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]

1

ACS Paragon Plus Environment


1

2

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Abstract

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

3

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

7

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

9

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

10

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

11

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

12

gastric conditions.

13

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

14

insulin.

In particular, the encapsulated insulin significantly increased Akt

15

16

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

19

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

20

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

21

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

22

otherwise they will not be eaten.

23

bioactive ingredients encapsulated within nanoparticles or microparticles, are

24

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

25

and medical foods.2

26

In addition,

Colloidal delivery systems, which consist of

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

27

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

28

lost economic activity.3-4

29

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

30

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

31

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

34

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

35

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

36

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

37

convenient and less painful than injections.10-11

38

At present, regular

Delivery of

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

39

development of successful oral insulin delivery systems.

First, insulin is highly

40

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

41

acidity of the gastric fluids.12-13

42

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

43

14

44

gastric stability of insulin.

45

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

46

such as nanoparticles or microparticles.11, 15-17

47

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

48

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

50

biopolymer microgels.

51

biopolymer microgels can modulate the gastrointestinal fate of encapsulated

52

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

53

and/or polysaccharides 18-21.

54

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

55

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

56

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

57

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

58

and cross-linking 23.

59

alginate microgels for encapsulating, protecting, and delivering insulin.

60

been encapsulated within calcium alginate microgels fabricated using an

61

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

62

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

63

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

64

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

68

24

69

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

70

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

71

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

72

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

75

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

76

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

77

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,

78

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

79

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

80

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

82

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

83

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

84

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

85

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

86

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

87

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

89

the encapsulated insulin.

90

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

91

in gastric fluids.

92

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

93

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

98

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

99

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

100

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

101

phosphatase inhibitor and protease inhibitor cocktail were from Thermo Scientific,

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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

111

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

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

113

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

114

solutions.

115

Microgels preparation

116

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

117

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

118

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

119

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

120

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

121

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

122

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

123

was homogeneous.

124

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

125

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

126

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

127

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

129

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

130

washed with buffer solution to remove any residual hardening solution.

131

Simulated gastrointestinal conditions

132

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

133

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

134

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

135

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

136

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

137

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

138

The resulting mixture was

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

139

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

140

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

141

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

142

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

145

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

146

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

147

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

148

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

149

The pH inside the biopolymer microgels was measured using a fluorescence

150

intensity method described previously.33

151

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

152

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

153

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

154

microgels were acquired using the confocal laser scanning fluorescence microscope

155

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

156

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

157

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

158

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

160

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

161

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

162

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

163

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

164

standard curve, as described previously.33

165

from at least eight measurements.

166

Particle size characterization

167

The average intensity ratio was calculated

Particle size distributions were carried out using a static light scattering

168

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

169

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

170

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

171

multiple scattering effects.

172

Insulin activity assay with L6 myotubes and immunoblotting

173

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

174

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

176

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

177

has been reported previously.36

178

mixing them with a calcium chelating agent (EDTA).

179

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

180

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

181

Cells were lysed with RIPA buffer supplemented with phosphatase inhibitor and

182

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

183

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

184

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

185

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

186

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

187

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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189

Statistical analysis

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

190

the results reported as averages and standard deviations calculated using Excel

191

(Microsoft, Redmond, VA, USA).

192

Results and Discussion

193

Preparation and characterization of insulin-loaded microgels

194

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

195

within the stomach phase was determined using confocal fluorescence microscopy

196

(Fig. 1).

197

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

198

systems 23, 31.

199

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

200

antacid-loaded microgels.

201

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

202

indicating that it had been successfully encapsulated.

203

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.

205

intensity of the insulin after exposure to the stomach phase.

206

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

207

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

208

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

209

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

210

(described later).

211

However, there was a significant decrease in the fluorescence

This effect can mainly

The confocal microscopy images indicated that the microgels had a roughly

212

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

213

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

214

measurements indicated that the microgels had a monomodal particle size distribution

215

both before and after exposure (Fig. 2).

216

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

217

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

218

µm after exposure.

219

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

221

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

222

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

223

the insulin molecules more effectively.

224

Mapping the pH inside the microgels

225

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

226

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

227

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

228

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

229

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

230

protease-degradation by maintaining a neutral pH inside them.

231

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

232

neutral should protect it from this type of degradation.

233

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

234

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

236

reducing the local pepsin activity.

237

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

238

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

239

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

240

based on a fluorescence intensity ratio method described earlier.33

241

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

242

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

243

ratio.

244

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

245

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

246

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

For the

247

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

248

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

249

phase.

250

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.

252

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

253

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

254

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

255

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

256

lactase-loaded alginate microgels.23, 31

257

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

258

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

259

neutral pH.

260

Influence of encapsulation on insulin activity

261

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

262

insulin after it had been exposed to simulated gastric conditions.

In its active form,

263

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

264

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

265

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

266

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

268

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

269

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

270

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

271

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

272

thereby inhibiting both acid- and pepsin-induced degradation.

273

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

274

insulin on its activity under gastric conditions.

Studies have shown that insulin can

275

be encapsulated in biopolymer nanoparticles fabricated from alginate and dextran

276

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

277

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

278

nanoparticles subcutaneously into diabetic rats.

279

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

280

administered to the rats.

281

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

282

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

284

encapsulating insulin, protecting it from degradation under gastrointestinal conditions,

285

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

286

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

287

plasma glucose levels after oral administration.44

288

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

289

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

290

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

291

.46

292

commercial viability of the ingredients and fabrication processes used to prepare

293

them, and their functional attributes.

294

Release of insulin in a simulated small intestine phase

295

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

296

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

297

systemic circulation.

298

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

300

small intestine.

301

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

313

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

316

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

317

phase, which would facilitate their subsequent absorption.

318

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

319

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

320

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

332

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

333

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

334

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

335

maintain their insulin activity under real life conditions.

336

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

337

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

338

incorporated into formulations intended for oral administration.

In addition, it will be

339 340

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References

342

1.

343

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

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2.

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Controlled Release System Based on Resistant Starch Acetate: Synthetization,

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

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2011, 59 (10), 5738-5745.

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3.

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

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

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4.

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and the Association with Clinical and Economic Outcomes. Clinical Therapeutics

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


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503

29



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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.

30


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(a)

(b)

31



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32


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Antacid-loaded microgels FITC

TMR

Initial

After stomach

(a) Antacid-free microgels FITC FITC

TMR TMR

Initial

After stomach

(b)

33



Page 34 of 37

34


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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

35



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Protein release (%)

40

30

20

10

0 0

20

40

60

80

100

120

Incubation Time (min)

36


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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

37


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