Green and Facile Preparation of Chitosan Sponges as Potential

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Green and Facile Preparation of Chitosan Sponges as Potential Wound Dressings Jimin Wu, Chen Su, Lei Jiang, Shan Ye, Xiufeng Liu, and Wei Shao ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01468 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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ACS Sustainable Chemistry & Engineering

Green and Facile Preparation of Chitosan Sponges as Potential Wound Dressings

Jimin Wu†, Chen Su†, Lei Jiang†, Shan Ye†, Xiufeng Liu‡* and Wei Shao†*

†College

of Chemical Engineering, Nanjing Forestry University, 159 Longpan

Road, Nanjing 210037, P. R. China ‡State

Key Laboratory of Natural Medicines, School of Traditional Chinese

Pharmacy, China Pharmaceutical University, 639 Longmian Road, Nanjing 210009, P. R. China

*Corresponding authors: *Fax: +86-25-85418873; Tel: +86-25-85427024; E-mail: [email protected] (W. Shao); [email protected] (X. Liu). ORCID: Wei Shao: 0000-0001-5666-1471; Xiufeng Liu: 0000-0002-3315-8571

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ABSTRACT The aim of this study was to prepare non-leaching chitosan based wound dressings via chemical modification. A green and facile method was applied to fabricate ampicillin grafted chitosan (CSAP) sponges. The morphology, porosity and swelling behavior of CSAP sponges were analyzed. Specifically, the antibacterial activity of CSAP sponges toward Staphylococcus aureus,

Candida albicans and Escherichia coli was assessed using a serious of assays, including bacterial growth curve, nucleic acids leakage and live/dead staining. As expected, CSAP sponges exhibited excellent antibacterial activity without any leaching. The cytotoxicity assay was carried out on HEK293 cells in vitro and the result proved the good biocompatibility of the developed sponges. Moreover, the wound healing ability was evaluated using a wound model in

vivo, and the result displays that the sponge could speed up wound healing efficiently. Thus, the chemical modified chitosan sponges exhibited great potential as promising wound dressings. Keywords: chitosan; sponge; non-leaching; antibacterial; wound dressing


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INTRODUCTION Skin, as the largest organ, is a protection for human body from external insults. It represents good self-regenerative ability for minor damages.1 However, it becomes a medical challenge to keep skin wounds sterile and dry in severe injuries. The undesired accumulation of wound exudates such as blood and fluid can promote bacterial growth, cause infections and lead to delay the wound healing process.2 Therefore, once the skin is damaged, wound dressings or ointments would be applied. However, the frequent change or reapplication on the wound could result in pain and burden for the patient.3 In order to avoid these, the wound dressings which are able to provide protection and help to recover the damaged skin are welcomed. 4 An ideal material for wound dressing should own some advantages, such as high absorbing ability, good oxygen permeability, pain alleviation, easy remove, anti-infection, wound healing promotion, and reduced scarring. Biopolymers based sponges are considered to be ideal candidates for wound dressings due to the good biocompability, high specific surface area, high porosity, and good absorb wound exudates ability.1, 5 Chitosan is a natural polysaccharide, consisting of randomly distributed N-acetyl-D-glucosamine and-(1,4)-linked D-glucosamine.6,

7

Chitosan is

derived from the deacetylation of chitin that can be obtained from the exoskeletons of crustaceans and insects, and cell walls of fungal.8 Chitosan has various biorelated applications because of its excellent unique features,



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such as large-scale availability, antitumor, antibacterial, antioxidant, various bioactivities, biodegradability, and biocompatibility.9-11 Since chitosan has unique chem-physical properties, it can be produced as drug delivery vehicles, artificial skin and scaffolds in the forms of coatings, beads, sponges, fibers and membranes. Recently, chitosan based sponges have attracted considerable attention in wound dressing applications due to its unique properties and easy preparation.8 It was reported that chitosan can affect macrophage function which could accelerate wound healing with higher angiogenic capability. 12 It can also stimulate cell proliferation and promote tissue granulation.3 However, chitosan can not inhibit bacterial growth effectively although it exhibits some antibacterial properties. Therefore, many researchers are working on improving antimicrobical property of chitosan.13 Chitosan can be functionalized by both physical and chemical methods since chitosan has some reactive functional groups, such as amine and hydroxyl groups leading to its polycationic property and extensive hydrogel bonding capacity. 14 It was reported that silver nanoparticle decorated chitosan cryogels were fabricated through a simple freeze-drying process for point-of-use water disinfection, showing an efficient bactericidal feature.15 Chitosan was successfully modified with 3,6-O-N-acetylethylenediamine which could be applied as wound dressing.16

Curcumin-loaded

chitosan

based

bio-nanocomposite

was

fabricated in order to reduce the dental bacterial biofilm formation. 17 Chitosan/ellagic acid films were prepared by solvent casting with high


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antioxidant and antimicrobial activities, showing great promise in food packaging materials.18 Thymol/chitosan composites with antibacterial and antioxidant activities were developed for the oral local delivery.19 As we all known, ampicillin is a-lactam antibiotic which belongs to the penicillin family. It is quite effective to treat a number of bacterial infections mediated by Gram-negative and Gram-positive strains.20 The extensive use of antibiotics usually leads to the development of the tolerance against the antibacterial action. Moreover, multi-drug resistant superbugs are becoming alarmingly common in recent years which is due to the excessive and improper usage of antibiotics.21-23 Thus, some policies establish in order to limit the use of antibiotics.24 It is crucial to develop novel effective antibacterial agents. Therefore, our study was to prepare ampicillin grafted chitosan (CSAP) sponges without any leaching in order to limit the usage of ampicillin. In this study, CSAP sponges were developed by a facile and green method. The CSAP sponges were analyzed by FTIR, SEM and XPS, respectively. The swelling behavior and porosity were tested. The antimicrobical activity of the CSAP sponges were investigated by S. aureus ATCC 6538, C. albicans CMCC(F) 98001 and E. coli ATCC 25922, respectively. The biocompatibility and cell morphology on HEK293 cells were investigated through MTT assay. Furthermore, the wound promotion efficacy of CSAP sponges was examined

in vivo. RESULTS AND DISCUSSION



Morphologies. The pictures of CA sponge and CA5 sponge are displayed in the Supporting Information (Figure S1). There is no obvious difference in appearance between chitosan and CA5 sponges that both of them display blue sponges. The porosity and surface morphology of CSAP sponges are important factors for wound dressing application, since they directly affect the absorption of exudates and blood capacities.25 Figure 1 shows the cross-section morphologies of the prepared CA (A) and CA5 sponges (B). As shown in Figure 1A, CA sponge presents well interconnected porous 3D network structure with quite smooth pore wall. The cross-section morphology and the structural integrity of the sponge were not influenced after being grafted with ampicillin (Figure 1B) at all that CA5 sponge still displays the interconnected 3D network structure. Besides, no obvious difference in the porosity between CA and CA5 sponges was observed as well.


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A

B

C

D

E

F

Figure 1 SEM images of CA (A) and CA5 sponges (B), element mappings of N (C) and Si (D), S (E) and EDS spectrum (F) of CA5 sponge. Moreover, the porosities of the fabricated sponges were calculated and the result is shown in Figure S2. It can be seen that the calculated porosity of CA sponge was 76.1%, and the porosities of CSAP sponges were in the range of 74.9%-75.5%. Although slightly reduced porosities were observed for the CSAP sponges, they still maintain very high porosity. These high porous structures can absorbe exudates and blood from wounds, which makes the fabricated sponges have great potentials in wound dressings. Figure 1C-F displays the element mappings of Si, N and S and EDS spectrum of CA5 Figure 1 SEM images of CS (A) and CS sponges (B), element mappings o 5



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sponge. The uniform distribution of Si, N and S elements confirmed ampicillin was successfully grafted onto CA sponge via APTES. XPS spectrum of CA5 sponge was shown in Figure S3. The successful fabrication of CSAP was further proved from the existence of Si, N and S elements. FTIR analysis. The successful chemical modification of CA sponge was confirmed by FTIR, as displayed in Figure 2A. FTIR spectra of free ampicillin, and chitosan-APTES were shown in Figure S4. For the FTIR spectrum of CA (Figure 2a), a broad peak existing between 3500 and 3100 cm-1 corresponds to the stretching vibrations of N-H and O-H.26 Two peaks at 1650 cm-1 and 1560 cm-1 are attributed to the C=O in amide group and N-H bending vibration of amine group, respectively.27 The bands between 1100 and 1000 cm−1 are assigned to C-N and C-O stretching vibrations.28 In the case of chitosan-APTES, two peaks at 1069 cm-1 and 1020 cm-1 appears, assigning to the siloxane groups (Si-O-Si) from APTES.29 FTIR spectra of ampicillin were shown in Figure S4b and S4c. Figure S4c shows the characteristic peaks of ampicillin between 2000 cm−1 and 1300 cm−1. Five main characteristic bands were seen at 1,770 cm−1 (C=O stretching of β lactam), 1,693 cm−1 (primary amide C=O stretching), 1603 cm−1 (C=C stretching of benzene ring), 1506 cm− 1

(both C-N stretching and N-H in-plane bending in secondary amide group)

and 1455 cm-1 (aromatic ring -C=C-C stretching), respectively.20,

27, 30

For

CSAP, some characteristic peaks of ampicillin (1693 cm-1 and 1455 cm-1, et al) are shown in the FTIR spectra (Figure 2A), the others are immerged into the


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large peaks of chitosan. The existence of characteristic peaks of ampicillin in the CSAP sponges reveals that ampicillin was grafted successfully onto CA sponges.

A

B

Figure 2 FTIR spectra (A) of CA (a), CA1 (b), CA2 (c), CA3 (d) CA4 (e) and CA5 sponges (f), and the swelling behaviors of CA and CSAP sponges at different time intervals (B). Swelling study. The abilities to hold and absorb water are essential features for wound dressings that can absorb exudates, metabolites and body fluids.31 The swelling performances of CA and CSAP sponges were displayed



in Figure 2B. The swelling ratio of CA sponge was 3240% after 24h. There are many hydrophilic groups including amino and hydroxyl groups, resulting in very high swelling ability.32 Similar swelling ability of CSAP sponges was observed after 24h, which is in the range of 3210-3250%. However, quite different swelling behaviors were observed for the CSAP sponges in the first 3h that the swelling ratios of CSAP sponges were much lower than that of CA sponge. This phenomenon could be due to the existence of APTES in the CSAP sponges that reduced the hydrophilic groups. Antibacterial Growth Behaviors. The antibacterial growth kinetics of S.

aureus, C. albicans and E. coli with CSAP sponges were studied (Figure 3A-C). The growth behavior was monitored by measuring the optical density at 600 nm (OD600) using a UV-vis spectrophotometer. There are similar antibacterial performances for CA and CSAP (CA1, d: CA2, e: CA3, f: CA4, g: CA5) sponges against the tested three strains. It can be seen that bacterial growth without any sponges (curve a) shows a typical bacterial growth curve with three phases in 8h. It is clearly observed that both CA and CSAP sponges delayed the progress of bacteria to exponential growth. There is stronger bacterial inhibition growth activity for CA grafted with higher amount of ampicillin due to the smaller OD600 value. Furthermore, the OD600 value remains almost zero after 8 h incubated with CA5 sponge. These results demonstrate that the incorporation of ampicillin into CA sponges can significantly enhance the antibacterial activity of CA. Moreover, disk diffusion method was conducted to


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demonstrate any leaching effect existence for the fabricated sponges against

S. aureus, C. albicans and E. coli. Pictures of the inhibition zones were shown in Figure S5. No zones of inhibition can be detected after 24 h incubation, which proves that there is no ampicillin leaching from the prepared sponges. Thus,

the

fabricated

CSAP

sponges

exhibited

great

antimicrobical

performance with no leaching. 2.5

a b c

A

a b

B

2.0

c d e

e 0.5

f g

0.0 0

1

D

2

3 4 5 Time (h)

6

7

8

OD 600

d 1.0

1.5 1.0

f

0.5

g

0.0 0

1

2

3

4 5 6 Time (h)

7

8

2.0

OD 600

1.5 OD 600

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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a b

C

1.5

c

1.0

d e

0.5

f g

0.0 0

E

1

2

3

4 5 6 Time (h)

7

8

F

Figure 3 Bacterial growth curves with/without fabricated sponges. A: S.aureus, B: C. albicans, C: E. coli. (a: blank, b: CA, C: CA1, d: CA2, e: CA3, f: CA4, g: CA5). OD260 values of bacteria treated with CA and CSAP sponges. D: S.

aureus, E: C. albicans, F: E. coli. Nucleic acids leakage. It is known to all that the UV light absorption capacity of nucleic acids is at the wavelength of 260 nm. The existence of nucleic acids in the bacterial suspension indicates the membranes of bacterial cells are destroyed. Therefore, the leakage of nucleic acids in the bacterial



suspension can be used to reveal the amount of bacterial membranes damage. Thus, the bacterial suspension was measured at the wavelength of 260 nm (OD260) in order to detect whether the inhibition effect on the bacterial growth was due to the cell membrane damage. The result is shown in Figure 3D-F. There was similar trend for the tested three strains. There was nearly no change in the OD260 values of the control group, which means little membrane damage happened. However, there were higher OD260 values for the bacterial suspension treated with CA sponge, indicating some leakage of intracellular contents occurred. It is stated that CA has some antibacterial performance due to the polycationic CA can bind to bacterial cell wall with negatively charged causing the leakage of nucleic acids and proteins.33 Significant differences of the OD260 values after treatment with CSAP sponges were observed. The OD260 values increased with increasing the initial additive amount of ampicillin, suggesting that more and more nucleic acids leak into the bacterial suspension through the damaged bacterial membrane. The OD260 values of S. aureus, C.

albicans and E. coli suspensions for the control were 0.014, 0.034 and 0.015, respectively, and that for treatment of CA5 sponges increased to 0.42, 0.557 and 0.658, respectively. These significant changes could be due to the existence of damages of bacterial membrane that higher OD260 values suggested the fabricated sponges have higher cell membrane damage capacity. The result revealed that the fabricated CSAP sponges have great damaging effect on the cell membranes of S. aureus, C. albicans and E. coli.


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This is consistence with the bacterial growth curves results. A

B

C

D

E

F

Figure 4 Live/dead fluorescent staining images of (A, D) S.aureus, (B, E) C.

albicans, and (C, F) E. coli cells were treated without (A, B, C) and with CA5 sponges (D, E, F). Live/dead assay. The live/dead assay was carried out using a fluorescence microscope based on LIVE/DEAD BacLight Bacterial Viability Kit (L13152) containing propidium iodide (PI) and SYTO 9. The strains treated with CSAP sponges were stained with PI and SYTO 9 for 15 min under dark condition. The bacteria which stains fluorescent green illustrates it is live bacteria with intact cell membrane, while the bacteria staining fluorescent red shows it is dead bacteria with damaged membranes.34 Representative fluorescence microscopy images are displayed in Figure 4, together with the control comprising untreated live bacteria. As expected, untreated S. aureus cells (the control) are almost alive (green), only a few S. aureus cells are dead (red) (Figure 4A). Most of S. aureus cells treated with CA5 sponges were dead with



only a little live bacteria being seen from Figure 4D. This result means CA5 sponges can induce S. aureus cell death. It can also be found similar results for both C. albicans and E. coli. Therefore, it provides a direct evidence that CSAP sponges exhibited great antimicrobial activity against S. aureus,

C.albicans and E. coli, which is consistence with antibacterial growth curves. Cytotoxicity Assay in Vitro. Biomaterials with good biocompatibility are considered to be the primary requirement for wound dressing application. MTT assay was conducted in order to test the effect of CSAP sponges on the growth of HEK293 cells. The MTT result in term of cell viability is shown in Figure 5. It can be found that cell growth was not inhibited by CSAP sponge at all after incubation for 24 h with the cell viabilities more than 95%. This result demonstrates that the fabricated CSAP sponges had good biocompatibility since no cytotoxic effect was detected at all. Cells morphologies treated with CA and CA5 sponges for 24h were stained and studied using confocal microscopy. The fluorescent microscopy pictures of HEK293 cells are shown in Figure 5. No difference in the cell shape and morphology among the cells treated with the control and the fabricated CA and CA5 sponges was observed. Therefore, the results show that the CSAP sponges are non-toxic to cell morphology, making them promising candidates for wound dressing applications.


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Figure 5 Cell viability percentages treated with CSAP sponges for 24 h (top), cell morphologies treated with CA and CA5 sponges (bottom). Wound healing capacity in vivo. The wound healing capacity of the sponges were determined on the back of mice. Figure 6 displays the optical pictures of the wound healing processes after being treated with CA and CA5 sponges for 2, 4, 6, 8 and 10 days, respectively. The wounds without being treated with any dressings were used as control. The wound covered with CA sponge heals better than the control, although the wound healing ability of CA sponge is slower than CA5 sponge. The wound contraction for CA5 sponge had the advantage over CA and the control group that CA5 sponge exhibited the best wound healing capacity after 10 days. The percentages of wound area



closure at different healing time were calculated. As it is shown in Figure 6, the wound size tends to contract with the increase of the healing time. The CA5 sponge exhibited a good wound healing capacity with 25% wound closure ratio after 2 days treatment, while the wound healing closure is 15% and that of the control (bare wound) is only 10%. On day 6, the wound treated with CA5 sponge appears significant closure to 85%, whereas the wounds treated with CA and the control shows 60% and 45% wound closure, respectively. After 10 days treatment with CA5 sponge, the wound was healed completely without any scar that the wound contraction is 100%, which shows a highly improved wound healing ability. This indicates that CA5 sponge could be beneficial and effective for accelerating wound healing. Chitosan based biomaterials are reported to promote wound healing, because the depolymerization of chitosan gradually occurs to release N-acetylglucosamine, which is beneficial for the deposition of ordered collagen, initiating fibroblast proliferation and stimulating the production of hyaluronic acid at the wound site.35 This great wound healing capacity of CA5 sponge may be attributed to the excellent antibacterial performance, biocompatible and good swelling ability based on CA sponge.


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Figure 6 Optical photographs of wounds healing process for 10 days covered with CA and CA5 sponges (top) and wound area closure percentage of wounds (bottom). Histopathological study was carried out to further compare the wound healing efficiencies of the control, CA and CA5 sponges. Figure 7 presents the histological result of wound tissues of the control and treated with CA and CA5 sponges. There are still some inflammatory cells exhibiting on the wound site without any treatment after 10 days. While, the inflammatory cells were completely diminished at day 10 treated with CA and CA5 sponges. Interestingly, the group treated with CA5 sponge clearly regenerated more



blood vessels than that of CA sponge. These results indicate that the CA5 sponge can effectively enhance wound healing process. These results demonstrate that the CA5 sponge have excellent wound healing ability.

Figure 7 Images of H&E-stained histopathological sections after 10 days of treatment. Black arrows: inflammatory cell; white arrows: microvessels. CONCLUSION. In summary, novel chitosan based sponges were successfully fabricated via a green and facile method. There is no significant difference in porosity and swelling behaviors between CSAP and CA sponges. It was observed that the fabricated CSAP sponges showed excellent antimicrobial activity towards S. aureus, C. albicans, and E. coli with non-toxic activity to HEK293 cells. Most importantly, CSAP sponges exhibited accelerated wound healing capacity in vivo. Therefore, the great antibacterial and wound healing performances of the developed CSAP sponges, as well as their good swelling behaviors, make them competitive candidates to be applied as potent wound dressings. EXPERIMENTAL SECTION Preparation of CSAP sponges. (3-aminopropyl)triethoxysilane (APTES) ethanol solution was added into chitosan acetic acid solution to achieve concentrations of 0.8 wt% and 2 wt%, respectively. Then it was stirred at 150


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rpm at 25ºC for 24 h and dialysis for 3 days. 10 mg/mL ampicillin-MES solution was prepared and different volumes were added into the above mixture, followed by adding EDC and NHS, with finial concentrations of 5 mM/L and 12.5 mM/L, respectively. After reacting for 4 h, 0.08% genipin solution was added and stirred for 0.5 h. The mixture was centrifugated at 4000 rpm for 3 min and kept at 45°C for 5 h to form hydrogels. The sponges were obtained by being freeze-dried at -40°C for 36 h. The final weight ratio of ampicillin to chitosan is 1 wt%, 2 wt%, 3 wt%，4 wt% and 5 wt%, respectively (named as CA1, CA2, CA3, CA4 and CA5, respectively). The chitosan sponge was prepared by the same method except no ampicillin addition (named as CA). Characterization. The morphologies of cross-section of chitosan and CSAP sponges were examined by JSM-7600F SEM. Spectrum Two Spectrometer (Perkin Elmer, USA) was used to record FTIR spectra. XPS analysis was performed with Thermo Escalab 250Xi instrument (Thermo Fisher, USA). Antibacterial activity. The antibacterial activity of CSAP sponges was carried out by measuring bacterial growth kinetics towards S. aureus, C. albicans and

E. coli according to previous methods.36 Bacteria suspension with concentration of 1 × 106 CFU/mL treated with sterilized CSAP sponges was performed and the growth behaviors were monitored at fixed interval at 150 rpm at 37°C by measuring OD600 value. All experiments were run in triplicate. Leaching of CSAP sponges was evaluated using disk diffusion method. About 1×104 CFU/plate of test strains were loaded on the TSA. The sterilized



chitosan and CSAP sponges with 1cm diameter were placed on top of the lawns. They were incubated at 37°C for 24 h. The inhibitory effect was evaluated. Live/Dead Bacterial Cell. Bacterial live/dead fluorescent staining tests were carried out in order to confirm bacterial death behaviors. 1×106 to 1×107 CFU/mL S. aureus, C. albicans and E . coli suspensions were incubated with CA5 sponges at 150 rpm at 37°C for 2 h. Then the mixture was stained with SYTO9 and PI (LIVE/DEAD™ BacLight™ Bacterial Viability Kit, L13152) and pictured by a Fuorescence Microscopy (Olympus, IX53). Cytotoxicity tests. MTT assay was carried out on HEK293 cells according to previous methods.37 Chitosan and CSAP sponges were cut into a square of 5 mm×5 mm. The cells treated with chitosan and CSAP sponges were fixed and stained with DAPI and FITC-phalloidin. The cell morphologies were obtained by confocal microscopy (Leica DM2500, Germany). In vivo wound healing evaluation. The wound healing ability was evaluated on mice according to our previous study.36 The wounds were treated using chitosan and CA5 sponges and the bare wounds were applied as the control. The sponges were replaced every 2 days, and the wound area was imaged and measured. The sizes of all images were the same. The area of wound closure (ARC) is calculated: ARC (%) =(A0− A)/A0 × 100%

Eq. (1)

Where A0 is the created wound area, and A is the area of wound at the


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dressing changing day. After day 10, the skin wound tissue samples were excised and fixed with 10% formalin. Then the samples were stained with hematoxylin eosin (H&E) and observed using an optical microscope (Eclipse CI, Nikon). NOTES The authors declare no competing financial interest. SUPPORTING INFORMATION Pictures of CA and CA5 sponges; porosity; XPS survey scan; FTIR spectra; inhibition zones against S. aureus, C. albicans and E. coli; live/dead fluorescent staining images; degradation rate.

Acknowledgements The work was financially supported by the Natural Science Foundation of Jiangsu Province (BK20161528, BK20160756), Ordinary University Graduate Student Scientific Research Innovation Projects of Jiangsu Province (KYZZ16-0320).

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Table of Contents Synopsis Novel non-leaching chitosan sponges were fabricated via a green and facile method and have great potentials in wound dressings.


Green and Facile Preparation of Chitosan Sponges as Potential

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