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Development of pH sensitive alginate/gum tragacanth based hydrogels for oral insulin delivery Sevil Cikrikci, Mecit Halil Oztop, and Behic Mert J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02525 • Publication Date (Web): 22 Oct 2018 Downloaded from http://pubs.acs.org on October 23, 2018

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Journal of Agricultural and Food Chemistry

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Development of pH sensitive alginate/gum tragacanth based hydrogels for oral insulin

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delivery

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Sevil Cikrikcia, Behic Mertb, Mecit Halil Oztopb

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a Food

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Corresponding author:

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Mecit Halil Oztop, Middle East Technical University (METU), Universiteler Mah. Dumlupinar

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Bulvari, No: 1 Cankaya-06800, Ankara, Turkey.

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E-mail: [email protected]

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Engineering Department, Middle East Technical University, Ankara, Turkey

[email protected] [email protected]

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Tel: +903122105632

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Fax: +903122102767

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ABSTRACT

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Insulin entrapped alginate-gum tragacanth (ALG-GT) hydrogels at different ALG replacement

16

ratios (100, 75, 50, 25) were prepared through an ionotropic gelation method, followed by

17

chitosan (CH) polyelectrolyte complexation. A mild gelation process without the use of harsh

18

chemicals was proposed to improve insulin efficiency. Retention of almost the full amount of

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entrapped insulin in simulated gastric environment and sustained insulin release in simulated

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intestinal buffer indicated the pH sensitivity of gels. Insulin release from hydrogels with

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different formulations showed significant differences (p < 0.05). Time Domain (TD) NMR

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relaxometry experiments also showed the differences for different formulations and the

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presence of CH revealed that ALG-GT gel formulation could be used as an oral insulin carrier 1 ACS Paragon Plus Environment


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at optimum concentrations. The hydrogels formulated from biodegradable, biocompatible and

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nontoxic natural polymers was seen as promising devices for potential oral insulin delivery.

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Keywords: Hydrogel; insulin release; NMR relaxometry; modelling

27 28

1. Introduction

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Hydrogels are three-dimensional polymeric networks that have great potential to be used as

30

scaffolds for tissue regeneration or as delivery vehicles for therapeutic agents, bioactive

31

components, in food systems 1. Having ability to absorb biological fluid or large amount of

32

water, hydrogels can provide excellent biocompatibility and biodegradability thus can be used

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for designing controlled release systems.

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Polymers from natural as well as synthetic sources have recently been taken place in hydrogel

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formulation 2. Some of the most commonly used synthetic polymers are poly (ethylene glycol)

36

(PEG), poly (vinyl alcohol) (PVA) or poly (2-hydroxyethyl methacrylate) (PHEMA). Natural

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polymers such as alginate, chitosan, agarose, hyaluronan, collagen, fibrin, pectin, xanthan gum,

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guar gum and gelatin were also utilized for this purpose 3–12. The advantage of natural polymers,

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as compared to their synthetic ones is their ability to degrade within the body over time which

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is usually considered as the ideal case for circulating drug delivery vehicles 6. Thus, attention

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would be given for the development of biocompatible and biodegradable hydrogels formulated

42

with polysaccharides, polypeptides and proteins.

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In recent years, hydrogels, particularly polysaccharides derived from seafood and agricultural

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wastes and surpluses, have been studied as delivery matrices for different compounds and active

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agents 13. Pharmaceutical and medical industries have been interested in the use of biopolymers

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like alginate owing to its unique biodegradability, biocompatibility, non-toxicity and

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immunogenicity

14

. Alginates which is a salt of alginic acid, a family of linear unbranched

2 ACS Paragon Plus Environment

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polysaccharides with varying amounts of 1,4′-linked β-D-mannuronic acid and α-L-guluronic

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acid residues is known to be an anionic polymer 15. It include carboxyl groups in each residue,

50

thereby could easily interact with polyvalent metal cations especially with Ca+2 for gel

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formation 16. It is very popular as a pH-responsive polymer due to its shrinkage at lower pH,

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which could enable encapsulated drug retention in the stomach protecting against enzymatic

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deactivation 17. Although calcium ions are good physical cross linkers of alginate, sometimes

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such kind of a strong interaction between calcium and the carboxylic groups of alginate

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suppress drug release 17. Its controllable degradation, ease of chemical modification and self-

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healing properties makes alginate a candidate polysaccharide for drug delivery

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other hand, enzymatic degradation, poor mechanical characteristics, uncontrolled drug release

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are among drawbacks of alginates so combining alginates with other polymers could overcome

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this problems improving favorable properties

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chitosan 20–22, gum arabic 23, pectin, carrageenan and psyllium 9 have been employed previously

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in the literature. To best of our knowledge, alginate (A) hydrogels combined with gum

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tragacanth have not been used as a carrier and the current study focused on this formulation.

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Gum tragacanth (GT) is an anionic, branched polymer with D-galacturonic acid, D-galactose,

64

D-xylose, L-arabinose and L-fucose units

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tragacanthic acid (water swellable) and tragacanthin (water soluble)

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fraction, has an approximate molar mass of 104 Da including highly branched arabinogalactan

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groups. Bassorin, the pectic component (60-70% of the total gum) with a molar mass of around

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105 Da has the ability to swell and form a gel

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immunogenic, nontoxic and biodegradable and exhibiting gel forming ability is another

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polysaccharide preferred for biomedical and oral drug delivery applications

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polycation obtained by alkaline deacetylation of chitin. While chitin, a linear homopolymer,

72

includes β-(1,4)-linked N-acetyl-glucosamine units, chitosan consists of copolymers of N-

24

9,14

18,19

. On the

. Synergistic interaction of alginate with

. It includes two major fractions as bassorin or

25

24

. Tragacanthin, neutral

. Chitosan (CH), being biocompatible, non-


26–28

. It is a


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29

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acetyl-glucosamine and glucosamine with the availability of free amino groups

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has strong mucoadhesive features owing to molecular attractive forces created by electrostatic

75

interaction between negatively charged mucosal surfaces and positively charged chitosan and

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thus it could act as permeation enhancer 30. Various studies have been reported about the use of

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chitosan in delivery formulations such as delivery of antioxidants

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polyphenols 31, curcumin 32, iron chelator 21, bacterial cells 33 and drugs 27,34,35.

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Therapeutic proteins are among such kind of agents that need a controlled release system since

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there are some limitations in their oral delivery. During passage through oral route, drug

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molecules encounter with several barriers, like rapid enzymatic degradation, harsh acidic

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environment and poor intestinal absorption 36. One such kind of a therapeutic protein is insulin

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as being polypeptide hormone that formed after elimination of C peptide by hydrolysis and

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regulating blood glucose level 37. Oral delivery of insulin is a challenging issue in drug industry.

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Diabetes is one of the most dangerous modern diseases and external insulin becomes one of the

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most effective treatment of diabetes 38. Since its discovery, diabetic patients take insulin solely

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through injection due to previously mentioned reasons but daily injections rise various concerns

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and discomfortability to patients

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patients lives and extensive amount of studies have been going on for this purpose.

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Although there are studies about formulation of pH responsive hydrogels for oral protein drug

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delivery such as chitosan based gels

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alginate hydrogels

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characteristics and more suitability for desired commercial usage 56–58. In order to enhance the

94

oral insulin bioavailability as well as to provide a biocompatible and stable environment for

95

entrapped drug, not all biopolymers show the same effectiveness. It is altered regarding to type

96

of biopolymer and the design techniques 39.

14,15,50–55

39

13

. Chitosan

, anthocyanins 8,

. Therefore, oral administration of insulin would relieve

2621,40–44

, synthetic polymer based gels

1,45–49

, composite

, there is still a need for design of hydrogel systems with better


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Hydrogels usually swell in water due to the penetration of a hydrophilic solvent caused by

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polymer chain relaxation and polymer-polymer and polymer-water interactions affect this

99

response

16

. Similarly, release ability also shows changes with regard to gel formulation.

100

Diffusion, swelling and chemical mechanisms could explain the release of active compounds

101

59

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mechanisms, there is also a need of rigorous mathematical modelling approach 60. In this paper,

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m release behavior of hydrogels was carried out using Fick's law of diffusion to predict effective

104

diffusivity of gels prepared with different polymer ratios. Since gels did not show a considerable

105

swelling, only diffusion as dominant mechanism was considered.

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Nuclear Magnetic Resonance (NMR) relaxometry is an non-invasive technique to examine

107

release and solvent uptake behaviors of polymer networks

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interactions of gums and water distribution in gels without any sample disruption 61. Since NMR

109

signal comes from protons in a sample, distribution and mobility of protons give information

110

about internal structure of the sample, particularly by measuring T1 (spin-lattice or longitudinal

111

relaxation time) and T2 (spin-spin or transverse relaxation time) values 62. While T1 is a measure

112

of the effectiveness of the magnetic energy transfer between spinning 1H protons and the

113

surrounding lattice, T2 is related to the effectiveness of energy transfer between neighboring

114

spins which is expected to be shorter for closer proximity between molecules 63. Therefore, pure

115

bulk water exhibits both higher T1 and T2 values than solids. In addition to these time constants,

116

self- diffusion coefficient (SDC), different than effective diffusivity, is another parameter that

117

could be analyzed by NMR relaxometry. NMR is an effective technique for analysis of

118

molecular mobility and SDC on a macroscopic scale 24.

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The main purpose of this study was to investigate the insulin release profiles from alginate gels

120

combined with GT at different replacement ratios. Additionally, it was aimed to analyze the

121

effect of polyelectrolyte complexation (PEC) with CH on gel behaviors. NMR relaxometry,

. For the successful design of the drug delivery systems and description of complete


24

. It helps to identify molecular


122

textural and microstructural analyses were performed to comprehend the gel characteristics.

123

Experimental data obtained from chromatographic measurements were also used for estimation

124

of D values. The findings of this study could be promising to develop new hydrogel

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formulations to fulfill therapeutic needs.

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2. Materials and methods

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2.1 Materials

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The polysaccharides sodium alginate with low viscosity (viscosity of 1% aq. solution: 75 % and

131

medium molecular weight) was provided from Sigma-Aldrich (St. Louis, MO) to be used in the

132

experiments. Absolute hydrochloric acid (37%, with density of 1.19 g/cm3 at 20 °C, boiling

133

point of 45 °C, melting point of -28 °C) and potassium dihydrogen phosphate (KH2PO4)

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(EMSURE® ISO) were obtained from Merck (Germany). Calcium chloride dihydrate, sodium

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chloride and sodium hydroxide were obtained from Sigma–Aldrich (St. Louis, MO). Standards,

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insulin human (≥27.5 units/mg, meets USP testing specifications) and m-cresol (99%) were

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purchased from Sigma–Aldrich (St. Louis, MO). Humulin R (100 IU/ml, Eli Lilly and

138

Company, France) was used as the insulin source in all experiments.

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Double distilled water from a water purification system (Nanopure Infinity, Barnstead

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International, IA) was used for preparation of HPLC phase solutions. All other chemicals used

141

were of analytical grade.

142

2.2 Gel preparation

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Hydrogels were prepared by immersing gelation solution including alginate (A) and gum

144

tragacanth (G) at different ratios into the gelation solution containing CaCl2 (1.5% (w/w)) and


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with/out chitosan solution (0.5% (w/v)). The total concentration of polymers (A and G) was

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kept at 1% (w/w). All formulations were presented in Table 1.

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Polymers alginate and gum tragancth in water were stirred at 10,000 rpm for 2 min using Ultra

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Turrax T-18 (IKA Corp., Staufen, Germany) in two separate beakers. After combining of them,

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they were left at the magnetic stirrer for 1 h at room temperature. Then 2.5 ml of humulin was

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added to the 100 g polymer solution and stirred at 5,000 rpm for 2.

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To prepare chitosan solution, same procedure given in the study of Xu & Dumont (2015) 64 was

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followed. Firstly 5.0 g of chitosan was dissolved in 500 mL 1% HCl solution. The pH of the

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solution was then adjusted to 5.5 with 1 M NaOH and diluted to give a final volume of 1 L 64.

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At 25 ˚C, A-G solution was poured into mesh plastic baskets by immersing into the binding

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solutions (with/out chitosan) at the same time. The resulting gels were allowed to harden in the

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gelation solution overnight and then washed with distilled water. Then they were cut into

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cylinders of 2 cm length and 1.3 cm in diameter.

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2.3

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Zeta potential of the solutions were measured by using nano ZS90 (Malvern Instruments, UK).

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Experiments were conducted at METU Central Laboratory.

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2.4

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The digestive media, SGF and SIF were prepared regarding to the method used by Xu &

163

Dumont (2015)

164

degradation of insulin in the release media, SGF and SIF were prepared with free enzyme. A

165

similar approach was also followed by other researchers

166

to the study of Xu & Dumont (2015) 64, SGF (pH 1.2) was prepared by dissolving 2.0 g of NaCl

167

in 7 mL of concentrated HCl and setting the final volume to 1 L after adjusting pH to 1.2. For

Zeta potential measurement

In vitro insulin release studies

64

. Since preliminary experiments showed that enzymes caused rapid

65,66


. For digestion mediums, similar


168

SIF (pH 6.8), 6.8 g of KH2PO4 was dissolved in 250 mL of distilled water, then 77 mL of 0.2

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N NaOH solution was added and final volume of 1 L was diluted adjusting pH to 6.8.

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After blotting the gels with a tissue paper as much as possible, each hydrogel was suspended in

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40 ml of digestive medium at 37 °C and stirred at 80 rpm for 24 h. At determined time intervals

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(0.5, 1, 2, 3, 4, 5, 6 and 24 h), an aliquot sample (1mL) was taken from the release medium to

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analyze insulin content by Reverse-Phase High Performance Liquid Chromatography (HPLC)

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(Shimadzu, Japan) using RP 18e Chromolith® HPLC Column (4.6 mm, 2 μm diameter, Merck,

175

Germany).

176

For HPLC experiments, the mobile phase was a premixed isocratic mixture of 0.2 M sodium

177

sulfate anhydrous solution adjusted to pH 2.3 with phosphoric acid and acetonitrile (72:28, v/v)

178

67

179

monitored at 214 nm 44. Calibration curve of insulin was prepared with insulin human standard

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solutions of known concentrations. All experiments were done at least duplicate.

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2.5

182

Effective diffusion coefficient for release of insulin from each gel samples were predicted by

183

using mass transport equation for diffusion in infinite plane and infinite cylinder in a stirred

184

solution of limited volume

185

geometry was found as given 24;

186

. Flow rate was 1.0 ml min−1 at 40 °C and the injection volume was 20 μl 68. The eluent was

Modelling release behavior of gel samples

69

combining using product rule. The solution for finite cylinder

∞

∞

𝑚=1

𝑚=1

2 𝑀𝑡 4𝛾 (1 + 𝛾) 𝐷𝑞𝑚 2𝛾 (1 + 𝛾) 4𝐷𝑝𝑛2 = 1 − (∑ 𝑒𝑥𝑝 (− 2 𝑡)) . ( ∑ 𝑒𝑥𝑝 (− 2 𝑡)) 2 𝑀𝑖𝑛𝑓 4 + 4𝛾 + 𝛾 2 𝑞𝑚 𝑅 1 + 𝛾 + 𝛾 2 𝑝𝑛2 ℎ

187

(Equation 1)

188

where Mt/Minf is the ratio the instantaneous uptake (Mt) to the uptake at long times (M∞). R is

189

radius and h is the height of the samples respectively. γ indicates the ratio of the volume of the


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solution to the volume of sample. Effective diffusion coefficient is represented as “D”. The

191

qm’s are the nonzero positive roots of

192

𝛾𝑞𝑚 𝐽0 (𝑞𝑚 ) + 2𝐽1 (𝑞𝑚 ) = 0

193

(Equation 2)

194 195

and pn’s are the nonzero positive roots of tan(𝑝𝑛 ) = −𝛾𝑝𝑛

196 197 198

(Equation 3)

199 200

Eqn. (1) was used to fit experimental data and then diffusion coefficients were estimated by

201

using Matlab function “lsqnonlin”.

202

2.6

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Textural property of gels were evaluated by using a texture analyzer (TA.XTPlus, U.K). 1 cm

204

diameter cylindrical probe was used for gel samples having dimensions of 3 cm x 3 cm x 2 cm.

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Gel samples were compressed to 80% of their initial height at a 1 mm/s pre-test speed. 1 mm/s

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test speed and 10 mm/s post-test speed with 60 s holding time were then used 70. From texture

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profile curve, firmness and springiness values were obtained. Six replicates from three different

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sets of gel solutions were measured to take averages of them for interpretation.

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2.7

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Morphological properties of hydrogels were analyzed with scanning electron microscope (FEI

211

Nova NanoSEM 430, Oregon, USA). Samples for SEM were first freeze dried (Christ, Alpha

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2-4 LD plus, Germany). A thin layer of gold was used for coating and then samples were

Texture profile analysis

Scanning Electron Microscopy (SEM)



213

analyzed at an acceleration voltage of 20 kV. Images were observed at magnification levels of

214

80X, 300X and 10000X. Analyses were done at Metallurgical and Materials Engineering

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Laboratory of METU (Ankara, Turkey).

216

2.8

217

FTIR spectra of hydrogels and also alginate, gum tragacanth and chitosan powders were

218

performed by IR Affinity-1 Spectrometer using Attenuated Total Reflectance (ATR)

219

attachment unit (Shimadzu Corporation, Kyoto, Japan). The range between 4000 and 400 cm-1

220

region at 4 cm-1 resolution for 32 scans was used. Before the analysis, the gel samples were

221

freeze dried (Christ, Alpha 2-4 LD plus, Germany) at 48h after refrigeration period 71.

222

2.9

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0.32 T NMR system (Spin Track SB4, Mary El, Russia) was used to conduct NMR

224

measurements. The parameters were set according to the study of Ozel et al. (2017)24. To

225

measure spin-lattice relaxation (T1) time, a saturation-recovery pulse sequence was used with

226

15s time of observation with 8 scans. Delay time was changed in the range of 0.5 ms-2 s with

227

1024 acquisition points. To measure spin-lattice relaxation (T2), Carr-Purcell- Meiboom-Gill

228

(CPMG) sequence was performed. 6s relaxation period with 32 scans, 1 ms echo time, 800

229

echoes were used. Self-diffusion coefficients (SDCs) of hydrogels were determined by a pulse

230

gradient spin echo sequence with 90° pulses, three 22 us, with acquisition time of 500 us. 2 ms

231

was used as the time interval between the first and the second pulses. The time interval between

232

the second and the third pulses were taken as 60 ms. The duration of the pulsed gradient field

233

and the gradient strength were set as 1 ms and 1.66e-2 T/m, respectively. All NMR

234

measurements of hydrogels were conducted at 0 h and 6 h and all NMR results were analyzed

235

using MATLAB.

Fourier Transform Infrared (FTIR) Spectroscopy

NMR relaxometry measurements

236


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2.10

Statistical analysis

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Statistical analyses to determine differences in gel characteristics were accomplished by

239

analysis of variance (ANOVA) using Minitab (Version 16). Tukey’s test at 5% significance

240

level was used for comparison.

241

3. Results and discussion

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3.1 Release profiles

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The HPLC chromatogram of Humulin (as two peaks) released from gels after incubation in the

244

biological fluids gave similar retention time with insulin and m-cresol standards (Fig. 1). It was

245

confirmed that first peak belonged to insulin while second peak represented m-cresol. The

246

insulin release study of gel samples was initially conducted in both SGF and SIF. Since initially

247

small amount of insulin in buffer was obtained but then no considerable cumulative release rate

248

was observed in SGF (Fig. 2), due to the acidic pH and gels not releasing out insulin at that pH,

249

release behavior SIF were just evaluated for further experiments (Figs. 3). In order to

250

understand whether the reason of not detecting insulin in SGF was either low release rate or

251

destruction of insulin due to low pH, insulin loaded gels were completely disintegrated in SGF

252

using a high shear homogenizer and HPLC analysis was conducted for the medium. Since the

253

samples gave identifiable insulin content, this implied that hydrogel created a controlled release

254

system protecting insulin from burst release in SGF, on the other side showing cumulative

255

release up to 70% in SIF. Although the release study was continued for 24 h, only 6 h release

256

was figured out due to insulin degradation after that period.

257

The intestine is usually seen as the main absorption site for oral formulations, thus it is

258

meaningful to focus on intestine-targeted delivery system that remains intact at acidic pH of

259

stomach but dissociates and releases the drug at alkaline pH of small intestine 72. In intestinal

260

media, calcium ions are also lost from the gels due to formation of calcium phosphate salts in



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SIF 64 causing weakening in the structure thereby increasing release. It is possible to list other

262

possible interactions that contribute to the three-dimensional cross-linked gel networks: the

263

guluronic and mannuronic acid units and interchain hydrogen bonds, the junction formed by

264

the calcium ion; electrostatic interactions (between opposite charges of the biopolymers), GT-

265

ALG interactions, interactions between insulin and polymers

266

carboxyl group due to the existence of galacturonic acid and this means that the charge

267

differences in GT are induced by this carboxyl group 75. This carboxyl group ( COO−) could

268

interact with the amino group ( NH3+) of cationic polymer (chitosan) and proceeds ionic

269

complexation between the two polymers via polyelectrolytes interactions depending on

270

medium pH 75,76.

271

The ionizable groups present in the polymer backbone are mainly responsible for pH responsive

272

behavior of the hydrogels. When gels are exposed to an aqueous solution of an appropriate pH

273

and ionic strength, these groups would ionize and emerge a fixed charge along the polymer 6.

274

Anionic hydrogel network had potential to swell in solutions at pH > pKa (dissociation

275

constant) due to presence and repulsions of ions, while cationic hydrogels were swollen at pH

276

< pKa since cationic pendant groups are protonated at pH less than pKa 6. Due to its structure,

277

alginate gel is stable in acidic pH of stomach, while it swells and begins dissolving in the

278

intestinal alkaline pH > 6 26. At SIF, pH (6.8) is higher than pKa of alginate (3.38-3.65) and GT

279

(at around 3) making them negatively charged. Thereby, hydrogel network composed of both

280

anionic and cationic polymers had a complex nature that differs regarding to composition. At

281

intestine media, both polysaccharides were strongly negatively charged thus steric and

282

electrostatic repulsions between the side chains played an important role in the release of

283

insulin. ALG, GT and CH underwent physical crosslinking owing to charge or hydrophobic

284

interactions, electrostatic repulsions, as well and their interactions shaped release mechanism.

285

Furthermore, isoelectric point (pI) of the target protein, insulin (pI of 5.5-6.4), also played an 12 ACS Paragon Plus Environment

73,74

. Like alginate, GT has a

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important role in gel response to environmental conditions. Insulin is a relatively small

287

hydrophilic, water soluble protein with molecular weight of approximately 6000

288

insulin is composed of 10 amino acid residues capable of attaching a negative charge and the 6

289

amino acid residues capable of attaining a positive charge, it has an amphiphilic character 79.

290

When pH > pI, charge status of insulin becomes negative

291

negatively charged at intestinal pH, it could still include a few positive charges in its own

292

structure. These properties were, thereby, possibly responsible for the entrapment of insulin in

293

the hydrogel network 26.

294

ALG-GT hydrogels without CH exhibited a similar release profile during early times.

295

Beginning from 3 h, distinction between samples began to appear in release percentages but

296

significant difference occurred at the end of 5 h and 6 h (p < 0.05). After the end of 5 h, 25A

297

hydrogels gave the highest release differing from other samples (p < 0.05). As insulin was

298

expelled from the gel, charge density differed and sample with high GT concentration, 25A,

299

promoted higher insulin release. On the other hand, PEC with CH created differences between

300

samples even at early times (p < 0.05). Unlike to standard hydrogels, PEC gels with CH showed

301

different a release pattern with changing gum ratios (p < 0.05). Similar to ALG-GT gels, 75A-

302

CH gels retarded release through all period and release of 50A-CH approached 25A-CH gels

303

giving high and closer results, especially exhibiting the most similar results with 25A-CH at the

304

end of 5 h (p < 0.05) (Fig.3b). Thereby, release time of 5 h was obtained as the critical time for

305

differentiation of hydrogels formulations both with CH and without CH. It has been

306

demonstrated that chitosan based formulations had easy paracellular transport of agents and

307

good mucoadhesive property due to electrostatic interaction between negatively charged sialic

308

acid of mucin and positively charged chitosan with abundant amino groups 27. Gel formulation

309

with chitosan and anionic polymer could protect therapeutic agent from gastric degradation but

310

allow the agent to release in intestine through electrostatic interaction. As gel moves to intestine, 13 ACS Paragon Plus Environment

39

78

. Since

. Although insulin becomes


311

at alkaline pH, negative charge of A and GT was higher sequestering positive charge of CH,

312

resulting in gel dissolution and acceleration of the drug release 80.

313

In order to understand the reasons these results, pH of each gel solutions during preparation

314

were also compared. They varied from 6.1 to 6.9 (from 25A to 100A) which showed that pH

315

increased as GT ratio decreased. According to this finding, it was expected to occur higher

316

repulsion due to higher negative charge in gel network as GT concentration decreased and so

317

higher release in lower GT ratios. However, opposite behavior was observed. This showed that

318

rather than the charge effect of polysaccharides, crosslinking between target protein-

319

polysaccharides and gel internal structure through electrostatic interactions predominated the

320

release pattern. GT is a branched and heterogeneous polysaccharide including both water

321

soluble and water swellable units 81. This high hydrophilicity of GT side residues could have

322

enhanced the interaction between gel and absorbed thus resulting in higher opening of the

323

network.

324

In overall, the ranges of insulin release from PEC with CH in intestine were observed to be

325

higher than that from gels without CH. PEC with CH weakened the gel structures leading

326

structural defects within the hydrogels. This promoted faster release rates. It shows that binding

327

solution with both calcium and CH was not strong enough to retain insulin in gel matrix as

328

solution with only calcium. Crosslinking density was reduced and caused erosion of polymers

329

in CH complex gels. At intestinal pH (> 6.5), CH could loose its positive charge and become

330

weaker 39. Electrostatic interaction between the protein and the polyanion might also favor the

331

retention of insulin within gel matrix in ALG-GT gels without CH complex 79. Opposite to our

332

results, Lim et al. (2014) 57, Reis et al. (2008) 73 and Martins et al. (2007) 79 revealed that CH

333

as additional binding could achieve sustained insulin release due to action of CH as a barrier

334

against insulin diffusion. Xu and Dumont (2015)

335

that PEC with CH served prolonged release matrices reducing impact of loss of calcium ions

64

also found in protein based alginate gels


Page 14 of 43

Page 15 of 43


336

on the hydrogel network. Although CH could have acted as a barrier, this did not overcome

337

other polysaccharide interactions in each gel formulations. Control sample, 100A (GT free),

338

exhibited immediate results in both gels with CH and without CH. This pointed out there was

339

an optimum concentration for the desired ALG-GT interactions. It was observed that the zeta

340

potential of binding solution with calcium chloride and CH was + 30.8 mV proving positive

341

charges in CH. As expected, opposite to CH solution, gelation solutions had the zeta potential

342

in the range of - 49.3 mV and - 42.4 mV.

343

3.3 NMR Relaxometry results

344

Non-destructive method, NMR for proton relaxation in gels, was conducted to comprehend

345

internal structure of samples. T1 and T2 are parameters giving crucial information about water

346

uptake and polymer-water/ polymer-polymer interactions, respectively. T1 relaxation time is

347

related to motional frequency. Since H protons in water have higher motional frequencies than

348

in solids, water has higher T1 values than solids 62. Vittadini et al. (2002) 82 reported that T1 and

349

T2 increased with moisture content due to higher mobility in samples. In the light of this

350

approach, it is applicable to say that samples absorbed higher amount of water could give higher

351

T1 results. Fig. 4 represented T1 values of all gels before and after immersed in SIF. Water

352

uptake in all samples after immersion into intestinal fluid could be clearly understood from T1

353

results of 6 h giving higher values than 0 h. However, this increase was lower in gels with CH

354

than in gels without CH. This could be the sign of action of CH as a barrier for water absorption.

355

Water uptake could decrease with the formation of ionic interaction between carboxylic groups

356

of ALG and amine groups of CH and with formation of covalent links between macromolecular

357

chains of ALG-GT and CH 26.

358

Another parameter, T2 relaxation time gives information about interaction between water and

359

polymer or between two polymers. As given in Fig.1, T2’s of gels was obtained before and after

360

immersion into SIF through 6 h. Initially, all gels had shorter T2 times and then they began to 15 ACS Paragon Plus Environment


361

increase after 6 h immersion in solution. This was consistent with T1 results. The main

362

contribution for this increase was the amount of water present in gels and interaction between

363

water and gel network

364

molecules led to larger T2.

365

0 h and 6 h T2 results differed significantly but the presence of CH did not cause significant

366

impact on T2 values (p < 0.05). In both samples with CH and without CH, T2 showed an increase

367

as GT ratio increased. While 100A gel gave T2 as 148.07 ms, T2 of 25A gel was 317.31 ms

368

giving significant difference at 0 h (p < 0.05). At the end of 6 h, T2 of 100A and 25A increased

369

to 155.93 ms and 287.11 ms, respectively. Although the results of gels with CH were higher

370

than samples without CH at the same ratio, they were not significantly different (p < 0.05).

371

Unlike to T1 results, CH complex gels gave closer profile with standard gels. Although the

372

presence of CH reduced fluid uptake regarding to T1 profile, it gave a similar increasing

373

tendency in the absence of CH. It could be hypothesized that CH complex gels had weaker

374

structures during uptake of water and led water molecules as free state diffusing in and out

375

through gels 24. This could have also caused weaker entrapment of insulin and easier release

376

from the matrix consistent with release measurements.

377

SDC of water molecules in gels varied from at around 2 x 10-9 to 1 x 10-9 m2/s (Table 2). They

378

represented average data of water molecules coming from different parts within gels. SDC is

379

related to self-diffusion of water in matrix and mobility of water molecules 24. All gels except

380

for 100 A-CH and 25 A-CH after 6 h immersion in intestine media gave similar SDC (p < 0.05).

381

Higher SDC value of 25 A-CH (2.04 x 10-9 m2/s) than 100 A-CH (1.53 x 10-9 m2/s) supported

382

T1 results indicating higher water uptake and more mobility of water molecules.

383

3.4 Texture profiles

384

Hardness and springiness of all fresh gel samples were shown in Table 3. Replacement of ALG

385

with GT reduced hardness of gels regardless of the presence of CH. This can be another reason

24

. Swelling of samples absorbing solvent and having more hydrogen


Page 16 of 43

Page 17 of 43


386

of both NMR relaxometry and chromatographic release results. With increase in GT ratio, a

387

weaker gel structure was obtained and tendency to absorb water became higher than tendency

388

for interactions between biopolymer chains. Hence, it was meaningful to obtain high T1 and T2

389

values in these samples. Similarly, weak interaction between ALG and GT may be another

390

reason for unsuccessful insulin entrapment in SIF. Nevertheless, these findings were different

391

than some studies such as reports of Belscak-Cvitanovic et al. (2015) 13. This may be due to

392

unique structure of GT and its characteristic interactions with other biopolymer materials.

393

Moreover, addition of CH decreased hardness values except for 25A formulation (p < 0.05) and

394

this pointed out again weak structure of gels prepared with CH as previously obtained in other

395

analyses.

396

Springiness (elasticity) is a measure of how much the gel structure is broken down by the initial

397

compression and is related to sensitivity of gel rubbery feeling in the mouth 83. In gels with/out

398

CH gave higher springiness as higher amount of ALG was replaced with GT up to 25A gel.

399

Optimum concentration was found as 50 A and after this concentration, springiness began to

400

decrease again (p < 0.05).

401

3.5 Release modelling of hydrogels

402

Fick’s second law was used to model insulin release from gels in intestinal fluid and to estimate

403

diffusion coefficient, D. Analytical solutions given in Eq. (1) was used to fit experimental data

404

for D estimation, using MATLAB nonlinear curve fitting subroutine. Since diffusion became

405

dominant rather than swelling in gels, diffusion-controlled mechanism has been demonstrated

406

in this study. Fitted D values for insulin release from the gel were in the order of 10-10 m2/s.

407

Results changed between 2.48 and 7.29 10-10 m2/s (Table 4).

408 409



410

3.6 FTIR

411

In order to characterize possible chemical interactions between polymers in gel structure, FTIR

412

measurements were conducted. Fig. 5 showed the FTIR spectra of each polymer and insulin

413

loaded gels at different ratios over the range of 400-4000 cm-1.

414

For common to all polysaccharides, the observed characteristic peak at around 1040 cm-1 was

415

contributed to stretching of C-O bonds

416

galacturonic and guluronic units, ALG-GT based gels were more likely to have strengthened

417

peaks 71,86. FTIR spectra of raw GT confirmed this hypothesis by giving considerable peak at

418

that region (Figure 4). Typical bands of polysaccharides in the spectral range of 3000-3680

419

cm−1 representing asymmetric stretching of the many hydroxyl groups were observed in both

420

ALG and GT. Since GT contains sugars like xylose, fucose and arabinose units, absorbance at

421

around 1040 and 1070 cm−1 representing the existence of galactose such as arabinogalactans

422

became higher in gels with higher GT content 87. Variations in pH could lead shifts of stretching

423

groups and so differences in zeta potential of each sample could be the reason of small shifting

424

in peaks but this did not affect absorbance values 75.

425

The broad bands at 1425 and 1640 cm−1 represent the symmetric and asymmetric stretching

426

mode of the carboxyl groups

427

galacturonic acid indicating carboxyl group ( COO− ) and it could interact with amino group

428

(NH3+) of cationic polymer (chitosan) forming ionic complex via polyelectrolytes interactions

429

89

430

about polymer interactions in gel network. FTIR spectra of insulin loaded gel samples were

431

different than spectra of pure polymers due to these interactions during and after gelation

432

process. After complexation with CH, especially the peak at range of 3000-3680 cm−1 belonging

433

hydroxyl groups enlarged and peak intensity decreased. In addition, the peaks related to amino

434

groups coming from CH and insulin at around 1153 cm-1, 1650 cm-1 and 1540 cm-1 can be seen

26,52,88

84,85

. Since this band could refer to the existence of

. Similar to ALG, GT as anionic polymer includes

. Hereby, monitoring changes in carboxyl group in spectra would be helpful to get information


Page 18 of 43

Page 19 of 43


64,76,89,90

435

in the spectra. These observations agreed with previous studies

. More interestingly,

436

comparison in peak intensities with respect to each gel formulation showed same trend with

437

release studies of these gel formulations. Since the sample with the high release rate also had

438

higher peak amplitude, use of FTIR response of the samples was efficient to study interactions

439

through the gels.

440

3.7 Microstructures of hydrogels

441 442

SEM was used to examine internal gel structure and surface morphology of hydrogels. Insulin

443

loaded freeze-dried hydrogels were displayed in Fig. 6. In the absence of CH, gels exhibited a

444

relatively smooth and homogenous surface while gels with CH revealed irregular shape with

445

fissures. Likely, the addition of GT created more heterogeneous structure in matrix. Plain ALG

446

gel, 100A, had the straightest and smooth surface and the most regular shape, without irregular

447

cavity and collapsed structure 9. These morphological changes in different samples pointed out

448

different physicochemical interactions in each gels

449

crosslinking of ALG with calcium might have been prevented with cavities which were in

450

agreement with the study of Belscak-Cvitanovic et al. (2015)

451

(2011) 91. Less denser and more porous and tortuous flake structure in gels with CH explains

452

the poor ability for slowing down insulin release and less firm gel structure obtained comparing

453

the gels without CH 23,79,92. These are also valid for GT effect in gel internal structures.

454

In conclusion, it can be stated that alginate can form cold set gel through two mechanisms. One

455

of them is polyelectrolyte complexation with an oppositely charged biopolymer like chitosan

456

and the latter is ionotropic gelation in the presence of cross-linkers like Ca2+

457

both of them were conducted and compared. The incorporation of another polyanions into

458

chitosan and alginate gel formulation has also been reported to improve insulin release and

459

entrapment efficiency

26

. With the presence of CH, strong

77

13

and Popa, Gomes, and Reis

57

. In this study,

. For this purpose, GT was used as additional polymer by replacing 19 ACS Paragon Plus Environment


460

ALG at different ratios. ALG-GT blend gels with/out CH were prepared at different

461

replacement ratios. Results of in-vitro studies showed that, all gels retained insulin in gastric

462

buffer however, PEC gels with CH showed more tendency to release entrapped insulin in

463

intestinal conditions. Increase of GT ratio in formulation also pronounced less firm gel structure

464

and weaker polymer-polymer interactions in gel network promoting release of insulin. The

465

current study could offer the use of GT as biodegradable and biocompatible natural polymer as

466

carrier for insulin and other therapeutic proteins.

467 468

4. Acknowledgement

469 470

This research was funded by Middle East Technical University, Faculty of Engineering,

471

Scientific Research Projects Funds with the proposal number BAP-03-14-2017-002. COST

472

Action CA 15209 European Network on Relaxometry is also acknowledged as some of the

473

findings are discussed in the action’s network meetings and suggestions were taken into

474

consideration in the final text. A part of the funding also came from Dr. Oztop’s award of

475

Science Academy’s Young Scientist Awards Program (BAGEP).

476 477

The authors are also thankful to the specialists working at ANT TEKNIK, Ankara, Turkey:

478

Akın Osanmaz, Selim Hasay and Hakan Aktas for their technical support. Authors would also

479

like to thank specialist at METU Food Engineering Department, Dr. Zinet Aytanga Gökmen

480

for her help in chromatographic analyses.

481 482 483 484 20 ACS Paragon Plus Environment

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760 761 762 31 ACS Paragon Plus Environment


763

Table 1. Composition of hydrogels

Sample

A : G ratio (%)

Binding solution

100A

100:0

1.5% CaCl2

100A CH

100:0

1.5% CaCl2 + 0.5% Chitosan (at equal ratio)

75A

75:25

1.5% CaCl2

75A CH

75:25


50A

50:50

1.5% CaCl2

50A CH

50:50


25A

25:75

1.5% CaCl2

25A CH

25:75


764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 32 ACS Paragon Plus Environment

Page 32 of 43

Page 33 of 43

785 786 787


Table 2. SDC values of gel samples. Results are mean values and errors are represented as standard deviation for at least two replicates in each gel sample. Lettering was done for each subgroup separately (p < 0.05). SDC 0 h (m2/s)

SDC 6 h (m2/s)

Hydrogels 100A 75A 50A 25A Hydrogels

(1.67 ± 0.24) x 10-9 a (2.06 ± 0.23) x 10-9 a (2.16 ± 0.05) x 10-9 a (2.13 ± 0.13) x 10-9 a

(1.86 ± 0.24) x 10-9 a (2.07 ± 0.26) x 10-9 a (2.14 ± 0.18) x 10-9 a (1.81 ± 0.09) x 10-9 a

100A CH 75A CH 50A CH 25A CH

(2.01 ± 0.17) x 10-9 a (2.30 ± 0.07) x 10-9 a (1.98 ± 0.13) x 10-9 a (2.25 ± 0.06) x 10-9 a

(1.53 ± 0.05) x 10-9 a (1.72 ± 0.08) x 10-9 ab (1.79 ± 0.13) x 10-9 ab (2.04 ± 0.13) x 10-9 b

788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806



807 808 809

Page 34 of 43

Table 3. Textural properties of fresh gel samples. Results are mean values and errors are represented as standard deviation for at least two replicates in each gel sample (n=5). Lettering was done for each subgroup separately (p < 0.05).

Hardness (g)

Springiness


888.27 ± 61.12a 406.31 ± 27.69b 241.75 ± 6.55c 73.87 ± 10.59d

3.94 ± 8.24 ± 9.68 ± 7.45 ±


505.05 ± 237.50 ± 157.82 ± 127.52 ±

8.29 ± 0.97a 12.73 ± 0.95b 15.25 ± 0.77c 6.52 ± 0.82d

54.82a 34.80b 12.70c 13.47d

810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829


0.18a 0.57b 0.51c 0.59d

Page 35 of 43

830 831 832


Table 4. Diffusion coefficients of gel samples. Results are mean values and errors are represented as standard deviation for at least two replicates in each gel sample. Lettering was done for each subgroup separately (p < 0.05). D (m2/s)

R2


(7.29 ± 0.95) x 10-10 b (3.34 ± 0.15) x 10-10 a (2.98 ± 0.25) x 10-10 a (2.48 ± 0.31) x 10-10 a

≥ 0.87 ≥ 0.90 ≥ 0.80 ≥ 0.70


(2.75 ± 0.92) x 10-10 a (3.94 ± 0.79) x 10-10 a (3.45 ± 0.21) x 10-10 a (2.73 ± 0.04) x 10-10 a

≥ 0.80 ≥ 0.70 ≥ 0.90 ≥ 0.80

833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 35 ACS Paragon Plus Environment


Page 36 of 43

854 m AU(x100) 1.2 214nm ,4nm (1.00) 4.342/236698

a)

3.0

b)

3.082/792656

m AU(x10) 214nm 4nm (1.00)

3.5

1.1 1.0 0.9

2.5

0.8 2.0

0.7 0.6

1.5 3.173/30406

0.5 1.0

0.5

0.4 0.3 0.2

0.0

0.1 -0.5

0.0 -0.1

0.0

2.5

5.0

m in

0.0

2.5

5.0

m in

mAU(x10) 2.00 214nm,4nm (1.00) 4.459/23466367

mAU(x1,000) 214nm4nm (1.00) 2.25

2.00

c)

d)

4.212/106839

855

1.75 1.50

1.75

1.25

1.25

1.00

1.00

0.75

0.75

0.50

0.50

3.001/44072

1.50

0.25

0.25

0.00 0.00

-0.25 -0.25

856 857

0.0

1.0

2.0

3.0

4.0

5.0

2.5

6.0 min

858 859 860 861 862 863 864 865 866 867

Figure 1 36 ACS Paragon Plus Environment

5.0

min

Page 37 of 43


20 18 Cumulative release (%)

16 14 12 10 8 6 4 2 0 0

1

2

3 Time (h)

868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889

Figure 2


4

5

6


Page 38 of 43

100 100A

Cumulative release (%)

90

75A

50A

25A

a)

80 70 60 50 40 30 20 10 0 0

1

2

3 Time (h)

50A CH

25A CH

2

3 Time (h)

4

5

6

890 891 100

100A CH

Cumulative release (%)

90

75A CH

b)

80 70 60 50 40 30 20 10 0 0

1

4

892 893 894 895 896 897 898

Figure 3

899 38 ACS Paragon Plus Environment

5

6

Page 39 of 43


900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921

Figure 4 39 ACS Paragon Plus Environment


Page 40 of 43

922 0.4 GT

a) Absorbance

0.3

ALG CH

b)

0.2 0.1 0 500

900

1300

1700

2100

2500

2900

3300

3700

Wavenumber (1/cm)

923

Absorbance

0.4

25A CH

0.35

50A CH

0.3

75A CH

0.25 0.2 0.15 0.1 0.05 0 500

1000

1500

2000

2500

3000

3500

4000

Wavenumber (1/cm)

924 925

926

0.4 25A

0.35

50A

c)

75A 100A

Absorbance

0.3

927 928 929

0.25

930

0.2

931 0.15

932

0.1

933

0.05

934 935

0 500

1000

1500

2000

2500

3000

Wavenumber (1/cm)

3500

4000

936 937

938 939 940 941

Figure 5


Page 41 of 43


a1)

b1)

a2)

b2)

a3)

b3)

942 943

944 945

946 947



a4)

948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984

b4)

Figure 6


Page 42 of 43

Page 43 of 43


985 986

Table of Contents

987 Absorbance

Alginate (ALG)

Gum Tragacanth (GT)

988

1500

2500

Wavenumber (1/cm)

Humulin

989

0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 500

Gel Ingredients

Gels

990 991 992 993 994 995 996 997 998 999 1000 1001


3500

Gum Tragacanth Based

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