g., with diabetic rats.

Overall, these results suggest

Nevertheless, it is also

In addition,

324

To conclude, insulin was encapsulated within calcium alginate microgels using an

325

injection-gelation method in the current study. The stability of insulin after exposure

326

to simulated gastric conditions was improved by co-encapsulating it with a basic

327

antacid: Mg(OH)2. This antacid is insoluble under neutral conditions, but dissolves

328

when hydrogen ions diffuse into the microgels, thereby ensuring that a neutral internal

329

pH is maintained (as long as some antacid remains). Gastric pepsin is rapidly

330

inactivated under neutral pH conditions, and so encapsulation of insulin in

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antacid-loaded microgels protects it from both acid- and protease-degradation. This

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study suggests that antacid-loaded microgels may be a suitable food-grade delivery

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system for acid and/or pepsin-sensitive bioactive proteins and peptides. However, in

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vivo animal and human studies are needed to confirm that these microgels can

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maintain their insulin activity under real life conditions.

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important to establish that the microgels can be fabricated economically on a

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sufficiently large scale for commercial applications, and that they can be successfully

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incorporated into formulations intended for oral administration.

In addition, it will be

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References

342

1.

343

functional ingredients into foods. Trends Food Sci. Technol. 2009, 20 (9), 388-395.

344

2.

345

Controlled Release System Based on Resistant Starch Acetate: Synthetization,

346

Characterization, and Preparation of Film-Coating Pellets. J. Agric. Food Chem.

347

2011, 59 (10), 5738-5745.

348

3.

349

Cost-Effectiveness of Interventions to Prevent and Control Diabetes Mellitus: A

350

Systematic Review. Diabetes Care 2010, 33 (8), 1872-1894.

351

4.

352

and the Association with Clinical and Economic Outcomes. Clinical Therapeutics

353

2011, 33 (1), 74-109.

354

5.

355

present and future. International Journal of Pharmaceutical Investigation 2016, 6 (1),

356

1-9.

357

6.

358

383 (9911), 69-82.

359

7.

360

Etiology, Immunology, and Therapeutic Strategies. Physiological Reviews 2011, 91

361

(1), 79-118.

362

8.

363

delivery of insulin. Expert Opinion on Drug Delivery 2017, 14 (6), 727-734.

364

9.

365

Mahmood, S.; Khan, S.; Farrukh, M. J., Evaluation of current trends and recent

366

development in insulin therapy for management of diabetes mellitus. Diabetes &

367

Metabolic Syndrome-Clinical Research & Reviews 2017, 11, S833-S839.

368

10. Silva, C. M.; Ribeiro, A. J.; Figueiredo, I. V.; Goncalves, A. R.; Veiga, F.,

369

Alginate microspheres prepared by internal gelation: development and effect on

370

insulin stability. International journal of pharmaceutics 2006, 311 (1-2), 1-10.

371

11. Wong, C. Y.; Martinez, J.; Dass, C. R., Oral delivery of insulin for treatment of

372

diabetes: status quo, challenges and opportunities. Journal of Pharmacy and

373

Pharmacology 2016, 68 (9), 1093-1108.

Day, L.; Seymour, R. B.; Pitts, K. F.; Konczak, I.; Lundin, L., Incorporation of

Pu, H. Y.; Chen, L.; Li, X. X.; Xie, F. W.; Yu, L.; Li, L., An Oral Colon-Targeting

Li, R.; Zhang, P.; Barker, L. E.; Chowdhury, F. M.; Zhang, X. P.,

Asche, C.; LaFleur, J.; Conner, C., A Review of Diabetes Treatment Adherence

Shah, R. B.; Patel, M.; Maahs, D. M.; Shah, V. N., Insulin delivery methods: Past,

Atkinson, M. A.; Eisenbarth, G. S.; Michels, A. W., Type 1 diabetes. Lancet 2014,

Van Belle, T. L.; Coppieters, K. T.; Von Herrath, M. G., Type 1 Diabetes:

Guo, X. H.; Wang, W., Challenges and recent advances in the subcutaneous

Nawaz, M. S.; Shah, K. U.; Khan, T. M.; Rehman, A. U.; Rashid, H. U.;

24

ACS Paragon Plus Environment

Page 25 of 37

Journal of Agricultural and Food Chemistry

374

12. Sood, A.; Panchagnula, R., Peroral route: an opportunity for protein and peptide

375

drug delivery. Chemical reviews 2001, 101 (11), 3275-303.

376

13. Cardenas-Bailon, F.; Osorio-Revilla, G.; Gallardo-Velazquez, T.,

377

Microencapsulation techniques to develop formulations of insulin for oral delivery: a

378

review. Journal of Microencapsulation 2013, 30 (5), 409-424.

379

14. He, H. N.; Ye, J. X.; Sheng, J. Y.; Wang, J. X.; Huang, Y. Z.; Chen, G. Y.; Wang,

380

J. K.; Yang, V. C., Overcoming oral insulin delivery barriers: application of cell

381

penetrating peptide and silica-based nanoporous composites. Frontiers of Chemical

382

Science and Engineering 2013, 7 (1), 9-19.

383

15. des Rieux, A.; Fievez, V.; Garinot, M.; Schneider, Y. J.; Preat, V., Nanoparticles

384

as potential oral delivery systems of proteins and vaccines: A mechanistic approach.

385

Journal of Controlled Release 2006, 116 (1), 1-27.

386

16. Fonte, P.; Araujo, F.; Silva, C.; Pereira, C.; Reis, S.; Santos, H. A.; Sarmento, B.,

387

Polymer-based nanoparticles for oral insulin delivery: Revisited approaches.

388

Biotechnology Advances 2015, 33 (6), 1342-1354.

389

17. Ma, G. H., Microencapsulation of protein drugs for drug delivery: Strategy,

390

preparation, and applications. Journal of Controlled Release 2014, 193, 324-340.

391

18. Gu, L. P.; Su, Y. J.; Zhang, Z. P.; Zheng, B. J.; Zhang, R. J.; McClements, D. J.;

392

Yang, Y. J., Modulation of Lipid Digestion Profiles Using Filled Egg White Protein

393

Microgels. Journal of Agricultural and Food Chemistry 2017, 65 (32), 6919-6928.

394

19. Li, W.; Luo, X. G.; Song, R.; Zhu, Y.; Li, B.; Liu, S. L., Porous Cellulose

395

Microgel Particle: A Fascinating Host for the Encapsulation, Protection, and Delivery

396

of Lactobacillus plantarum. Journal of Agricultural and Food Chemistry 2016, 64

397

(17), 3430-3436.

398

20. Wang, S. S.; Chen, Y. Y.; Lang, H.; Chen, Y. M.; Shi, M. X.; Wu, J. D.; Liu, X.

399

W.; Li, Z. S.; Liu, B.; Yuan, Q. P.; Li, Y., Intestine-Specific Delivery of Hydrophobic

400

Bioactives from Oxidized Starch Microspheres with an Enhanced Stability. Journal of

401

Agricultural and Food Chemistry 2015, 63 (39), 8669-8675.

402

21. Ji, N.; Hong, Y.; Gu, Z. B.; Cheng, L.; Li, Z. F.; Li, C. M., Binary and Tertiary

403

Complex Based on Short-Chain Glucan and Proanthocyanidins for Oral Insulin

404

Delivery. Journal of Agricultural and Food Chemistry 2017, 65 (40), 8866-8874.

25

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 26 of 37

405

22. van de Weert, M.; Hennink, W. E.; Jiskoot, W., Protein instability in

406

poly(lactic-co-glycolic acid) microparticles. Pharmaceutical research 2000, 17 (10),

407

1159-67.

408

23. Zhang, Z.; Chen, F.; Zhang, R.; Deng, Z.; McClements, D. J., Encapsulation of

409

Pancreatic Lipase in Hydrogel Beads with Self-Regulating Internal pH

410

Microenvironments: Retention of Lipase Activity after Exposure to Gastric

411

Conditions. J. Agric. Food Chem. 2016, 64 (51), 9616-9623.

412

24. Cui, J. H.; Goh, J. S.; Park, S. Y.; Kim, P. H.; Le, B. J., Preparation and physical

413

characterization of alginate microparticles using air atomization method. Drug

414

development and industrial pharmacy 2001, 27 (4), 309-19.

415

25. Anal, A. K.; Stevens, W. F., Chitosan-alginate multilayer beads for controlled

416

release of ampicillin. International journal of pharmaceutics 2005, 290 (1-2), 45-54.

417

26. Hari, P. R.; Chandy, T.; Sharma, C. P., Chitosan/calcium-alginate beads for oral

418

delivery of insulin. Journal of Applied Polymer Science 1996, 59 (11), 1795-1801.

419

27. El-Kamel, A. H.; Al-Gohary, O. M.; Hosny, E. A., Alginate-diltiazem

420

hydrochloride beads: optimization of formulation factors, in vitro and in vivo

421

availability. J. Microencapsul. 2003, 20 (2), 211-25.

422

28. Pillay, V.; Danckwerts, M. P.; Muhidinov, Z.; Fassihi, R., Novel modulation of

423

drug delivery using binary zinc-alginate-pectinate polyspheres for zero-order kinetics

424

over several days: experimental design strategy to elucidate the crosslinking

425

mechanism. Drug Dev. Ind. Pharm. 2005, 31 (2), 191-207.

426

29. Murata, Y.; Tsumoto, K.; Kofuji, K.; Kawashima, S., Effects of natural

427

polysaccharide addition on drug release from calcium-induced alginate gel beads.

428

Chemical & pharmaceutical bulletin 2003, 51 (2), 218-20.

429

30. Zhu, G.; Mallery, S. R.; Schwendeman, S. P., Stabilization of proteins

430

encapsulated in injectable poly (lactide-co-glycolide). Nature biotechnology 2000, 18

431

(1), 52-57.

432

31. Zhang, Z.; Zhang, R.; McClements, D. J., Lactase (β-galactosidase) encapsulation

433

in hydrogel beads with controlled internal pH microenvironments: Impact of bead

434

characteristics on enzyme activity. Food Hydrocolloid. 2017, 67, 85-93.

435

32. Tanaka, T.; Yada, R. Y., N-terminal portion acts as an initiator of the inactivation

436

of pepsin at neutral pH. Protein engineering 2001, 14 (9), 669-74.

26

ACS Paragon Plus Environment

Page 27 of 37

Journal of Agricultural and Food Chemistry

437

33. Zhang, Z.; Zhang, R.; Sun, Q.; Park, Y.; McClements, D. J., Confocal

438

fluorescence mapping of pH profile inside hydrogel beads (microgels) with

439

controllable internal pH values. Food Hydrocolloid. 2017, 65 (Supplement C),

440

198-205.

441

34. Deriy, L. V.; Gomez, E. A.; Zhang, G.; Beacham, D. W.; Hopson, J. A.; Gallan, A.

442

J.; Shevchenko, P. D.; Bindokas, V. P.; Nelson, D. J., Disease-causing mutations in the

443

cystic fibrosis transmembrane conductance regulator determine the functional

444

responses of alveolar macrophages. The Journal of biological chemistry 2009, 284

445

(51), 35926-38.

446

35. Chen, Y.; Arriaga, E. A., Individual acidic organelle pH measurements by

447

capillary electrophoresis. Analytical chemistry 2006, 78 (3), 820-6.

448

36. Hwang, S.-L.; Yang, B.-K.; Lee, J.-Y.; Kim, J.-H.; Kim, B.-H.; Suh, K.-H.; Kim,

449

D.-Y.; Kim, M. S.; Song, H.; Park, B.-S.; Huh, T.-L., Isodihydrocapsiate stimulates

450

plasma glucose uptake by activation of AMP-activated protein kinase. Biochem.

451

Bioph. Res. Co. 2008, 371 (2), 289-293.

452

37. Brange, J.; Langkjaer, L., Chemical-stability of insulin .3. Influence of excipients,

453

formulation, and pH. Acta Pharmaceutica Nordica 1992, 4 (3), 149-158.

454

38. Reis, C. P.; Ribeiro, A. J.; Neufeld, R. J.; Veiga, F., Alginate microparticles as

455

novel carrier for oral insulin delivery. Biotechnology and bioengineering 2007, 96 (5),

456

977-89.

457

39. Deat-Laine, E.; Hoffart, V.; Cardot, J. M.; Subirade, M.; Beyssac, E.,

458

Development and in vitro characterization of insulin loaded whey protein and alginate

459

microparticles. International journal of pharmaceutics 2012, 439 (1-2), 136-44.

460

40. Chong, A. S. C.; Hashim, R.; Chow-Yang, L.; Ali, A. B., Partial characterization

461

and activities of proteases from the digestive tract of discus fish (Symphysodon

462

aequifasciata). Aquaculture 2002, 203 (3), 321-333.

463

41. Lopes, M. A.; Abrahim-Vieira, B.; Oliveira, C.; Fonte, P.; Souza, A. M. T.; Lira,

464

T.; Sequeira, J. A. D.; Rodrigues, C. R.; Cabral, L. M.; Sarmento, B.; Seica, R.; Veiga,

465

F.; Ribeiro, A. J., Probing insulin bioactivity in oral nanoparticles produced by

466

ultrasonication-assisted emulsification/internal gelation. International Journal of

467

Nanomedicine 2015, 10, 5865-5880.

468

42. Zhao, K.; Li, W.; Zhang, Y.; Wei, H. X.; Wang, X. H.; Hong, K.; Jin, Z.; Wang,

469

W. F., Preparation, Characterization and Hypoglycaemic Effects of Orally Delivered 27

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 28 of 37

470

Insulin-Loaded PLGA Nanoparticles in Diabetic Rats. Science of Advanced Materials

471

2015, 7 (6), 1114-1124.

472

43. Zhang, Y. L.; Wei, W.; Lv, P. P.; Wang, L. Y.; Ma, G. H., Preparation and

473

evaluation of alginate-chitosan microspheres for oral delivery of insulin. European

474

Journal of Pharmaceutics and Biopharmaceutics 2011, 77 (1), 11-19.

475

44. Situ, W.; Chen, L.; Wang, X.; Li, X., Resistant starch film-coated microparticles

476

for an oral colon-specific polypeptide delivery system and its release behaviors. J.

477

Agric. Food Chem. 2014, 62 (16), 3599-609.

478

45. Ji, N.; Hong, Y.; Gu, Z.; Cheng, L.; Li, Z.; Li, C., Binary and Tertiary Complex

479

Based on Short-Chain Glucan and Proanthocyanidins for Oral Insulin Delivery. J.

480

Agric. Food Chem. 2017, 65 (40), 8866-8874.

481

46. Chaturvedi, K.; Ganguly, K.; Nadagouda, M. N.; Aminabhavi, T. M., Polymeric

482

hydrogels for oral insulin delivery. Journal of Controlled Release 2013, 165 (2),

483

129-138.

484

47. Kamei, N.; Morishita, M.; Eda, Y.; Ida, N.; Nishio, R.; Takayama, K., Usefulness

485

of cell-penetrating peptides to improve intestinal insulin absorption. J. Control.

486

Release 2008, 132 (1), 21-25.

487

48. Sarmento, B.; Ribeiro, A.; Veiga, F.; Sampaio, P.; Neufeld, R.; Ferreira, D.,

488

Alginate/chitosan nanoparticles are effective for oral insulin delivery. Pharmaceutical

489

research 2007, 24 (12), 2198-206.

490

49. Zhang, Z.; Zhang, R.; Zou, L.; McClements, D. J., Protein encapsulation in

491

alginate hydrogel beads: Effect of pH on microgel stability, protein retention and

492

protein release. Food Hydrocolloid. 2016, 58 (Supplement C), 308-315.

493

50. Gombotz, W. R.; Wee, S. F., Protein release from alginate matrices. Advanced

494

Drug Delivery Reviews 2012, 64, 194-205.

495

51. Sood, N.; Bhardwaj, A.; Mehta, S.; Mehta, A., Stimuli-responsive hydrogels in

496

drug delivery and tissue engineering. Drug Delivery 2016, 23 (3), 758-780.

497 498

Funding

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This material was partly based upon work supported by the National Natural

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Science Foundation of China (No. 31741104) and the National Institute of Food and

501

Agriculture, USDA, Massachusetts Agricultural Experiment Station (MAS00491),

502

USDA, AFRI Grants (2013-03795, 2014-67021, 2016-25147, and 2016-08782). 28

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Figure captions

Figure 1. Confocal microscopy images of antacid-loaded microgels (a) before and (b) after exposure to simulated gastric conditions.

Figure 2. The particle size distribution of antacid-loaded microgels (a) before and (b) after exposure to simulated gastric conditions.

Figure 3. Fluorescent microscopy images of microgels (a) with or (b) without 0.15% Mg(OH)2 before and after exposure to simulated gastric conditions.

The intensity of

the TMR signal was enhanced using Image J to improve contrast.

Figure 4. Effects of diluted digestion solution with insulin (beads free, encapsulated in beads and encapsulated in microgels with antacid) on Akt phosphorylation in L6 myotubes. Akt, Protein kinase B; pAkt T308, phosphorylation of Akt at Threonine 308; pAkt S473, phosphorylation of Akt at serine 473. The diluted digestion solution was obtained by diluting original digestion solution by 100 times with cell culture medium. Protein expression levels were determined after treatment with or without diluted digestion solution (100 nM) for 15 min) by western blotting.

Figure 5. The ratio of (a) pAkt/Akt T308 and (b) pAkt/Akt S473, representing the phosphorylation of Akt, which is due to insulin stimulation in muscle cells. The higher ratio of beads + antacid group suggests that insulin was better protected under gastric conditions than the other groups.

Figure 6. The release profile of insulin from antacid-loaded biopolymer microgels during incubation in a simulated small intestine phase.

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(a)

(b)

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Antacid-loaded microgels FITC

TMR

Initial

After stomach

(a) Antacid-free microgels FITC FITC

TMR TMR

Initial

After stomach

(b)

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pAkt T308/Akt

(a)

Fold increase

2.0

a

1.5 b

b

1.0

0.5

0.0 Free insulin

(b)

Microgels

Microgels +Antacid

pAkt S473/Akt 2.5

Fold increase

a 2.0

1.5 b

b

Free insulin

Microgels

1.0

0.5

0.0 Microgels +Antacid

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Protein release (%)

40

30

20

10

0 0

20

40

60

80

100

120

Incubation Time (min)

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Graphic for table of contents pAkt T308/Akt

(a) 2.0

Antacid-loaded microgels

Antacid-loaded microgels

a

TMR

Fold increase

FITC

Stomach digestion

Intestinal digestion

1.5 b 1.0

0.5

0.0 Free insulin

(b)

Microgels

TMR TMR

a

Fold increase

Antacid-free microgels

Microgels +Antacid

pAkt S473/Akt 2.5

Antacid-free microgels FITC FITC

b

2.0

1.5 b

b

Free insulin

Microgels

1.0

0.5

0.0

pH measurements

Microgels +Antacid

Insulin activity

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