fish gelatin composite hydrogel as antimicrobial wound dressing

Journal Pre-proof Fabrication and characterization of matrine-loaded konjac glucomannan/fish gelatin composite hydrogel as antimicrobial wound dressing

Liping Zhou, Tingwei Xu, Jicheng Yan, Xin Li, Yuqing Xie, Hao Chen PII:

S0268-005X(19)32220-9

DOI:

https://doi.org/10.1016/j.foodhyd.2020.105702

Reference:

FOOHYD 105702

To appear in:

Food Hydrocolloids

Received Date:

29 September 2019

Accepted Date:

21 January 2020

Please cite this article as: Liping Zhou, Tingwei Xu, Jicheng Yan, Xin Li, Yuqing Xie, Hao Chen, Fabrication and characterization of matrine-loaded konjac glucomannan/fish gelatin composite hydrogel as antimicrobial wound dressing, Food Hydrocolloids (2020), https://doi.org/10.1016/j. foodhyd.2020.105702

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Journal Pre-proof

Journal Pre-proof Fabrication

and

characterization

of

matrine-loaded

konjac

glucomannan/fish gelatin composite hydrogel as antimicrobial wound dressing Liping Zhou1,2, Tingwei Xu2, Jicheng Yan2, Xin Li2, Yuqing Xie2 and Hao Chen1, 2 1

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

Technology and Business University (BTBU), Beijing 100037, China； 2

Marine collage, Shandong University, weihai, weihai 264209, China

Corresponding author (Hao Chen) Tel. /Fax: +86-0631-5688079, E-mail: [email protected] Affiliation addresses: Marine College Shandong University (Weihai), Wenhua Wes t Road, Gao Strict, Weihai, Shandong Province, 264209, P. R. China

Journal Pre-proof Abstract The konjac glucomannan (hereafter referred to simply as konjac) hydrogel has favorable gel-forming ability and swelling capacity, but the insufficient flexibility and poor water retention have limited its utilization. In this study, composite gels based on konjac and fish gelatin (FG) were prepared by alkali processing and thermal treatment subsequently. The influence of FG contents on the gel fraction, swelling behavior, biodegradation. was researched. Matrine was incorporated into the hydrogel and its effect on the hemocompatibility and antibacterial activity of gels were evaluated. According to the results, all gels presented at least 270% swelling degree and 72.7%~81.5% equilibrium water content. Pure konjac hydrogel presented 83.9% gel fraction and 96.9% water evaporation rate, while those of the composite gels were 84.8%~93.0% and 75.0%~91.9% respectively. As the doses of matrine increased from 0 to 40 mg/mL, hemolysis values decreased from 1.56% to 0.12%, indicating the improved hemocompatibility. Furthermore, the antimicrobial activity of gels was enhanced steadily with the increase of matrine concentration. When matrine concentration was 40 mg/mL, the inhibitory zones for Escherichia coli ATCC25922 and Staphylococcus aureus CMCC(B) 26003 were 12 and 11.5 mm, respectively. The overall results suggested that matrine-loaded konjac/FG hydrogel could not only maintain physiological environment for wound healing but also inhibit the growth of bacterial on the wound surface.

Journal Pre-proof Key words：konjac glucomannan, fish gelatin, matrine, antibacterial activity, wound dressing 1. Introduction Medical wound dressing, a temporary covering on wound surface, plays an important role in physical barrier to protect the wound from infection. An ideal wound dressing should maintain optimal moisture, absorb excess exudate and be removed easily without trauma to the wound. Many endeavors have been exerted to develop advanced wound dressing during the past ten years. Several dressing materials such as hydrogels, foams, hydrocolloids, and fills have been recommended as prominent dressings for wounds and burns (Kamoun, Kenawy, & Chen, 2017). Hydrogels are vastly hydrophilic macromolecular networks produced by chemical or physical crosslinking of soluble or hydrophilic polymers (Dragan, 2014). They are deemed as best choice for wound dressings with peculiar tissue-like structure and function of cooling the wound (L. Wang, et al., 2018). Their significant disadvantage is the limited mechanical strength at swollen state. This drawback could be overcome by fabricating “composite or hybrid hydrogel” system (Akhtar, Hanif, & Ranjha, 2016). A variety of materials have been adopted to develop hydrogels, ranging from natural polymers e.g. gelatin and chitosan (Alizadeh, Abbasi, Khoshfetrat, & Ghaleh, 2013) to synthetic polymers such as polyvinyl pyrrolidone and polyvinyl alcohol (Razzak, Darwis, Zainuddin, & Sukirno, 2001). Konjac is a natural macromolecular

Journal Pre-proof polysaccharide extracted from the tubers of konjac, a perennial plant of the genus Amorphophallus belonging to the family Araceae (Devaraj, Reddy, & Xu, 2018). It consists of 1,4-linked-β-D-mannopyranose and β-D-glucopyranose units in a molar ratio of 1.6:1 with a low degree of acetyl groups at the side chain C-6 position (Pal, Banthia, & Majumdar, 2007). At present, there are mainly three types of konjac gels-thermally irreversible gels prepared in the presence of alkaline coagulant, thermally reversible gels formed with boron, and synergistic reversible gels fabricated by complexing with gums such as xanthan, carrageenan, etc. The properties of konjac gels can be improved by alkaline treatment because of the deacetylation. Konjac gels prepared in the presence of alkaline coagulant have been reported to possess high absorbency and thermal irreversibility ability which would promote wound healing (L. Fan, Zhu, Zheng, Xu, & Zhang, 2007; Huang, Chu, Huang, Wu, & Tsai, 2015; Huang, Yang, Chu, Wu, & Tsai, 2015). FG gels have been applied in wound care on account of its good biocompatibility (Gu, Wang, Ren, & Zhang, 2009), biodegradability, low antigenicity (Wen, Cao, Yin, Wang, & Zhao, 2009), as well as the function of activating macrophages and hemostasis. The single utilization of konjac or FG gels in the field of wound dressing has been extensively investigated by many researchers (L. Fan, et al., 2007; Gu, et al., 2009; Huang, Chu, et al., 2015; Huang, Yang, et al., 2015; Pal, et al., 2007; T. Wang, et al., 2012; Wen, et al., 2009). However, the inferior mechanical properties of the two single network gels impose restrictions on their

Journal Pre-proof widespread application. The combination of konjac and FG might be a promising way to get over their own shortcomings and reinforce the gel properties. Though the mechanical strength of konjac and FG can be enhanced by the mixed modification, they still show limitations in terms of promoting healing process (Biranje, et al., 2019). Incorporation of bioactive agents into hydrogels has been reported to be a highly beneficial way for wounds healing (Boateng & Catanzano, 2015). Matrine is the main active ingredient of many Chinese herbal medicines such as sophora flavescens, sophora alopecuroides, radix sophora, etc. It has been adopted for the treatment of vaginitis, chronic cervicitis, senile vaginitis and pelvic inflammatory disease clinically, since it possesses a variety of pharmacological effects, such as bacterial activity, anti-inflammatory, anti-tumor, etc. (Kucukboyaci, Ozkan, Adiguzel, & Tosun, 2011). In the present research, we aim to fabricate composite hydrogels with konjac and FG. Alkali processing and thermal treatment were successively carried out to develop the hydrogels. Then matrine was added to improve the antimicrobial properties. Eventually the optimum gelation-fabrication condition was obtained. Matrine encapsulation efficiency and the influence of matrine on biological properties were evaluated. 2. Materials and methods 2.1 Materials

Journal Pre-proof Konjac glucomannan was purchased from Cargill Asia Pacific food system (Beijing) co. LTD. Fish gelatin was a kind gift from Vinhwellness company, Vietnam. Matrine was obtained from Ningxia Yanchi bauhinia pharmaceutical co., LTD. Anticoagulant sheep blood was supplied by Shanghai Yuduo biotechnology co., LTD. Other chemicals were analytical grade without further purification unless otherwise described. 2.2 Preparation of hydrogels 2.2.1

Preparation of konjac/FG hydrogels

The fabrication process of hydrogels was conducted as follows: firstly, a certain amounts of FG was dissolved in distilled water at 45°C for 30 min under magnetic stirring. Then, the FG solution was adjusted to pH 11 with 0.5 mol/L NaOH solution. After that, konjac powder was added into FG solution at room temperature for 2 h under magnetic stirring to allow sufficient dissolution of konjac. The composition of konjac and FG solution for hydrogels is given in Table 1. Subsequently, the konjac/FG solution (30 mL) was poured into a glass container (diameter 94 mm) and defoamed by an ultrasonic cleaner (KQ-300B, Kunshan ultrasonic instrument co. LTD, China) with the frequency of 30 kHz for 30 min to remove the trapped air bubbles. Then the upper solution with some bubbles was removed and the retained solution (3 mm thick) was heated in the thermostatic water bath at 60°C for 4 h to form gels.

The obtained hydrogels were cooled to room temperature by running

water and then rinsed several times in 0.1 mol/L PBS (pH 5.8) to neutralize.

Journal Pre-proof Following that, the hydrogels were stored in a refrigerator at 4°C for 24 h to reinforce their mechanical strength. Finally, the hydrogels were taken out from the refrigerator and restored to room temperature for further appearance and gel characterization evaluation. Table 1 Composition of konjac/FG hydrogels. Samples

KF0

KF1

KF2

KF3

KF4

FG % (w/v)

0.0

1.0

2.0

3.0

4.0

Konjac % (w/v)

2.0

2.0

2.0

2.0

2.0

2.2.2

Preparation of matrine-loaded konjac/FG hydrogels

In the matrine-encapsulated konjac/FG hydrogel, a certain amount of matrine crystalloid was dissolved into FG solution. The final concentration of matrine in the mixed solution was 10, 20, 30, and 40 mg/mL, respectively. The subsequent steps were consistent with the fabrication process of konjac/FG hydrogels. 2.3 The physicochemical properties of konjac/FG hydrogels 2.3.1

Rheological experiments

The gelation processes of konjac, FG and konjac/FG hydrogels were characterized by dynamic viscoelastic measurements, which were carried out on a rheometer (Haake Mars 3, Thermofisher, Germany) with a parallel plate geometry (35 mm in diameter, gap 1 mm) and a strain of 0.1%. In brief, 0.5% (w/v) konjac solution, 4% (w/v) FG solution and 0.5% konjac+4% FG (w/v) composite solutions (2 mL) was pipetted onto the plate of the instrument respectively. Then the edge of the geometry was covered with a layer of cedar oil to prevent water evaporation (Gu, et al., 2009).

Journal Pre-proof Time dependence of G' and G'' for different samples was carried out at the frequency of 1 rad/s. The pH values of all samples were adjusted to 11 with 0.5 mol/L NaOH. The temperature increased from 40°C to 80°C at a rate of 5 °C/min initially and subsequently maintained at 80°C for 40 min (Tomczynska-Mleko, Brenner, Nishinari, Mleko, & Kramek, 2014). 2.3.2

Determination of gel fraction

The pieces of hydrogels with a thickness of 3 mm were cut into squares of 2×2 cm and dried in an oven at 37°C for 48 h to a constant weight. The dry samples were left in deionized water at room temperature for 4 days to extract leachable sol fraction or unconnected gel matrix from polymer matrix and then dried at 37°C for 48 h to a constant weight (Gonzalez, Maiolo, Hoppe, & Alvarez, 2012; Wen, et al., 2009). The Gel fraction (GF) were calculated using equation (1): Wf

GF = Wi × 100%

(1)

Where Wf and Wi represent the weights of the dried hydrogel after and before immersed, respectively. 2.3.3

Determination of swelling behavior

The hydrogels with a thickness of 3 mm were cut into squares of 3× 3 cm, then dried in the oven at 37°C for 48 h and weighed immediately. Subsequently, the dried gels were soaked in saline solution (0.90% w/v of NaCl in water) at room temperature. At specific time interval (every hour), the hydrogels were taken out, gently blotted with filter paper to remove the surface water and weighed again (Gonzalez, et al.,

Journal Pre-proof 2012; Hago & Li, 2013; Oliveira, McGuinness, Ramos, Kajiyama, & Thire, 2016; Wen, et al., 2009). The swelling degree (SD), equilibrium degree of swelling (EDS) and equilibrium water content (EWC) were calculated as follows:

SD =

Mt ― M1

EDS =

M1

× 100%

M2 ― M1

EWC =

M1

× 100%

M2 ― M1 M2

× 100%

(2) (3) (4)

Where M1 is the weight of dried gel, Mt is the weight after swelling for predetermined time and M2 is the weight of equilibrium swelling hydrogels. 2.3.4

Measurement of water evaporation rate

The equilibrium swelling hydrogels were cut into squares of 3 × 3 cm and then kept in an incubator at 37°C (relative humidity of 50%) for 24 h. The hydrogels were taken out and weighed every 2 h until reached the constant weight (Li, Fan, Yang, Yang, Peng, & Hu, 2016). The water evaporation rate was calculated by the following equation: W1 ― Wt

Water lost = W1 ― W3 × 100% (5) Where W1, Wt, W3 are, respectively, the initial weight, the measured weight after evaporating for predetermined time and the final weight of the hydrogels. 2.3.5

In vitro degradation

The hydrogels were cut into 2×2 cm squares and dried in an oven at 37°C for 48 h. Followed that, the dry hydrogels were weighed before being immersed in 0.1 mol/L PBS solution (pH 7.4) at room temperature. Every 4 days, the samples were taken out

Journal Pre-proof from the solution and washed thoroughly with deionized water. Then they were dried in an oven at 37°C for 48 h and weighed again (Adali, Kalkan, & Karimizarandi, 2019; Biranje, et al., 2019; L. Wang, et al., 2018). Repeat the foregoing operations 4 times. The degradation rate is expressed by the weight loss and calculated as follow equation:

Weight loss =

W1 ― Wt W1

× 100% (6)

Where W1 is the initial weight of the dry hydrogels and Wt is the weight of dry hydrogels after degrading for 4, 8, 12 and 16 days respectively. 2.4 Bio evaluation characteristics of matrine-loaded hydrogels 2.4.1 Determination of matrine encapsulation efficiency and loading efficiency Matrine-loaded gel (0.5 g) was ground and immersed in 50 mL 0.1 mol/L PBS buffer (pH 7.4) at 37±0.5°C, with vigorously stirring for 24 h to extract matrine from the hydrogels. The dissolution medium was filtered and poured 100 μL into a 60 mL separation funnel, then 6 mL of 0.1 mol/L PBS solution (pH 7.6) with 2×10-4 mol/L bromothymol blue and 6 mL of chloroform were added successively. Subsequently, the separation funnel was inverted and vigorously shaken for 2 minutes, followed by standing for 2 h to make the two phases stratified. The chloroform was separated and then assayed using UV spectrophotometer at 420 nm. The matrine encapsulation efficiency (EE) and loading efficiency (LE) were calculated by the following equations: Wc

EE = Wo × 100% (7)

Journal Pre-proof Wc

LE = Wi × 100% (8) Where Wc is the weight of matrine actually loaded in the hydrogel, Wo is the total amount of matrine added and Wi represent the weight of hydrogel. 2.4.2 In vitro drug release test The in vitro drug release test was carried out by immersing 0.5 g matrine-loaded hydrogel in 100 mL phosphate buffer (pH 7.4) at 37°C under continuous stirring. At scheduled time intervals (15, 30, 45, 60, 90, 120, 240 and 360 min), 5 mL dissolution medium was withdrawn and filtered using a 0.45 μm syringe filter. The removed volume of the medium was supplemented with the same amount of fresh medium each time. The filtered solution (200 μL) was transferred into a 60 mL separation funnel, then 6 mL of 0.1 mol/L phosphate buffer solution (pH 7.6) with 2×10-4 mol/L bromothymol blue and 6 mL of chloroform were added successively. Subsequently, the separation funnel was inverted and vigorously shaken for 2 minutes, followed by standing for 2 h to make the two phases stratified. The chloroform was separated and then assayed using UV spectrophotometer at 420 nm. The amount of released matrine was calculated according to the standard curve. 2.4.3 Antibacterial activity test The antibacterial efficacy of the matrine-encapsulated hydrogel against E. coli (ATCC25922, gram-positive) and S. aureus (CMCC(B)26003, gram-negative) was evaluated according to the inhibition zone. All bacterial strains were cultured on an agar plate at 37°C for 24 h. The inocula were prepared by selecting 3~5 isolated

Journal Pre-proof colonies of bacteria into 5 mL of Luria–Bertani medium (LB medium) (1% tryptone, 0.5% yeast extract, and 1% NaCl, sterilization at 120°C for 20 min). Then they were incubated at 37°C for 24 h. The obtained bacteria suspension was diluted by the same LB medium solution and the concentration of bacteria was determined by a UV/VIS spectrometer at 625 nm. Afterwards, 0.1 mL inoculums containing approximately 106~107 CFU/mL of tested bacteria were seeded on the surface of the solid LB media. Finally, the matrine-encapsulated hydrogels were cut into a disc form with a 6 mm diameter mold and then placed on the LB plates. The plates were incubated at 37°C for 24 h before the diameters of inhibitory zones were measured. 2.4.4 Hemocompatibility determination of matrine-loaded hydrogels In order to perform hemolysis tests, matrine-loaded hydrogels were placed into flat-bottom beakers and equilibrated with saline solution (NaCl, 0.9 w/v at 37°C for 24 h). Anticoagulant sheep blood (2 mL) was diluted by 2.5 mL saline solution and incubated at 37°C. A certain amount of matrine-loaded hydrogel was transferred into polypropylene test tubes containing 10 mL of normal saline. All the tubes were incubated at 37°C for 30 minutes. Subsequently, 0.1 mL of diluted blood sample was pipetted into the tubes. The mixed solution was incubated at 37°C for 60 minutes. Positive and negative controls were prepared by adding the same volume of anticoagulant sheep blood to 10 mL of distilled water and saline solution, respectively. Each tube was incubated and gently inverted twice every 30 min to guarantee the continuous contacting between hydrogels and anticoagulant sheep blood. The

Journal Pre-proof mixtures were transferred to a centrifuge tube and then centrifuged at 1237.4 g for 20 min. The hemolysis values, a result of hemoglobin release, were measured by detecting the optical densities (OD) of the supernatant at 540 nm using a UV spectrophotometer (Li, Fan, et al., 2016; Kamoun, Kenawy, Tamer, El-Meligy, & Eldin, 2015). The percentage hemolysis was calculated by equation (9). As the results of hemolysis test, the materials were classified into three types according to their hemolytic index as follows: (a) hemolytic materials: hemolysis (%) >5%, (b) slightly hemolytic materials: hemolysis (%) is between 2% and 5%, and (c) non-hemolytic materials: hemolysis (%) <2%. 𝐴1 - 𝐴3

Hemolysis = 𝐴

2

- 𝐴3

× 100%

(9)

Where A1 is absorbance of tested hydrogel sample, A2 and A3 represent absorbance of positive control and negative control respectively. 2.5 Statistical analysis All the analyses were carried out in triplicates to find mean and standard deviation values. Figures were generated with Origin 8.5. Microsoft Excel Data Analysis Centre (2016) and IBM SPSS software (version 21) were used for statistical analysis. Primary statistical analysis of data was performed with the analysis of a nonparametric comparison test (Kruskal–Wallis Test), to determine mean differences in every tested group, followed by one-way ANOVA test for multiple comparisons. Variance homogeneity was examined using the Levene test. 3. Results and Discussion

Journal Pre-proof 3.1 Appearance characteristics of hydrogels Fig.1 shows the photographic appearance of fabricated hydrogels. All samples could be removed from the glass container easily and showed smooth surface. The hydrogels with a thickness of 3 mm exhibited extremely slight yellow color except KF0 (without FG). The original slight yellow color of FG might be responsible for this. All hydrogels were transparent, which was evident from the apparent letter of A on the white background.

Fig.1 Images of hydrogels as a function of FG concentration. 3.2 Rheological properties It has been reported that the gelation time corresponding to the intersection of storage modulus (G') and loss modulus (G'') was suggested to mark gelation point (Gao & Nishinari, 2004). In this research, the gelation process of konjac, FG and konjac/FG hydrogels were characterized by rheological test. Fig.2 shows the time dependence of G' and G'' for 0.5% (w/v) konjac solution, 4% (w/v) FG solution and KF (0.5% konjac+4% FG) at a constant frequency of 1 Hz. Obviously, the values of G' and G'' of FG solution were almost constant, and its G'' kept larger than G', suggesting that no gelation process took place in pure FG solution. Konjac sol showed a decrease in both G' and G'' during the heating process and the G'' maintained larger

Journal Pre-proof than G'. Then both G' and G'' increased sharply with time, meanwhile, the intersection of G' and G'' was observed, which corresponded to the gelation point. Then both G' and G'' climbed to a plateau after all the cross-links were formed and the gelation process was completed. Composite sol exhibited a similar trend with konjac sol over gel formation curves. Nevertheless, G' and G'' of the konjac/FG sol increased slower and last longer than pure konjac sol while the plateau value of G' was higher, indicating the presence of FG decelerated the gelation process thus lengthened the time needed for gelation but enhanced the elasticity of the gel. This phenomenon could be contributed to the deacetylation of konjac when exposed to alkaline and thermal treatment. The removal of acetyl groups caused the konjac molecular chain to change from semi-crimping to self-crimping (Yang, et al., 2017). Then the gelation of the konjac molecules occurred through the formation of a network structure supported by hydrogen bonds (Chen, Chen, Yan, Liu, & Iop, 2017). Konjac could provide with a large number of hydroxyl groups, which can easily form hydrogen bonding with konjac molecules and FG molecules. Generally, more intermolecular interactions contribute to the increase of density of physically cross-linked points (Li, et al., 2015). Thus, the existence of FG was in favor of increasing the number of cross-linked points, which resulting in the enhancement of elastic modulus.

Journal Pre-proof

1000

G' G'' T

KF KF

KGM KGM

FG FG

80

100

60 10 40 1

0.1

T (℃ )

G', G'' (Pa)

100

20

0

0 500 1000 1500 2000 2500 3000 3500 4000

t (s) Fig.2 Time dependence of G' (solid), G'' (open) for 0.5% konjac sol, 4% FG solution and KF (0.5% konjac＋4% FG) at the frequency of 1 Hz. 3.3 Gel fraction of hydrogels The weight ratio of dried hydrogels in rinsed and unrinsed conditions can be assumed as an index of the degree of crosslinking (Hago, et al., 2013). A dependency of gel fractions to the quantity of FG is given in Table 2. As seen, the gel fractions of samples varied in the range of 84%~93% as FG concentration increased from 0% to 4%. The gel fraction with 3% FG was about 93% which was significantly higher (p＜ 0.05) than pure konjac hydrogels (Sung, et al., 2010). This might be attributed to that the presence of FG enhanced the cross-linking extent, thus formed denser network in the three-dimensional structure (Kokabi, Sirousazar, & Hassan, 2007). When FG and konjac cross-linked with each other, additional intermolecular interactions including

Journal Pre-proof hydrogen bonding and hydrophobic interaction would be developed between functional groups of konjac and FG (Chen, et al., 2017). These interactions induced entanglement, which caused an increase in gel fraction. Table 2 The effect of FG concentration on gel fraction (GF), equilibrium degree of swelling (EDS), equilibrium water content (EWC), matrine encapsulation efficiency (EE) and loading efficiency (LE)（*p＜0.05, **p＜0.01 compared to KF0). Samples

KF0

KF1

KF2

KF3

KF4

GF (%)

83.98±0.24

84.86±3.27

89.67±1.8

92.95±0.67*

91.75±3.06

EDS (%)

460.63±4.36

328.55±5.35**

296.31±7.69**

269.56±8.91**

287.47±2.84**

EWC (%)

81.54±1.11

77.30±1.02**

75.07±0.74**

72.72±0.59**

74.22±0.21**

EE (%)

70.87±0.37

71.44±0.88

81.98±0.71**

89.46±0.77**

82.99±0.74**

LE (%)

2.34±0.0035

2.49±0.0265**

2.74±0.0221**

2.81±0.0093**

2.51±0.0124**

3.4 Swelling properties of hydrogels Swelling properties of hydrogels were investigated to evaluate their capacity to absorb wound exudation fluid. The equilibrium degree of swelling (EDS) and equilibrium water content (EWC), as important swelling characteristics of hydrogels, were calculated using equation (3) and (4). Fig.3 displays the swelling degree of hydrogels in saline solution at room temperature. The swelling degree increased sharply at the first 3 h and then leveled off. Table 2 demonstrates EDS and EWC of hydrogels with a function of FG content. Both parameters showed nearly similar decreasing trends with the increasing FG concentration. All gels presented at least 270% swelling degree and 72.7%~81.5% equilibrium water content. The samples

Journal Pre-proof based on konjac and FG were found to have significantly lower (p＜ 0.05) swelling capacity than pure konjac hydrogels. Besides, it could be figured out that higher gel fractions led to lower EDS and EWC. This might be attributed to that some molecules generated entanglement on account of hydrogen bonds thus less free hydroxyl groups were left to play an important role in water uptake (Pal, et al., 2007). In addition, the incorporation of the second network increased the crosslinking density of the interpenetrating polymer networks, which led to denser network structure (Xu, et al., 2013). Moreover, the distance between adjacent crosslinking points was shortened and thus restricted the extension of macromolecular chains during swelling process (Rathna & Chatterji, 2001). These factors together led to the lower swelling ratio of composite gels compared with single konjac gel. Although the swelling characteristics of konjac/FG hydrogels decreased due to the presence of FG, the swelling capacity was still high enough as wound dressing (Hago, et al., 2013; Sung, et al., 2010).

Journal Pre-proof

KF0 KF1 KF2 KF3 KF4

500

Swelling degree (%)

450 400 350 300 250 200 150 100

0

1

2

3

4

5

6

7

8

9

10

11

Time (h) Fig.3 Swelling degree of konjac/FG gels with different FG concentrations. 3.5 Water evaporation rate analysis The hydrogel dressings with smaller water evaporation rate can reduce the replacing times leading to quicker healing, less pain and great cost savings (Huang, Yang, et al., 2015). Fig.4 presents the effects of FG amount on the water evaporation rate of hydrogels. As shown, the higher the FG concentration, the lower the water evaporation rate. The water loss of pure konjac hydrogel was 96.9% in 24 h while those of the composite hydrogels ranging from 75.0% to 91.9%. This result could be ascribed to that the addition of FG increased the crosslinking density, led to denser network structure and smaller pores of hydrogel eventually (Hwang, et al., 2010).

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Water evaporation rate (%)

120

KF0 KF1 KF2 KF3 KF4

100 80 60 40 20 0

0

2

4

6

8

10 12 14 16 18 20 22 24

Time (h) Fig.4 Water evaporation rate of gels with different FG concentrations. 3.6 In vitro degradation analysis In order to evaluate the degradation properties of hydrogels, weight loss of each hydrogel was recorded during a period of 16 days. Fig.5 shows the percentage of weight loss occurred in 0.1 mol/L PBS (pH 7.4) at room temperature. It turned out that the degradation of hydrogels increased steadily during this period. After 16 days, the degradation degrees of the samples containing 3% and 4% (w/v) FG were 50% and 65% respectively, while

pure konjac hydrogel was about 80%. It could be

concluded that FG content exerted significantly influence on the degradation rate of hydrogels. The higher degradation rate of FG might contribute to this phenomenon (Mahnama, Dadbin, Frounchi, & Rajabi, 2017). The degradation of konjac/FG hydrogels were predominantly the cleavage of entanglement segments of konjac and

Journal Pre-proof FG in three-dimensional network (Hago, et al., 2013). FG possesses a considerable number of hydrophilic carbonyl groups, which is beneficial to the occurrence of degradation behavior (T. Wang, et al., 2012). Thus, the addition of FG resulted in faster degradation of hydrogels.

KF0 KF1 KF2 KF3 KF4

70

Weight loss (%)

60 50 40 30 20 10 0

4

8

12

16

Time (day)

Fig.5 In vitro degradation of gels with different FG concentrations. 3.7 Matrine encapsulation efficiency and loading efficiency The effect of FG on matrine encapsulation efficiency (EE) and loading efficiency (LE) was studied and the results are tabulated in Table 2. Encapsulation and loading efficiency were calculated according to matrine standard curve (A=0.05812C-0.0628, R2 =0.9972, linear range, 3.33~16.67 mg/L in chloroform). As shown in table 2, EE of these samples ranged from 70.87% to 89.46% with LE values varying in the range of 2.3%~2.8% (w/w to hydrogel). Compared with single konjac hydrogel, EE and LE of

Journal Pre-proof composite hydrogels were significantly higher and the maximum was obtained with 3% (w/v) FG. This result further affirmed the preceding assumption that the gel based on FG and konjac possessed denser network structure and smaller pores than the single konjac gel. Based on this, matrine could be trapped in the composite gels more effectively. To sum up, the more entangled structure and intermolecular interaction were conducive to embed matrine within the gels. 3.8 In vitro drug release The in vitro cumulative release profiles of matrine from different hydrogels in phosphate buffers of pH 7.4 at 37°C are shown in Fig.6. Two phases could be clearly observed in the release curves of all samples. In the initial 45 min, a rapid release ranging from 31% to 90% could be found. This could be mainly attributed to the rapid diffusion of matrine on the hydrogel surface as well as the swelling behavior of hydrogels. In the following 45 to 360 min, matrine kept a relatively slow release. This might be due to the swelling equilibrium of hydrogels. Furthermore, with the increase of FG concentration, the drug release rate became slower, particularly in the rapid release phase. It could be concluded that the presence of FG molecules could effectively improve the sustained release of matrine from konjac/FG gels.

Journal Pre-proof

100

Accumulative release (%)

90 80 70 60 50 40

KF0 KF1 KF2 KF3 KF4

30 20 10 0

0

60

120

180

240

300

360

Time (min) Fig.6 The effect of FG contents on the accumulative release of matrine from konjac/FG hydrogel in phosphate buffer solution (at 37°C, pH 7.4). 3.9 Antibacterial activity The antimicrobial activity is an important property for the hydrogel dressing. In order to obtain excellent antibacterial activity, various concentrations of matrine were added to evaluate the antibacterial activity of matrine-loaded hydrogels. From the photographs shown in Fig.7, the hydrogels exhibited good antimicrobial activity against gram-positive bacteria (Staphylococcus aureus CMCC(B)26003) and gram-negative bacteria (Escherichia coli ATCC25922). It can be seen that increasing doses of matrine assisted enhancing effective antibacterial property against E. coli and S. aureus. The largest average diameters of inhibition zone were 12 and 11.5 mm

Journal Pre-proof respectively, which can be seen with 40 mg/mL matrine incorporated. The microbial clear zone was not observed at the absence of matrine.

The diameter of inhibition zone (mm)

(C) E.coli S.aureus

12 10 8 6 4 2 0

5

10

15

20

25

30

35

40

45

The concentration of matrine (mg/mL) Fig.7 Inhibitory effect against E. coli (A), S. aureus (B) and the inhibition zone size (C) of matrine-loaded gels as a function of matrine concentration. 3.10

Hemocompatibility of matrine-loaded hydrogels

The chemical composition of a hydrogel is the key factor affecting blood compatibility of the proposed biomaterials (Chhatri, Bajpai, & Bajpai, 2011). In this

Journal Pre-proof research, the in vitro blood compatibility of the prepared matrine-loaded konjac/FG hydrogels was determined by the method of hemocompatibility test. All prepared hydrogels showed excellent blood compatibility, as evident shown in Fig.8. The results indicated that the addition of matrine could improve the blood compatibility of gels significantly. Hemolysis values dropped from 1.56% to 0.12% with the matrine content increased from 0% to 3%. The result demonstrated that matrine in moderate content could significantly modify the blood compatibility of hydrogels. According to the classification of hemolytic tendency of polymeric materials (Kamoun, et al., 2015), the obtained hydrogels could be considered as non-hemolytic materials since their hemolysis values were all below 2%. The observed fairly good blood compatibility may due to that the naturally-sourced konjac, FG and matrine are all biocompatible constituents.

2.0

Hemolysis (%)

1.5

*

1.0

0.5

**

**

20

30

0.0 0

10

The concentration of matrine (mg/ml)

40

Journal Pre-proof Fig.8 Hemolysis of gels with different concentration of matrine (*p＜0.05 and **p＜ 0.01compared to 0）. 4. Conclusion In current research, the konjac/FG composite hydrogels loaded with matrine were developed using alkali processing and thermal treatment subsequently. The effect of FG content on various gel properties and the influence of matrine on biological properties were evaluated. The obtained hydrogels possessed great transparency, smooth surface, favorable swelling capacity and moderate water evaporation rate, which qualified the essential requirements for ideal medical wound dressing applications. FG played an effective role in enhancing gel elasticity, reducing water evaporation rate and adjusting biodegradation rate. Furthermore, both matrine encapsulation efficiency and loading efficiency were significantly raised with the presence of FG. Matrine could enhance the blood compatibility and antibacterial activity against Escherichia coli and Staphylococcus aureus of gels constructively. The overall results suggested that matrine-loaded konjac/FG hydrogel could not only maintain physiologically environment beneficial to wound healing but also inhibit the growth of bacterial on the wound surface. Acknowledgement We thank Jiajun Xian (Octogone (Guangzhou) Trading Co., Limited) for his assistance to supply FG. The authors gratefully thank Shandong Provincial Natural Science Foundation, China (NO. ZR2019BC053), Beijing Food Nutrition and Human

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Liping Zhou: Visualization, Writing- Original draft preparation, Investigation. Methodology. Tingwei Xu: Writing- Original draft preparation, Investigation, Formal analysis. Jicheng Yan: Investigation, Formal analysis. Xin Li: Investigation, Visualization. Yuqing Xie: Investigation. Hao Chen: WritingReviewing and Editing, Supervision, Funding acquisition.

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Liping Zhou, Tingwei Xu, Jicheng Yan, Xin Li, Yuqing Xie and Hao Chen

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Highlights  Konjac/FG hydrogels were fabricated by alkali processing and thermal treatment.  Composite hydrogels showed excellent matrine encapsulation efficiency.  The incorporated matrine improved the antibacterial activity of Konjac/FG gels.