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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
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Preparation and characterization of quaternary ammonium chitosan hydrogels with significant antibacterial activity Lihong Fan a,∗ , Jing Yang a , Huan Wu a , Zhihai Hu a , Jiayan Yi a , Jun Tong a , Xiaoming Zhu b a b
College of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, China Hubei Collaboration Innovation Center of Non-power Nuclear, China
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a r t i c l e
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Article history: Received 5 December 2014 Received in revised form 6 April 2015 Accepted 8 April 2015 Available online xxx
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Keywords: Quaternary ammonium chitosan hydrogel Gamma radiation Antibacterial activity
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1. Introduction
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Quaternary ammonium chitosan (HACC)/polyvinyl alcohol (PVA)/polyethylene oxide (PEO) hydrogels were prepared using gamma radiation. The chemical structure of the hydrogels was characterized using FT-IR. The results revealed that HACC, PVA and PEO were perfectly compatible and interacted via the hydrogen bonds. As revealed by SEM, scaffolds with a homogeneous interconnected pore structure were obtained after lyophilizing the hydrogels. The influence of different radiation doses and weight ratios on properties including gel content, swelling ability, water evaporation rate and mechanical properties were investigated. It indicated that the hydrogels had the good swelling ability, water evaporation rate and mechanical properties. In vitro antibacterial activity assessment, the hydrogels exhibited a pronounced inhibitory effect against two bacteria (Staphylococcus aureus and Escherichia coli). Therefore, the hydrogels showed a promising potential to be applied as wound dressing. © 2015 Published by Elsevier B.V.
The skin forms a protective barrier against the external environment. An intact barrier of this kind is of critical importance following the occurrence of a wound [1]. The healing of a wound requires the protection of the skin surface including providing, retaining moisture and inhibiting the growth of bacterial [2]. Recently, hydrogels in a three-dimensional network structure crosslinked by linear polymer materials have attracted a lot of attentions for their potential applications in medical and pharmaceutical areas. Hydrogel dressings with high water content, as well as the ability to swell, are able to absorb leaking body fluids and facilitate the creation of a moist environment. The moist environment encourages rapid granulation tissue formation and re-epithelialization that accelerates wound healing [3,4]. Compared with a traditional dressing, a hydrogel dressing has several advantages and may indeed be considered an ideal dressing: it is relatively soft and can reduce pain that is often caused when dressings are changed. Also, a hydrogel dressing make it possible to maintain a moist environment to promote wound healing [5]. At present, most hydrogels are prepared via radical polymerization in the presence of crosslinking agents. However, the use of a chemical crosslinker can lead to structural non-homogeneity.
∗ Corresponding author. Tel.: +86 27 13018011218; fax: +86 27 87859019. E-mail address: [email protected] (L. Fan).
These agents can affect the integrity of the substance to be entrapped and make the substance toxic [6–8]. In this study, quaternary ammonium chitosan (HACC) combined with polyvinyl alcohol (PVA) and polyethylene oxide (PEO) were mixed to form a HACC/PVA/PEO hydrogel by employing ␥-radiation as the free radical initiator. It is an environmental approach to prepare hydrogels for biomaterial applications. Chitosan hydrogels have been studied widely. However, until recently no reports have been found in the literature on the radiation crosslinking of quaternary ammonium chitosan hydrogels. Chitosan is composed of N-acetyl-d-glucosamine and dglucosamine units linked by -(1–4) bonds. It is insoluble in neutral aqueous and alkaline solutions [9]. This biopolymer is known for its wide biological properties [10]. Chitosan has always been used as a wound healing promoter. The main biochemical effects of chitosan in wound healing are fibroblast activation, cytokine production, giant cell migration and stimulation of type IV collagen synthesis [11,12]. Physical or chemical modification of chitosan can lead to new products with significantly functional properties. It is reported that quaternization is an efficient means of imparting new functional properties to polysaccharides [13]. So far, quaternization has been widely applied to a variety of polysaccharides, such as pectin [14], konjac glucomannan [15], starch [16], alginate [17] and cellulose [18]. In previous reports [19], the antibacterial ability of natural polysaccharides is shown to be improved by quaternization. As a polymer with a quaternary ammonium salt (QP) group, Chitosan possesses potential antibacterial properties. Hyaluronic acid
http://dx.doi.org/10.1016/j.ijbiomac.2015.04.013 0141-8130/© 2015 Published by Elsevier B.V.
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CH2OR1
CH2OH ClCH2CH(OH)CH2N+(CH3)3Cl-
O
O O
O
NHR2
R1O
NHR
HO
Quatemary ammonium chitosan Chitosan, R= H, Ac R1= H,ClCH2CH(OH)CH2N+(CH3)3ClR2= H,ClCH2CH(OH)CH2N+(CH3)3Cl-,Ac
Scheme 1. Synthesis of quaternary ammonium chitosan.
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(HA), an important functional ingredient in cosmetics, is unique for its excellent moisture-absorption and moisture-retention abilities. But the total amount of HA is limited, and the price is high. The results showed that QP group could replace HA for its better moisture-absorption and moisture-retention abilities. Although moisture facilitates the immigration of epithelium cells, it may also brings more risks of infection, which may cause excessive scar formation and chronic clinical problems [20,21]. Chitosan derivatives achieved by quaternization may solve the contradiction between moist healing and infection, due to its good antibacterial activity [22]. In addition to the water-solubility and antibacterial activity, quaternary ammonium chitosan is promising for the use in wound dressings. However, chitosan and its derivatives are degradable polymers and difficult to form hydrogels in conditions involving a high radiation dose. The introduction of PVA and PEO can solve this problem and can greatly improve the mechanical strength of the hydrogels [23]. Both PVA and PEO are biocompatible synthetic polymers that do not cause any toxicity or stimulation to the tissue cells of the body. The prepared hydrogels were verified by Fourier transform infrared (FTIR) spectroscopy and Surface morphology (SEM). The influence of different radiation doses and weight ratios on the properties including gel content, swelling ability and mechanical properties were investigated. Finally, the HACC/PVA/PEO hydrogel was found to have antibacterial activity against two bacteria. It suggests that this additive-free and sterilized hydrogels may have applications as a wound dressing.
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2. Experimental
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2.1. Materials
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pressure-equalizing dropping funnel. After that, magnetic stirring was done continuously for 72 h at 78 ◦ C. The solution was then purified by dialysis through a 10,000–8000 molecular weight cut-off dialysis tubing for three days and dried overnight in an oven at 50 ◦ C. The reaction is shown in Scheme 1. 2.3. Preparation of HACC/PVA/PEO hydrogels HACC solution was completely dissolved and mixed with PVA/PEO solution at 80 ◦ C under the stirring for 6 h to form a homogenous solution. The total polymer concentration was fixed at 10% (different weight ratios between HACC and PVA/PEO were: 1:2, 1:1 and 2:1. The mixtures were then poured into test tubes and the solutions were made free from oxygen by purging nitrogen gas for 5 min. They were then subjected to ␥-radiation with an absorbed dose of 20, 30, 40, 50 and 60 kGy using a 60 Co facility. The radiation crosslinking was performed at room temperature with a dose rate of 20 Gy/min. After copolymerization, the polymeric cylinders were removed by breaking the test tubes and cut into discs of 2 mm of thickness. All samples were washed in excess water to remove the non-reacted components, and then air-dried at room temperature. 2.4. Calculation of gel content Dried hydrogels were extracted with distilled water for 24 h at 100 ◦ C to extract the insoluble parts of the hydrogels. The insoluble or gelled parts were taken out and washed with hot distilled water to remove the soluble parts and then were dried and weighed. This extraction cycle was repeated until the weight became constant. The gel yield of hydrogels were determined as follows:
W
Gel % =
110
Chitosan (degree of deacetylation = 93.23%) was purchased from Zhejiang Yuhuan Ocean Biochemistry Co. Ltd. (China). Monochloro acetic acid, sodium bisulfite, sodium nitrite and other reagents used in this investigation were of analytical grade and without further purification. CHPTAC (N-(3-chloro-2-hydroxypropyl) trimethyl ammonium chloride) was purchased from Guofeng Fine Chemical Co. Ltd. (Shandong, China) and it was used as an etherifying reagent without further purification. Polyvinyl alcohol (PVA-1788) and polyethylene oxide (PEO, Mv ∼300,000) were purchased from Aladding Industrial Corporation Co. Ltd. (Shanghai, China). Radiation sources used in the experiment were provided by the Radiation Center of Academy of Agricultural Sciences of Hubei Province.
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2.2. Synthesis of quaternary ammonium chitosan
Swelling % =
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In a typical reaction procedure, chitosan (8 g) was dissolved in NaOH/CHPTAC aqueous solution (85 mL) through a
e
Wd
× 100
2.5. Swelling measurement The clean, dried hydrogels of known weight was immersed in distilled water at room temperature for 48 h. The hydrogels were removed and the excess solvent on the surface was removed by blotting quickly with absorbent paper and were weighed. The water uptake was calculated as follows:
md
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where Wd and We represent the weights of the dry hydrogels and the gelled part after extraction, respectively.
m − m s d
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× 100
where md and ms are the weights of dry and wet films, respectively.
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2.6. Water evaporation rate Prepared hydrogels after extraction were immersed in distilled water. Taking a certain weight of the hydrogel after reaching its reaching swelling equilibrium, then the hydrogels were placed in an incubator at 50 ◦ C for 24 h in a humid atmosphere of 50%. Weight of the hydrogel was measured at regular intervals until completely dry. The water evaporation rate was calculated by the following equation: Water lost % =
(W1 − W2 ) × 100 (W1 − W3 )
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where W1 , W2 and W3 are the starting weight, measured weight and last weight of the hydrogels, respectively.
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2.7. Mechanical property test
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Several hydrogel samples were prepared and cut into a dumbbell shape with a length of 75 mm and a width of 13 mm. The depression of the middle portion of the sample was a rectangle shape, and the sides of the shank portion were in a dumbbell shape with a length of 30 mm and a width of 4 mm. The thickness of the hydrogels samples was determined using a caliper. The tension (N) and elongation (mm) at break of the samples was determined by a machine at a speed of 200 operating at room temperature. The tensile strength of the hydrogels was determined as follows:
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F Tensile strength (MPa) = (W × H) S Elongation % = × 100 L
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where F represents the tension of the samples at the break, S represents the elongation of the samples at break, and W, H and L are the width, height and length of the samples.
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2.8. Characterization of the hydrogels by FT-IR
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FT-IR spectra of powdered discs of prepared CMC/PVA/PEO hydrogels were recorded over the range 400–4000 cm−1 with a Fourier transform infrared spectrophotometer (Nicolet 170SX, Thermo Fisher Scientific Inc., USA). One milligram of dry sample was mixed with 100 mg of dry KBr, and the mixture was pressed into a disk for spectrum recording. In fact, FT-IR spectra were utilized to confirm the occurrence of the polymerization reactions on the polymer chains.
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2.9. Scanning electron microscopy (SEM)
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For morphological characterization, the hydrogels after swelling in water (to an equilibrium state) were freeze-dried using a freeze drier (Christ, Germany, Alpha 1-2) at −52 ◦ C for 6 h. Transverse sections were cut from the freeze-dried film samples using a cold knife. Samples were then examined with a Jeol JSM-5400 scanning electron microscope (JEOL, Tokyo, Japan).
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2.10. Antibacterial assay
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Two kinds of bacteria were used as test microorganisms in this study. Test bacteria included Staphylococcus aureus (RCMB 000106) (as Gram positive bacteria) and Escherichia coli (RCMB 000107) (as Gram negative bacteria). The strains were stored at 4 ◦ C. The medium used for growing bacteria was universal nutrient agar. Antibacterial activity was determined by the agar well-diffusion method. Briefly, agar plates were seeded with test microorganisms and kept for 30 min until the medium solidified. Circular pieces of
1:1(HACC:PVA/PEO) 2:1 1:2
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Gel content(%)
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Radiation dose(KGy) Fig. 1. Gel content of HACC/PVA/PEO hydrogel prepared at different radiation doses and weight ratios.
approximately 1 cm in diameter of the test hydrogels were added. Plates were kept for 12 h at 4 ◦ C prior to incubation at the appropriate temperature for the bacteria. Plates were examined on a daily basis to check for the development of growth inhibition zones around the loaded hydrogels.
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3. Results and discussion
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To quantitatively evaluate the degree of cross-linking, the gel content of the hydrogels was investigated. The solution of HACC/PVA/PEO (10%) composite was exposed to various radiation doses. In Fig. 1, we can see that the gel content of all of the compositions, ascended rapidly initially, while it declined slightly at higher radiation doses. This phenomenon is universal for the formation of hydrogels via radiation crosslinking. Initially, the amount of free radicals generated by radiation is not high enough for recombination to form a physically stable hydrogel. However, when the radiation dose reaches a critical dose (the gelation dose), the intermolecular recombination of free radicals readily occurs and finally leads to the formation of a three-dimensional network. Further increase of the absorbed dose would cause increased crosslinking density. At a sufficient high absorbed dose, the intramolecular free radical recombination tends to dominate over the intermolecular radical recombination, which would terminate the crosslinking and begin to degrade the network. Hence, a slightly decrease is observed in the gel content. The influence of different ratios of quaternary ammonium chitosan on the gel content was also studied in this study. Polysaccharides are degraded by ionization radiation in solid state and low concentration solutions. However, a large variety of polysaccharide hydrogels are prepared in concentrated solutions (these have a so-called, “paste-like status”) by radiation crosslinking [24,25]. Quaternary ammonium chitosan may be crosslinked and degraded concurrently in the presence of radiation. Although a physically stable hydrogel can be formed, the maximum gel content of the pure quaternary ammonium chitosan hydrogel is rather low, i.e. 40%. On the contrary, the radiation crosslinking efficiency of PVA/PEO was much higher than that of the quaternary ammonium chitosan. Consequently, the gel content of the HACC/PVA/PEO hybrid hydrogel
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Fig. 2. (a) Swelling percentage of HACC: PVA/PEO(1:1) hydrogel prepared at different irradiation doses. (b) Swelling percentage of HACC/PVA/PEO hydrogel (40 kGy) prepared at different weight ratios.
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increased with the decreasing content of quaternary ammonium chitosan. 3.2. Swelling percentage The influence of different radiation doses and weight ratios on the swelling percentage of the prepared hydrogels is shown in Fig. 2. All of the hydrogels showed a swelling percentage at the range from 1000% to 4000%, which is sufficient for an application as a hydrogel wound dressing [26]. HACC/PVA/PEO hydrogels had a very high swelling percentage, due to the fact that quaternary HACC, PVA and PEO are hydrophilic polymers which contain a large amount of hydrophilic groups such as OH and NH2 . In Fig. 2(a), we can see that the swelling percentage decreased when the radiation dose increased from 20 kGy up to 60 kGy. The reason for this might be that when the radiation dose increases, the crosslink degree of hydrogel also increases too. Thus, the polymer network structure becomes more compact, which makes it difficult to stretch the macromolecular chains, and the pore size of the hydrogel becomes smaller [27]. In Fig. 2(b), we can see that
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3.3. Water evaporation rate
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Generally, a smaller water evaporation rate can provide a moister wound healing environment. The maintenance of a moist
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90
swelling percentage increased as the weight ratio of quaternary ammonium chitosan increased under the same radiation dose. It is believed that the addition of quaternary ammonium chitosan could loosen the structure of the hydrogel, which would make the macromolecular chain easier to stretch. It can be also seen from Fig. 2 that the swelling percentage increased sharply over 6 h and reached a constant swelling percentage in 24 h. In the wound healing process, excess exudates are among the most difficult aspects of wound management. They can distress patients extremely and often causes maceration and bacterial overgrowth in the wound site. An ideal wound dressing should be able to absorb sufficient wound exudates and to provide a wet environment for the wound. In the Figure below, we can see that all the hydrogels show great swelling ability that may help accelerate healing.
Water lost (%)
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Water lost (%)
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15
20
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Time (h)
Fig. 3. (a) Water lost of HACC:PVA/PEO (1:1) hydrogel prepared at different radiation doses. (b) Water lost of HACC/PVA/PEO hydrogel (40 kGy) prepared at different weight ratios.
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The mechanical properties of the hydrogel are important requirements for a wound dressing. And the tensile strength is an important parameter to use to evaluate the mechanical properties of the hydrogels. The tensile strength of HACC/PVA/PEO hydrogels at different radiation doses is given in Fig. 4(a). It can be seen that the tensile strength reached the highest: 34 MPa under the radiation dose of 40 kGy. When the dose is less than 40 kGy, the tensile strength enhanced as the radiation dose increased. However, when the radiation dose was more than 40 kGy, the tensile strength decreased as the radiation dose increased. The reason for this phenomenon can be explained as follows: when the radiation dose increases, the crosslink density of the polymer molecules also increases, which effectively improves the mechanical properties of the hydrogels. However, when the dose is more than 40 kGy, the radiation degradation effects will be enhanced. The hardness of the hydrogels increases, and the hydrogel becomes brittle, causing the tensile strength to decrease. In addition, Fig. 4 also shows that the tensile strength and elongation of the hydrogel decreased with the increase of the weight ratio of quaternary ammonium chitosan.
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3.5. FT-IR
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In Fig. 5, there are the FT-IR spectra of HACC, PVA, PEO and HACC/PVA/PEO hydrogel. The FT-IR spectrum of PVA exhibits several characteristic bands. It showed a characteristic broad band at 3450 cm−1 that corresponds to the O H stretching vibration of PVA-hydroxyl group. The sharp band at 1635 cm−1 corresponds to the C O stretching of the acetate group of PVA. The sharp band at 2910 cm−1 is due to the stretching vibration of backbone aliphatic C H. The strong absorption peak at 1100 cm−1 has been assigned to the C O in stretching mode for PVA and the bands observed at 1355 cm−1 have been attributed to combination frequencies of CH-OH [28]. PEO has a characteristic absorbance band at 1100 cm−1 due to the C O stretching vibration [29]. The absorption band at 1656 cm−1 in native chitosan is referred as an amide I band, and the absorption band at 1600 cm−1 is ascribed to the N H bending mode in the primary amine. The absorption band of NH2 in quaternary ammonium chitosan almost disappeared, which suggests that N-alkylation is occurs in native chitosan. Compared with native chitosan, quaternary ammonium chitosan shows a new band at 1484 cm−1 , which is attributed to the methyl groups of ammonium [30]. It could be expected that the hydrogel reserves the methyl groups of ammonium supported by the absorption band at 1480 cm−1 . It can also prove that between the hydroxyl groups of PVA, PEO and the amide groups of HACC exist hydrogen bond supported by the shape distortion of the hydrogen-bonded hydroxyl group (3420 cm−1 ) of the hydrogel matrixdgdm, as well as a shift of the C O peak (1645 cm−1 ). Based upon the above experimental observations, it can be stated that
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2:1
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Tensile strenth (MPa)
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Elongation (%)
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wound bed has been widely accepted as the most ideal environment for effective wound healing. Many clinical studies attest to the benefits of moist wound healing and demonstrate that hydrogel dressings lead to quicker healing, less pain and great cost savings when compared to saline dressings [27]. Fig. 3(a) is the variation of the water lost percentage of different hydrogels after equilibrium swelling. It can be seen from the figure that the water lost percentage increased sharply in 6 h and reached a constant swelling percentage in 24 h. The water lost percentage increased as the radiation dose increased. It can be seen in Fig. 3(b) that water loss increased with an increase of the weight ratio of the quaternary ammonium chitosan.
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Radiation dose (KGy) Fig. 4. (a) Tensile strength of HACC/PVA/PEO hydrogel prepared at different radiation doses and weight ratios. (b) Elongating of HACC/PVA/PEO hydrogel prepared at different radiation doses and weight ratios.
PVA, PEO and HACC are compatible and interact via the hydrogen bond. 3.6. Scanning electron microscopy (SEM) SEM can provide information on the material surface and is capable of imaging large samples up to several square centimeters in area. SEM of the hydrogel of 40 kGy is investigated in Fig. 6(a) and (b). It can be seen that the hydrogels have a good three-dimensional network structure. Moreover, it shows that the microstructure of the HACC/PVA/PEO hydrogel exhibits many micro-pores, and the pores are distributed uniformly on the surface of the materials. The SEM of the hydrogel of 30 kGy is investigated in Fig. 6(c). It shows that the size of the pores of the materials decreased with the increase of the radiation dose, and the density of the pores on the surface of the materials increased with the increase of the radiation dose.
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PEO 1100
Transmittance
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Hydrogel
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PVA
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Fig. 5. FT-IR spectra of PVA, PEO, HACC and HACC/PVA/PEO hydrogel prepared at radiation dose: 40 kGy.
Table 1 Antimicrobial activity of HACC/PVA/PEO hydrogel prepared at irradiation dose: 40 kGy. Bacterials
S. aureus E. coli
PVA/PEO hydrogel
HACC/PVA/PEO hydrogel
– –
++ ++
–: no inhibition; +: inhibition.
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Fig. 7. Effect HACC/PVA/PEO hydrogel on growth of S. aureus (a) and E. coli (b) strains.
Antibacterial activity
3.7. Antibacterial study Wounds often provide a favorable environment for the colonization of microorganisms which may both delay healing and cause infection. As a wound dressing, a hydrogel must constitute a barrier against external contaminating microorganisms and exhibit broad spectrum antibacterial activity to control the colonization of microorganisms in the wound. The above research indicated that the hydrogel prepared under 40 kGy and the weight ratio between HACC and PVA/PEO was 1:1 had the best mechanical properties. And it also showed swelling ability and water evaporation rate. Here, we chose this kind of hydrogels as the antibacterial experiment object. The capability of PEO/PVA hydrogel and HACC/PEO/PVA hydrogel in inhibiting the growth of the tested bacterials is shown in Table 1. The PEO/PVA hydrogel was
devoid of antibacterial activity, and the HACC/PEO/PVA hydrogel performed great antibacterial activity against S. aureus and E. coli as shown in Fig. 7. This reflected that the antibacterial activity of the hydrogels was due to the introduction of quaternary ammonium chitosan which is relatively safe and exhibits antibacterial activity. In the case of the antibacterial mechanism against S. aureus and E. coli, it is generally believed that the following occurred: the positive charge of the quaternized group results in a polycationic structure that is absorbed onto the negatively-charged cell surface of the bacteria, which leads to a significant alteration of the structure of the outer membranes and causes the release of a large proportion of proteinaceous material out from the cell. In addition, it would also releases cytoplasmic constituents, such as K+ ions, DNA and RNA, ultimately leading to bacterial death [31–33]. Recently, hydrogel wound dressings that contained nanosilver which have been synthesized and exhibited excellent antibacterial activity. Nanosilver is increasingly being applied as an antibacterial agent in health care products. Nanocrystalline silver results in a very large specific surface area for the release of ionic silver, and even a small amount of silver provides a bactericidal action. Moreover, the silver ion is released into the wound for long-lasting
Fig. 6. SEM picture of HACC/PVA/PEO hydrogel prepared at radiation dose: (a) 40 kGy, (b) 40 kGy, and (c) 30 kGy.
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bactericidal effect in a controlled fashion. However, domestic and international research on the toxicity of nanosilver mainly relied on single cell toxicity testing such as the morphological method and cytotoxic activity method so far. The biological effects of nanoscale, especially in terms of toxicology and safety issues, have no obvious conclusion. Its shape, particle size and the dose should be considered. Therefore, the nanosilver dressings need biological evaluation at the level of nanoscience. In this study, we introduced quaternary ammonium chitosan (HACC) with antibacterial properties to the PVA/PEO to form the hydrogel and get a new antibacterial and safe dressing. As a derivative of chitosan, quaternary ammonium chitosan not only retains the characteristics of chitosan but also shows excellent water-solubility and antibacterial activity. HACC/PVA/PEO hydrogel is promising to be use as a safe wound dressing.
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4. Conclusion
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The goal of wound therapy is to keep the wound microbial content of the wound as low as possible in order to prevent infection and to stimulate the repair process. The hydrogels have the potential to mimic the extra cellular matrix, which may lead to tissue regeneration and is necessary for wound healing [34]. To design a matrix, which can promote wound healing, it is important to provide adequate antibacterial protections [35]. HACC with excellent antimicrobial activity applied in hydrogel dressing has not been well reported so far. In this study, various HACC/PEO/PVA hydrogels were prepared by radiation technology for the use in wound dressing applications. Gamma radiation was found suitable as a technique for the incorporation of HACC in the PVA/PEO polymer matrix. The results demonstrate that the fluid-handling properties and antibacterial activity of the HACC/PEO/PVA hydrogels meet the requirements of an ideal wound dressing. HACC/PEO/PVA hydrogels prepared by radiation have great clinical potential to be applied as a wound dressing.
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Acknowledgements
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The work was supported by the National Natural Science Foundation of China (Foundation No. 51173143, No. 51273156), The Special Funds Project of Major New Products of Hubei Province (Foundation No. 20132h0040), University-industry Cooperation Projects of The Ministry of Education of Guangdong province (Foundation No. 2012B091100437), The innovation fund project of the Ministry of Science and Technology of Small and Medium-sized Enterprises (Foundation No. 11C26214202642,
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No. 11C26214212743), Zhuhai Science and Technology Plan Projects (Foundation No. 2011B050102003), Wuhan Science and Technology Development (Foundation No. 201060623262), The Fundamental Research Funds for the Central Universities (Foundation No. 2014-zy-220). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35]
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
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Preparation and characterization of quaternary ammonium chitosan hydrogels with significant antibacterial activity Lihong Fan a,∗ , Jing Yang a , Huan Wu a , Zhihai Hu a , Jiayan Yi a , Jun Tong a , Xiaoming Zhu b a b
College of Chemical Engineering, Wuhan University of Technology, Wuhan 430070, China Hubei Collaboration Innovation Center of Non-power Nuclear, China
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a r t i c l e
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Article history: Received 5 December 2014 Received in revised form 6 April 2015 Accepted 8 April 2015 Available online xxx
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Keywords: Quaternary ammonium chitosan hydrogel Gamma radiation Antibacterial activity
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1. Introduction
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Quaternary ammonium chitosan (HACC)/polyvinyl alcohol (PVA)/polyethylene oxide (PEO) hydrogels were prepared using gamma radiation. The chemical structure of the hydrogels was characterized using FT-IR. The results revealed that HACC, PVA and PEO were perfectly compatible and interacted via the hydrogen bonds. As revealed by SEM, scaffolds with a homogeneous interconnected pore structure were obtained after lyophilizing the hydrogels. The influence of different radiation doses and weight ratios on properties including gel content, swelling ability, water evaporation rate and mechanical properties were investigated. It indicated that the hydrogels had the good swelling ability, water evaporation rate and mechanical properties. In vitro antibacterial activity assessment, the hydrogels exhibited a pronounced inhibitory effect against two bacteria (Staphylococcus aureus and Escherichia coli). Therefore, the hydrogels showed a promising potential to be applied as wound dressing. © 2015 Published by Elsevier B.V.
The skin forms a protective barrier against the external environment. An intact barrier of this kind is of critical importance following the occurrence of a wound [1]. The healing of a wound requires the protection of the skin surface including providing, retaining moisture and inhibiting the growth of bacterial [2]. Recently, hydrogels in a three-dimensional network structure crosslinked by linear polymer materials have attracted a lot of attentions for their potential applications in medical and pharmaceutical areas. Hydrogel dressings with high water content, as well as the ability to swell, are able to absorb leaking body fluids and facilitate the creation of a moist environment. The moist environment encourages rapid granulation tissue formation and re-epithelialization that accelerates wound healing [3,4]. Compared with a traditional dressing, a hydrogel dressing has several advantages and may indeed be considered an ideal dressing: it is relatively soft and can reduce pain that is often caused when dressings are changed. Also, a hydrogel dressing make it possible to maintain a moist environment to promote wound healing [5]. At present, most hydrogels are prepared via radical polymerization in the presence of crosslinking agents. However, the use of a chemical crosslinker can lead to structural non-homogeneity.
∗ Corresponding author. Tel.: +86 27 13018011218; fax: +86 27 87859019. E-mail address: [email protected] (L. Fan).
These agents can affect the integrity of the substance to be entrapped and make the substance toxic [6–8]. In this study, quaternary ammonium chitosan (HACC) combined with polyvinyl alcohol (PVA) and polyethylene oxide (PEO) were mixed to form a HACC/PVA/PEO hydrogel by employing ␥-radiation as the free radical initiator. It is an environmental approach to prepare hydrogels for biomaterial applications. Chitosan hydrogels have been studied widely. However, until recently no reports have been found in the literature on the radiation crosslinking of quaternary ammonium chitosan hydrogels. Chitosan is composed of N-acetyl-d-glucosamine and dglucosamine units linked by -(1–4) bonds. It is insoluble in neutral aqueous and alkaline solutions [9]. This biopolymer is known for its wide biological properties [10]. Chitosan has always been used as a wound healing promoter. The main biochemical effects of chitosan in wound healing are fibroblast activation, cytokine production, giant cell migration and stimulation of type IV collagen synthesis [11,12]. Physical or chemical modification of chitosan can lead to new products with significantly functional properties. It is reported that quaternization is an efficient means of imparting new functional properties to polysaccharides [13]. So far, quaternization has been widely applied to a variety of polysaccharides, such as pectin [14], konjac glucomannan [15], starch [16], alginate [17] and cellulose [18]. In previous reports [19], the antibacterial ability of natural polysaccharides is shown to be improved by quaternization. As a polymer with a quaternary ammonium salt (QP) group, Chitosan possesses potential antibacterial properties. Hyaluronic acid
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CH2OR1
CH2OH ClCH2CH(OH)CH2N+(CH3)3Cl-
O
O O
O
NHR2
R1O
NHR
HO
Quatemary ammonium chitosan Chitosan, R= H, Ac R1= H,ClCH2CH(OH)CH2N+(CH3)3ClR2= H,ClCH2CH(OH)CH2N+(CH3)3Cl-,Ac
Scheme 1. Synthesis of quaternary ammonium chitosan.
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(HA), an important functional ingredient in cosmetics, is unique for its excellent moisture-absorption and moisture-retention abilities. But the total amount of HA is limited, and the price is high. The results showed that QP group could replace HA for its better moisture-absorption and moisture-retention abilities. Although moisture facilitates the immigration of epithelium cells, it may also brings more risks of infection, which may cause excessive scar formation and chronic clinical problems [20,21]. Chitosan derivatives achieved by quaternization may solve the contradiction between moist healing and infection, due to its good antibacterial activity [22]. In addition to the water-solubility and antibacterial activity, quaternary ammonium chitosan is promising for the use in wound dressings. However, chitosan and its derivatives are degradable polymers and difficult to form hydrogels in conditions involving a high radiation dose. The introduction of PVA and PEO can solve this problem and can greatly improve the mechanical strength of the hydrogels [23]. Both PVA and PEO are biocompatible synthetic polymers that do not cause any toxicity or stimulation to the tissue cells of the body. The prepared hydrogels were verified by Fourier transform infrared (FTIR) spectroscopy and Surface morphology (SEM). The influence of different radiation doses and weight ratios on the properties including gel content, swelling ability and mechanical properties were investigated. Finally, the HACC/PVA/PEO hydrogel was found to have antibacterial activity against two bacteria. It suggests that this additive-free and sterilized hydrogels may have applications as a wound dressing.
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2. Experimental
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2.1. Materials
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pressure-equalizing dropping funnel. After that, magnetic stirring was done continuously for 72 h at 78 ◦ C. The solution was then purified by dialysis through a 10,000–8000 molecular weight cut-off dialysis tubing for three days and dried overnight in an oven at 50 ◦ C. The reaction is shown in Scheme 1. 2.3. Preparation of HACC/PVA/PEO hydrogels HACC solution was completely dissolved and mixed with PVA/PEO solution at 80 ◦ C under the stirring for 6 h to form a homogenous solution. The total polymer concentration was fixed at 10% (different weight ratios between HACC and PVA/PEO were: 1:2, 1:1 and 2:1. The mixtures were then poured into test tubes and the solutions were made free from oxygen by purging nitrogen gas for 5 min. They were then subjected to ␥-radiation with an absorbed dose of 20, 30, 40, 50 and 60 kGy using a 60 Co facility. The radiation crosslinking was performed at room temperature with a dose rate of 20 Gy/min. After copolymerization, the polymeric cylinders were removed by breaking the test tubes and cut into discs of 2 mm of thickness. All samples were washed in excess water to remove the non-reacted components, and then air-dried at room temperature. 2.4. Calculation of gel content Dried hydrogels were extracted with distilled water for 24 h at 100 ◦ C to extract the insoluble parts of the hydrogels. The insoluble or gelled parts were taken out and washed with hot distilled water to remove the soluble parts and then were dried and weighed. This extraction cycle was repeated until the weight became constant. The gel yield of hydrogels were determined as follows:
W
Gel % =
110
Chitosan (degree of deacetylation = 93.23%) was purchased from Zhejiang Yuhuan Ocean Biochemistry Co. Ltd. (China). Monochloro acetic acid, sodium bisulfite, sodium nitrite and other reagents used in this investigation were of analytical grade and without further purification. CHPTAC (N-(3-chloro-2-hydroxypropyl) trimethyl ammonium chloride) was purchased from Guofeng Fine Chemical Co. Ltd. (Shandong, China) and it was used as an etherifying reagent without further purification. Polyvinyl alcohol (PVA-1788) and polyethylene oxide (PEO, Mv ∼300,000) were purchased from Aladding Industrial Corporation Co. Ltd. (Shanghai, China). Radiation sources used in the experiment were provided by the Radiation Center of Academy of Agricultural Sciences of Hubei Province.
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2.2. Synthesis of quaternary ammonium chitosan
Swelling % =
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In a typical reaction procedure, chitosan (8 g) was dissolved in NaOH/CHPTAC aqueous solution (85 mL) through a
e
Wd
× 100
2.5. Swelling measurement The clean, dried hydrogels of known weight was immersed in distilled water at room temperature for 48 h. The hydrogels were removed and the excess solvent on the surface was removed by blotting quickly with absorbent paper and were weighed. The water uptake was calculated as follows:
md
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where Wd and We represent the weights of the dry hydrogels and the gelled part after extraction, respectively.
m − m s d
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× 100
where md and ms are the weights of dry and wet films, respectively.
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2.6. Water evaporation rate Prepared hydrogels after extraction were immersed in distilled water. Taking a certain weight of the hydrogel after reaching its reaching swelling equilibrium, then the hydrogels were placed in an incubator at 50 ◦ C for 24 h in a humid atmosphere of 50%. Weight of the hydrogel was measured at regular intervals until completely dry. The water evaporation rate was calculated by the following equation: Water lost % =
(W1 − W2 ) × 100 (W1 − W3 )
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where W1 , W2 and W3 are the starting weight, measured weight and last weight of the hydrogels, respectively.
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2.7. Mechanical property test
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Several hydrogel samples were prepared and cut into a dumbbell shape with a length of 75 mm and a width of 13 mm. The depression of the middle portion of the sample was a rectangle shape, and the sides of the shank portion were in a dumbbell shape with a length of 30 mm and a width of 4 mm. The thickness of the hydrogels samples was determined using a caliper. The tension (N) and elongation (mm) at break of the samples was determined by a machine at a speed of 200 operating at room temperature. The tensile strength of the hydrogels was determined as follows:
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F Tensile strength (MPa) = (W × H) S Elongation % = × 100 L
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where F represents the tension of the samples at the break, S represents the elongation of the samples at break, and W, H and L are the width, height and length of the samples.
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2.8. Characterization of the hydrogels by FT-IR
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FT-IR spectra of powdered discs of prepared CMC/PVA/PEO hydrogels were recorded over the range 400–4000 cm−1 with a Fourier transform infrared spectrophotometer (Nicolet 170SX, Thermo Fisher Scientific Inc., USA). One milligram of dry sample was mixed with 100 mg of dry KBr, and the mixture was pressed into a disk for spectrum recording. In fact, FT-IR spectra were utilized to confirm the occurrence of the polymerization reactions on the polymer chains.
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2.9. Scanning electron microscopy (SEM)
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For morphological characterization, the hydrogels after swelling in water (to an equilibrium state) were freeze-dried using a freeze drier (Christ, Germany, Alpha 1-2) at −52 ◦ C for 6 h. Transverse sections were cut from the freeze-dried film samples using a cold knife. Samples were then examined with a Jeol JSM-5400 scanning electron microscope (JEOL, Tokyo, Japan).
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2.10. Antibacterial assay
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Two kinds of bacteria were used as test microorganisms in this study. Test bacteria included Staphylococcus aureus (RCMB 000106) (as Gram positive bacteria) and Escherichia coli (RCMB 000107) (as Gram negative bacteria). The strains were stored at 4 ◦ C. The medium used for growing bacteria was universal nutrient agar. Antibacterial activity was determined by the agar well-diffusion method. Briefly, agar plates were seeded with test microorganisms and kept for 30 min until the medium solidified. Circular pieces of
1:1(HACC:PVA/PEO) 2:1 1:2
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Radiation dose(KGy) Fig. 1. Gel content of HACC/PVA/PEO hydrogel prepared at different radiation doses and weight ratios.
approximately 1 cm in diameter of the test hydrogels were added. Plates were kept for 12 h at 4 ◦ C prior to incubation at the appropriate temperature for the bacteria. Plates were examined on a daily basis to check for the development of growth inhibition zones around the loaded hydrogels.
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3. Results and discussion
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To quantitatively evaluate the degree of cross-linking, the gel content of the hydrogels was investigated. The solution of HACC/PVA/PEO (10%) composite was exposed to various radiation doses. In Fig. 1, we can see that the gel content of all of the compositions, ascended rapidly initially, while it declined slightly at higher radiation doses. This phenomenon is universal for the formation of hydrogels via radiation crosslinking. Initially, the amount of free radicals generated by radiation is not high enough for recombination to form a physically stable hydrogel. However, when the radiation dose reaches a critical dose (the gelation dose), the intermolecular recombination of free radicals readily occurs and finally leads to the formation of a three-dimensional network. Further increase of the absorbed dose would cause increased crosslinking density. At a sufficient high absorbed dose, the intramolecular free radical recombination tends to dominate over the intermolecular radical recombination, which would terminate the crosslinking and begin to degrade the network. Hence, a slightly decrease is observed in the gel content. The influence of different ratios of quaternary ammonium chitosan on the gel content was also studied in this study. Polysaccharides are degraded by ionization radiation in solid state and low concentration solutions. However, a large variety of polysaccharide hydrogels are prepared in concentrated solutions (these have a so-called, “paste-like status”) by radiation crosslinking [24,25]. Quaternary ammonium chitosan may be crosslinked and degraded concurrently in the presence of radiation. Although a physically stable hydrogel can be formed, the maximum gel content of the pure quaternary ammonium chitosan hydrogel is rather low, i.e. 40%. On the contrary, the radiation crosslinking efficiency of PVA/PEO was much higher than that of the quaternary ammonium chitosan. Consequently, the gel content of the HACC/PVA/PEO hybrid hydrogel
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Fig. 2. (a) Swelling percentage of HACC: PVA/PEO(1:1) hydrogel prepared at different irradiation doses. (b) Swelling percentage of HACC/PVA/PEO hydrogel (40 kGy) prepared at different weight ratios.
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increased with the decreasing content of quaternary ammonium chitosan. 3.2. Swelling percentage The influence of different radiation doses and weight ratios on the swelling percentage of the prepared hydrogels is shown in Fig. 2. All of the hydrogels showed a swelling percentage at the range from 1000% to 4000%, which is sufficient for an application as a hydrogel wound dressing [26]. HACC/PVA/PEO hydrogels had a very high swelling percentage, due to the fact that quaternary HACC, PVA and PEO are hydrophilic polymers which contain a large amount of hydrophilic groups such as OH and NH2 . In Fig. 2(a), we can see that the swelling percentage decreased when the radiation dose increased from 20 kGy up to 60 kGy. The reason for this might be that when the radiation dose increases, the crosslink degree of hydrogel also increases too. Thus, the polymer network structure becomes more compact, which makes it difficult to stretch the macromolecular chains, and the pore size of the hydrogel becomes smaller [27]. In Fig. 2(b), we can see that
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Generally, a smaller water evaporation rate can provide a moister wound healing environment. The maintenance of a moist
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swelling percentage increased as the weight ratio of quaternary ammonium chitosan increased under the same radiation dose. It is believed that the addition of quaternary ammonium chitosan could loosen the structure of the hydrogel, which would make the macromolecular chain easier to stretch. It can be also seen from Fig. 2 that the swelling percentage increased sharply over 6 h and reached a constant swelling percentage in 24 h. In the wound healing process, excess exudates are among the most difficult aspects of wound management. They can distress patients extremely and often causes maceration and bacterial overgrowth in the wound site. An ideal wound dressing should be able to absorb sufficient wound exudates and to provide a wet environment for the wound. In the Figure below, we can see that all the hydrogels show great swelling ability that may help accelerate healing.
Water lost (%)
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Water lost (%)
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20
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Time (h)
Fig. 3. (a) Water lost of HACC:PVA/PEO (1:1) hydrogel prepared at different radiation doses. (b) Water lost of HACC/PVA/PEO hydrogel (40 kGy) prepared at different weight ratios.
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The mechanical properties of the hydrogel are important requirements for a wound dressing. And the tensile strength is an important parameter to use to evaluate the mechanical properties of the hydrogels. The tensile strength of HACC/PVA/PEO hydrogels at different radiation doses is given in Fig. 4(a). It can be seen that the tensile strength reached the highest: 34 MPa under the radiation dose of 40 kGy. When the dose is less than 40 kGy, the tensile strength enhanced as the radiation dose increased. However, when the radiation dose was more than 40 kGy, the tensile strength decreased as the radiation dose increased. The reason for this phenomenon can be explained as follows: when the radiation dose increases, the crosslink density of the polymer molecules also increases, which effectively improves the mechanical properties of the hydrogels. However, when the dose is more than 40 kGy, the radiation degradation effects will be enhanced. The hardness of the hydrogels increases, and the hydrogel becomes brittle, causing the tensile strength to decrease. In addition, Fig. 4 also shows that the tensile strength and elongation of the hydrogel decreased with the increase of the weight ratio of quaternary ammonium chitosan.
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3.5. FT-IR
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In Fig. 5, there are the FT-IR spectra of HACC, PVA, PEO and HACC/PVA/PEO hydrogel. The FT-IR spectrum of PVA exhibits several characteristic bands. It showed a characteristic broad band at 3450 cm−1 that corresponds to the O H stretching vibration of PVA-hydroxyl group. The sharp band at 1635 cm−1 corresponds to the C O stretching of the acetate group of PVA. The sharp band at 2910 cm−1 is due to the stretching vibration of backbone aliphatic C H. The strong absorption peak at 1100 cm−1 has been assigned to the C O in stretching mode for PVA and the bands observed at 1355 cm−1 have been attributed to combination frequencies of CH-OH [28]. PEO has a characteristic absorbance band at 1100 cm−1 due to the C O stretching vibration [29]. The absorption band at 1656 cm−1 in native chitosan is referred as an amide I band, and the absorption band at 1600 cm−1 is ascribed to the N H bending mode in the primary amine. The absorption band of NH2 in quaternary ammonium chitosan almost disappeared, which suggests that N-alkylation is occurs in native chitosan. Compared with native chitosan, quaternary ammonium chitosan shows a new band at 1484 cm−1 , which is attributed to the methyl groups of ammonium [30]. It could be expected that the hydrogel reserves the methyl groups of ammonium supported by the absorption band at 1480 cm−1 . It can also prove that between the hydroxyl groups of PVA, PEO and the amide groups of HACC exist hydrogen bond supported by the shape distortion of the hydrogen-bonded hydroxyl group (3420 cm−1 ) of the hydrogel matrixdgdm, as well as a shift of the C O peak (1645 cm−1 ). Based upon the above experimental observations, it can be stated that
3.5
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Tensile strenth (MPa)
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wound bed has been widely accepted as the most ideal environment for effective wound healing. Many clinical studies attest to the benefits of moist wound healing and demonstrate that hydrogel dressings lead to quicker healing, less pain and great cost savings when compared to saline dressings [27]. Fig. 3(a) is the variation of the water lost percentage of different hydrogels after equilibrium swelling. It can be seen from the figure that the water lost percentage increased sharply in 6 h and reached a constant swelling percentage in 24 h. The water lost percentage increased as the radiation dose increased. It can be seen in Fig. 3(b) that water loss increased with an increase of the weight ratio of the quaternary ammonium chitosan.
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Radiation dose (KGy) Fig. 4. (a) Tensile strength of HACC/PVA/PEO hydrogel prepared at different radiation doses and weight ratios. (b) Elongating of HACC/PVA/PEO hydrogel prepared at different radiation doses and weight ratios.
PVA, PEO and HACC are compatible and interact via the hydrogen bond. 3.6. Scanning electron microscopy (SEM) SEM can provide information on the material surface and is capable of imaging large samples up to several square centimeters in area. SEM of the hydrogel of 40 kGy is investigated in Fig. 6(a) and (b). It can be seen that the hydrogels have a good three-dimensional network structure. Moreover, it shows that the microstructure of the HACC/PVA/PEO hydrogel exhibits many micro-pores, and the pores are distributed uniformly on the surface of the materials. The SEM of the hydrogel of 30 kGy is investigated in Fig. 6(c). It shows that the size of the pores of the materials decreased with the increase of the radiation dose, and the density of the pores on the surface of the materials increased with the increase of the radiation dose.
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Fig. 5. FT-IR spectra of PVA, PEO, HACC and HACC/PVA/PEO hydrogel prepared at radiation dose: 40 kGy.
Table 1 Antimicrobial activity of HACC/PVA/PEO hydrogel prepared at irradiation dose: 40 kGy. Bacterials
S. aureus E. coli
PVA/PEO hydrogel
HACC/PVA/PEO hydrogel
– –
++ ++
–: no inhibition; +: inhibition.
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Fig. 7. Effect HACC/PVA/PEO hydrogel on growth of S. aureus (a) and E. coli (b) strains.
Antibacterial activity
3.7. Antibacterial study Wounds often provide a favorable environment for the colonization of microorganisms which may both delay healing and cause infection. As a wound dressing, a hydrogel must constitute a barrier against external contaminating microorganisms and exhibit broad spectrum antibacterial activity to control the colonization of microorganisms in the wound. The above research indicated that the hydrogel prepared under 40 kGy and the weight ratio between HACC and PVA/PEO was 1:1 had the best mechanical properties. And it also showed swelling ability and water evaporation rate. Here, we chose this kind of hydrogels as the antibacterial experiment object. The capability of PEO/PVA hydrogel and HACC/PEO/PVA hydrogel in inhibiting the growth of the tested bacterials is shown in Table 1. The PEO/PVA hydrogel was
devoid of antibacterial activity, and the HACC/PEO/PVA hydrogel performed great antibacterial activity against S. aureus and E. coli as shown in Fig. 7. This reflected that the antibacterial activity of the hydrogels was due to the introduction of quaternary ammonium chitosan which is relatively safe and exhibits antibacterial activity. In the case of the antibacterial mechanism against S. aureus and E. coli, it is generally believed that the following occurred: the positive charge of the quaternized group results in a polycationic structure that is absorbed onto the negatively-charged cell surface of the bacteria, which leads to a significant alteration of the structure of the outer membranes and causes the release of a large proportion of proteinaceous material out from the cell. In addition, it would also releases cytoplasmic constituents, such as K+ ions, DNA and RNA, ultimately leading to bacterial death [31–33]. Recently, hydrogel wound dressings that contained nanosilver which have been synthesized and exhibited excellent antibacterial activity. Nanosilver is increasingly being applied as an antibacterial agent in health care products. Nanocrystalline silver results in a very large specific surface area for the release of ionic silver, and even a small amount of silver provides a bactericidal action. Moreover, the silver ion is released into the wound for long-lasting
Fig. 6. SEM picture of HACC/PVA/PEO hydrogel prepared at radiation dose: (a) 40 kGy, (b) 40 kGy, and (c) 30 kGy.
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bactericidal effect in a controlled fashion. However, domestic and international research on the toxicity of nanosilver mainly relied on single cell toxicity testing such as the morphological method and cytotoxic activity method so far. The biological effects of nanoscale, especially in terms of toxicology and safety issues, have no obvious conclusion. Its shape, particle size and the dose should be considered. Therefore, the nanosilver dressings need biological evaluation at the level of nanoscience. In this study, we introduced quaternary ammonium chitosan (HACC) with antibacterial properties to the PVA/PEO to form the hydrogel and get a new antibacterial and safe dressing. As a derivative of chitosan, quaternary ammonium chitosan not only retains the characteristics of chitosan but also shows excellent water-solubility and antibacterial activity. HACC/PVA/PEO hydrogel is promising to be use as a safe wound dressing.
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The goal of wound therapy is to keep the wound microbial content of the wound as low as possible in order to prevent infection and to stimulate the repair process. The hydrogels have the potential to mimic the extra cellular matrix, which may lead to tissue regeneration and is necessary for wound healing [34]. To design a matrix, which can promote wound healing, it is important to provide adequate antibacterial protections [35]. HACC with excellent antimicrobial activity applied in hydrogel dressing has not been well reported so far. In this study, various HACC/PEO/PVA hydrogels were prepared by radiation technology for the use in wound dressing applications. Gamma radiation was found suitable as a technique for the incorporation of HACC in the PVA/PEO polymer matrix. The results demonstrate that the fluid-handling properties and antibacterial activity of the HACC/PEO/PVA hydrogels meet the requirements of an ideal wound dressing. HACC/PEO/PVA hydrogels prepared by radiation have great clinical potential to be applied as a wound dressing.
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Acknowledgements
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The work was supported by the National Natural Science Foundation of China (Foundation No. 51173143, No. 51273156), The Special Funds Project of Major New Products of Hubei Province (Foundation No. 20132h0040), University-industry Cooperation Projects of The Ministry of Education of Guangdong province (Foundation No. 2012B091100437), The innovation fund project of the Ministry of Science and Technology of Small and Medium-sized Enterprises (Foundation No. 11C26214202642,
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No. 11C26214212743), Zhuhai Science and Technology Plan Projects (Foundation No. 2011B050102003), Wuhan Science and Technology Development (Foundation No. 201060623262), The Fundamental Research Funds for the Central Universities (Foundation No. 2014-zy-220). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35]
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