- Email: [email protected]
Mechanically magnified chitosan-based hydrogel as tissue adhesive and antimicrobial candidate
International Journal of Biological Macromolecules 125 (2019) 109–115
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Mechanically magnified chitosan-based hydrogel as tissue adhesive and antimicrobial candidate Swati Sharma a, Rajesh Kumar a,⁎, Puja Kumari b, Ravindra Nath Kharwar b, Amarish Kumar Yadav c, Srikrishna Saripella c a b c
Organic Polymer Laboratory, Centre of Advanced Studies in Chemistry, Institute of Science, Banaras Hindu University, Varanasi 221005, UP, India Department of Botany, Institute of Science, Banaras Hindu University, Varanasi 221005, UP, India Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi 221005, UP, India
a r t i c l e
i n f o
Article history: Received 21 August 2018 Received in revised form 23 November 2018 Accepted 1 December 2018 Available online 3 December 2018 Keywords: Chitosan Mechanical strength Tissue adhesive Antimicrobial activity
a b s t r a c t The present article reports the development of chitosan (CS) based hydrogel series by varying the concentration of cross-linking agent i.e. N,N′-methylenebisacrylamide (MBA) (0.8–1.4 wt%) via free-radical polymerization in aqueous medium. SEM image analysis confirmed the presence of porous 3D-network in the hydrogel. Prepared hydrogel series exhibited good tissue adhesive property along with antimicrobial activity against E. coli, K. pneumonia, S. aureus, C. albicans & M. gypseum bacteria with the good MIC (4–20 mm). The adhesive strength of hydrogel was found 14 kPa, which seems to be quite efficient in tissue adhesiveness applications, which was also validated and tested on Drosophila (Oregon-R) tissues, results were promising. Magnified mechanical strength i.e. storage modulus (G′) and loss modulus (G″) were found 106 Pa and 104 Pa, respectively, which makes the hydrogel a potential candidate in the biomedical field. Moreover, CS hydrogel showed good swelling ratio in aqueous medium up to 390% at room temperature. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Hydrogels have a 3D network which is similar in structure with soft tissues and can be engineered to resemble an extracellular matrix, therefore, hydrogel has various applications in the biomedical field [1–4]. Generally, hydrogels have a tendency to absorb a large amount of water inside the hydrogel matrix due to which it has brittle in nature, and it's the biggest challenge to develop biomimetic hydrogels with efficient mechanical properties [5]. Numerous work had been done to develop tough hydrogels such as interpenetrating network [6], nanocomposite and double network hydrogels [7], but they generally consist of interpenetrated brittle [8] and ductile networks [9], that can effectively dissipate energy. A reported hydrogel showed lack of selfhealing ability of natural tissues after damage because they are unable to reform covalent bond [10]. To overcome this challenge the sacrificial covalent bonds were replaced in the brittle networks with easily reformable non-covalent bonds like ionic-bond in cross-linked alginate [11] and electrostatic cross-linked networks [12]. The incorporation of the non-covalent bond which has the ability to reform also enhances the self-healing ability in the hydrogels [13]. In addition, excellent cell affinity and tissue adhesiveness enable integration with surrounding tissue after implantation. However, it is challenging to integrate these ⁎ Corresponding author. E-mail address: [email protected] (R. Kumar).
https://doi.org/10.1016/j.ijbiomac.2018.12.018 0141-8130/© 2018 Elsevier B.V. All rights reserved.
characteristics into one hydrogel. Hydrogels require for tissue repair must be designed in such a manner where hydrogel should allow tissues and cells to attach for acceleration after implantation [14]. Most of the reported hydrogel have poor cell affinity, therefore, unable to show adhesive property [15–17]. Tough, hydrogel does not have an affinity to adhere to the tissue during surgical operation. Poly-dopamine (PDA) based hydrogel has excellent adhesiveness and cell affinity property along with the self-healable ability due to the presence of catechol groups [18–24]. Most of the reported PDA based hydrogel were produced via metal chelation, therefore, toxicity may be the concern for the biomedical application. In general, the adhesive property of the hydrogel is weakened due to the water uptake property of hydrogel because water molecules interact with the adhesive groups of the hydrogel resulting diminish adhere property [25]. The multipurpose and strong adhesion is more applicable to complicated interfaces like surgical operations, wound dressing and bioglue [25]. Tissue-adhesive materials are clinically used for local hemostasis in surgery, for stopping body fluids and air leaks that may be resistant to conventional suture or stapling techniques. In the past decades, numerous work has been reported regarding the tissue adhesive hydrogels which include collagen sheets with fibrin glues [26], fibrillar collagen [27], collagen with citric acid derivative [28], gelatin with resorcin and formalin (GRF glue™) [29], albumin with glutaraldehyde [30], cyanoacrylate [31], and synthetic polymers [32–35]. But the reported hydrogel is not suitable for surgical operations due to the presence of glutaraldehyde inside the
110
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
matrix because of its toxic nature. Hydrogels having tissue adhesive, self-healing property and the antimicrobial activity plays a significant role in the biomedical field. Hydrogel having antimicrobial activity reduces the risk of infection over the wound dressing, surgical operation and other biological applications. Recently, chitosan attracted attention towards its usage in the food industry as a preserving agent [36]. Bio-films based on chitosan combined with materials such as proteins, polysaccharides and antimicrobial peptides have been successfully probed at an experimental level on food products such as eggs, fruits, vegetables, dairy products and meat [37]. One of the effective biological applications of chitosan is found in the biomedical field for gene delivery because of its ability to interact with anionic DNA [38]. Researchers also developed chitosan coupled with AMP which enhances its solubility in water and facilitates it's binding with DNA, which ultimately enhances the delivery of DNA into the cancer cells [38]. Although, Chitosan (CS) is a naturally occurring stable, safe, bio-adhesive and pH-sensitive biopolymers, which is used in food and pharmaceutical industry [36,37]. It possesses a linear straight-chain, composed of randomly distributed β-(1 → 4)-linked Dglucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) [39]. Chitosan-based hydrogels were found to be a very potential candidate in the field of medicine because of its unique properties such as non-toxic, biocompatibility, biodegradability, stimuli-responsive etc. [37,39]. Chitosan-based hydrogel found to be very potent because of its optical properties as well as a biomedical application such as antimicrobial behaviour [40,41]. The hydrogels could provide the warm and moist micro-environments, its good biological properties could diminish the risk of bacterial infection, and therefore hydrogels are promising candidates used as soft tissue adhesives [42]. In view of the above shortcomings of above-discussed hydrogel and chitosan, the developed hydrogel series revealed a remarkable responsiveness in our study. Thus, in the present article, we explored the biomedical application such as tissue adhesiveness on tissues of flies of Drosophila and antimicrobial activity of CS-PAP hydrogel in a different environment. In the earlier work, we reported the CS-PAP hydrogel as drug carrier vehicle and also proved that hydrogel was not toxic and it was biocompatible but mechanical strength was not up to expectation [39]. An ideal hydrogel for biomedical application should have similar intrinsic properties of natural tissue, especially moderate mechanical strength and self-healing ability in order to increase the lifespan of the polymeric material. Therefore, herein we developed a series of CS-PAP hydrogel for good adhesiveness along with good antimicrobial activity against E. coli, K. pneumonia, S. aureus, C. albicans & M. gypseum. Further, the study reveals that the synthesized series of hydrogel shown very good viscoelastic properties/mechanical strength.
2. Experimental section
2.2. Instrumentation The shape and morphology of the hydrogel were analyzed by AFM (Make: NT-MDT Model: Solver NEXT) and HR-SEM (HRSEMSUPRA 40, ZEISS (Germany)), stereomicroscope (Magnus MSZBI). The rheology studies were carried out on Malvern Kinexus Pro Rheometer. The adhesive strength of the hydrogel with different concentration of crosslinking agent was measured on the Instron Materials Test system (MTS Criterion 43, MTS Criterion). 2.3. Preparation of tissue adhesive hydrogel A well-developed procedure [39] with some modification was chosen to prepare the hydrogel. We reported the synthesis of chitosan-based multi-responsive biocompatible hydrogel via free radical polymerization [FRP] using ammonium persulfate as initiator and N,N′-methylenebisacrylamide (1 wt%) as a cross-linking agent. In the present article, we developed a series of the CS-based hydrogel by varying the concentration of cross-linking agent i.e. N,N′methylenebisacrylamide from 0.8 to 1.4 wt% using ammonium persulfate as FRP initiator. Briefly, chitosan solution was prepared by dissolving CS (1.5 wt%) in aqueous acetic acid solution, the solution was stirred for 2 h for homogenization and then acryloyl phenylalanine [39] was added to the CS solution. Further, a known amount of ammonium persulfate (0.5 wt%) was added to the above solution and stirred the reaction mixture for an additional few minutes for homogenization. To move ahead, the N,N′-methylenebisacrylamide was added as a cross-linking agent in the reaction mixture and stirred the reaction mixture for 15 min. After 15 min a soft jelly type polymeric hydrogel material was obtained and left it for few hours in a vacuum oven for drying. By following the abovedescribed method, a series of hydrogel with different concentration of N,N′-methylenebisacrylamide was prepared (Scheme 1). The characterization data such as FTIR, NMR, XRD, were same as reported elsewhere [39]. 2.4. Water content measurement Since the developed polymeric material was a hydrogel, so its water uptake property was studied to understand its water holding and releasing behaviour. To investigate this, the hydrogels were vacuumdried to obtain a constant weight and then immersed in distilled water at 37 ± 0.1 °C. The polymeric material was swollen in water at different pH (3 to 10) and swollen gels were removed from the water at a determined interval of time and weighed until equilibrium was reached. The water content of the hydrogels was calculated using the Eq. (1) [37,39]. Swelling Ratio ð%Þ ¼
Ws −Wo 100 Wo
ð1Þ
2.1. Materials Chitosan (CS), the degree of deacetylation was determined as reported elsewhere [39] and it was found 85.8% and molecular weight was 96,950 Da. Phenylalanine and N,N′-methylenebisacrylamide (MBA) were purchased from Sigma-Aldrich Co. Ltd. (Gillingham, Dorset, SP8 4XT, UK.) Beef extract, casein hydrolysate, starch, Dextrose, peptone, cornmeal agar media, agar all were purchased from Himedia (Mumbai, India) and used without any purification. Drosophila (Oregon-R) stock was procured from Bloomington Drosophila Stock Centre, USA. Staphylococcus aureus (IMS/GN7), Escherichia coli (ATCC 25922), Salmonella typhi (MTCC 3216), Aeromonas hydrophila (IMS/GN11), Klebsiella pneumonia, Pseudomonas aeruginosa (clinical isolates), Shigella boydii (IMS/GN2), Microsporum gypseum, Trichophyton mentagrophytes and Candida albicans were obtained from Institute of Medical Sciences Banaras Hindu University, India.
where Ws: The weight of the swollen sample. Wo: The original weight of the sample. 2.5. Rheology experiment The viscoelastic property of CS-PAP hydrogel was 140 times better than the earlier report [39] of CS-PAP hydrogel with a fixed amount of cross-linking agent. In the present study, the effect of N,N′methylenebisacrylamide from 0.8 to 1.4 wt% concentration was studied on storage (G′) as well as loss modulus (G″) values by varying the different parameters like time sweep study, strain amplitude sweep, and frequency sweep and found up to 140 × 103 Pa as storage modulus. All the experiments were carried out at 37 ± 0.1 °C using a 40 mm
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
111
Scheme 1. Cross-linked CS based tissue adhesive, mechanically strong and soft polymeric hydrogel.
parallel plate with plate gap of 1.0 mm. The hydrogel was placed between the parallel plate and the platform with special care to avoid evaporation of water. The storage modulus (G′) and loss modulus (G″) were measured as a function of frequency (ω), time, and strain amplitude.
2.6. Tissue adhesiveness property To examine the adhesive property of CS-PAP hydrogel, adhesion strength test was carried out on Instron Materials Test system (MTS Criterion 43, MTS Criterion) by varying the concentration of cross-linking
Fig. 1. The swelling ratio of hydrogel with different concentration of MBA in distilled water at 37 °C (i), SEM images showing the porous behaviour of hydrogel and more compact network formation on increasing MBA concentration.
112
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
Fig. 2. Rheology analysis of hydrogel, (a) more stability of CS-based hydrogel series as G′ was found higher than G″, (b) mechanical strength of hydrogel was performed by strain amplitude sweep study, (c) frequency sweep study of CS-based hydrogel.
agent (0.8% to 1.4 wt%). Furthermore, the adhesive nature of CS-based hydrogel on the tissue was also investigated by applying a definite/ known amount of hydrogel on Drosophila flies. Drosophila flies were cultured on standard cornmeal agar media and the stock was maintained in BOD with alternative light-dark cycle at 24 ± 1 °C. In each group of the experiment, fifteen virgin female flies of the same age were taken and under the stereomicroscope, with the help of fine sterile surgical blade (Pro-care, No. 11) small cut/wound was made on the abdomen (ventral side) of each anaesthetized fly. The hydrogel was applied immediately over the wound area in the one set of the group.
Experimental group flies were kept in fresh food vials and monitored for their survival till thirty days and adhesive nature of CS-based hydrogel was analyzed. 2.7. Antimicrobial assay 2.7.1. Antibacterial activity The antibacterial activity of chitosan-based hydrogel was tested against six different human pathogenic bacteria named as Staphylococcus aureus (IMS/GN7), (Gram +ve) and Escherichia coli (ATCC 25922),
Fig. 3. Representing the adhesion property of CS-PAP hydrogel, wounded fly and hydrogel applied over the wounded fly. Plot represents percent survival of wounded flies and wounded flies with hydrogel (n = 15).
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
113
Fig. 4. MHA plates showing the antibacterial activity and inhibition zone against E. coli and S. aureus performed by the Well diffusion method. (1) - The synthesized amino acid-based hydrogel, (2) - chitosan, (3) - acryloyl phenylalanine and (4) - cross-linking agent.
Salmonella typhi (MTCC 3216), Aeromonas hydrophila (IMS/GN11), Klebsiella pneumonia (clinical isolates), Shigella boydii (IMS/GN2) and a clinical isolate Pseudomonas aeruginosa (all Gram −ve) taking the pure chitosan as control through well diffusion method on MHA (Mueller Hinton Agar) plates. The MHA plates for antibacterial assay were prepared by dissolving beef extract (2.0 g), casein hydrolysate (17.5 g), starch (1.5 g), agar (17.0 g) in 1000 mL of distilled water. Wells (5 mm) were prepared by borer on media plates and the plate was swabbed separately with different organisms to be tested. The MHA plates were then incubated for 18–24 h at 37 °C and the zone of inhibition was calculated. 2.7.2. Antifungal activity To investigate the antifungal activity of synthesized CS based hydrogel against different human pathogenic fungi viz. M. gypseum, T. mentagrophytes and C. albicans in SDA (Sabouraud Dextrose Agar) plates were screened via a well diffusion method. Sabouraud's Dextrose agar, SDA (dextrose 40 g; peptone 10 g; agar 15 g; distilled water 1 L) were used. Wells (5 mm) were prepared by borer on media plates and the plate was swabbed separately with different organisms to be tested. SDA plates were incubated for 2 to 3 days at 27 °C and the zone of inhibition was measured. 3. Results and discussion 3.1. Preparation and characterization of CS-based adhesive hydrogel A series of CS-based tissue adhesive hydrogel was successfully synthesized via free radical polymerization by varying the concentration of cross-linking agent from 0.8 to 1.4 wt%. Effect of concentration of cross-linking agent on the mechanical strength of hydrogel was carried out on rheometer. Morphology of the synthesized hydrogel was
examined by performing scanning electron microscopy. The prepared hydrogel was further explored as tissue adhesive as well as an antimicrobial agent. 3.2. Swelling ratio and morphology of hydrogel Swelling behaviour of the hydrogel series was carried out by immersing the hydrogels in distilled water having different pH (3 to 10) at 37 ± 0.1 °C (Fig. S1). As shown in Fig. 1, the equilibrium state of swelling was reached after 60 min and hydrogel showed different swelling degree with a different concentration of the cross-linking agent. As the concentration of N,N′-methylenebisacrylamide from 0.8 to 1.4 wt% has increased the rate of swelling was increased from 190 to 390% (Fig. 2i). The swelling property of the hydrogel was might be due to its porous network development in the hydrogel, on increasing the concentration of cross-linking agent from 0.8 to 1.4 wt%, leading towards the formation of more tougher hydrogel network and provided more space for water diffusion [36,39]. On increasing the cross-linker concentration, the matrix of the hydrogel become more compact and more strengthen which reflects its water absorbance capacity i.e. absorbs more amount of water and show the higher swelling rate at maximum cross-linker concentration. When the concentration of the cross-linking agent was 0.8 wt% only 190% swelling ratio was obtained while when the concentration of cross-linking agent was 1.4 wt%, its swelling ratio was 390%, which confirms the vital role of the concentration of the cross-linking agent in the compactness, strength, as well as toughness of the hydrogel. The porous behaviour of the hydrogel was further confirmed by analysis of images taken by a SEM microscope. The films were developed with varying concentration of N,N′-methylenebisacrylamide. The SEM images shown that hydrogel network becomes more and more compact on increasing the concentration of cross-linking agent [43], the porous structure becomes non-porous due to the formation of the
Table 1 Antibacterial activity test of CS-based hydrogel. S. no.
Sample
E. coli
K. pneumonia
S. aureus
++(12 mm) ++(13 mm) − −
++(17 mm) +(7 mm) ++(14 mm) +(4 mm)
P. aeruginosa
S. boydii
A. hydrophila
− − − −
− − − −
− − − −
Inhibition zone (mm) 1. 2. 3. 4.
CS-PAP AP CS Cross-linking agent
++(20 mm) +(10 mm) ++(11 mm) −
Note: Table representing the result of the antibacterial activity test on the basis of the inhibition zone. (+ denotes weak activity (less than or equal to 10 mm), ++ denotes strong activity (more than 11 mm), and − denotes no activity).
114
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
Fig. 5. SDA plates showing the antifungal activity and inhibition zone against C. albicans & M. gypseum performed by the Well diffusion method. (A)-Chitosan, (B)-amino acid-based hydrogel.
compact network (Fig. 1A–D). When the concentration of cross-linking agent was minimum i.e. 0.8 wt% hydrogel showed porous network. Hydrogel with a maximum concentration of crosslinking agent i.e. N,N′methylenebisacrylamide (1.4 wt%) shown more compact type network rather than porous hydrogel matrix but seems to be more strengthen network which was further confirmed by rheology mechanical experiment. 3.3. Rheology behaviour of CS-based adhesive hydrogel Regardless of its porous attributes, the synthesized CS based hydrogel series with different concentration of MBA exhibited tough mechanical properties. To investigate the effect of cross-linker with different ratios on the mechanical properties of CS-based hydrogels, dynamic rheological measurements were carried out. The viscoelastic properties were studied by rheological experiments on Malvern Kinexus Pro instrument. The storage modulus (G′) and loss modulus (G″) were indicated that the amount of energy stored and energy dissipated under the oscillatory stress, respectively. Synthesized soft polymeric material exhibited gel behaviour, which was revealed by the wide linear viscoelastic region in the dynamic frequency sweep study and confirmed from the storage modulus (G′) i.e. 72 × 103 Pa which was higher than loss modulus (G″) i.e. 48 × 103 Pa (Fig. 2) when cross-linker concentration was minimum (0.8 wt%). On increasing the concentration of crosslinking agent from 0.8 to 1.4 wt% (Fig. 2a), both the storage and loss modulus was increased up to 132 × 103 Pa and 85 × 103 Pa, respectively, and found G′ was 61% higher than G″, proved the gel behaviour of hydrogel [44,45]. Stability of the hydrogel series was checked by time sweep experiment at constant strain amplitude % i.e. 50%. Fig. 2b reveals the more stability of hydrogel as storage and loss modulus was found almost constant i.e. 138 × 103 Pa in the time range 20 to 400 s, when hydrogel had maximum cross-linker concentration (1.4%wt) storage modulus(G′) was 138 × 103 Pa which was found to be 49% higher than loss modulus (G″) i.e. 65 × 103 Pa. On increasing the concentration of MBA, same results were obtained i.e. storage modulus was found to be more than loss modulus in each case, shown that stability was
Table 2 Antifungal activity test of CS-based hydrogel against human pathogenic fungi. Sample name
C. albicans
M. gypseum
T. mentagrophytes
CS-PAP CS AP
+17 mm +15 mm −
14 mm 9 mm −
− − −
“+” represents the positive response of pathogen against the test. While “−” represents the negative response against the test.
increased on increasing the MBA concentration in hydrogel matrix [43]. The high mechanical strength of hydrogel was investigated by performing strain amplitude% study (Fig. 2b) from where it was found that CS-PAP hydrogel exhibited 850% strain value at storage modulus 140 × 103 Pa and loss modulus 80 × 103 Pa value, respectively. It was confirmed that CS based hydrogel exhibited more G′ & G″ than the previous report [39], therefore, it was observed that on increasing the concentration of cross-linker from 0.8 to 1.4 wt%, storage as well as loss modulus both were increased appreciably (Fig. 2c). Therefore, this study supports that hydrogel exhibited excellent mechanical properties. This enhances mechanical strength which was due to more crosslinking taken place between the polymeric hydrogel networks and resulted in the stiffer network on increasing concentration of crosslinking agent [44,45]. Improved mechanical strength in hydrogel was obtained by varying the concentration of MBA. 3.4. Adhesive property of hydrogel To know the strength of adhesion of the synthesized hydrogel series, the adhesion test was carried out on the Instron Materials Test system (MTS Criterion 43, MTS Criterion). From the observation, it was found that on increasing the concentration of cross-linking agent i.e. MBA from 0.8 to 1.4 wt%, the adhesion strength of hydrogel decreases (Fig. 3A), while pure chitosan solution did not show any adhesion strength, the reason behind it might be due to the formation of compact hydrogel network, which restricted the mobility of polymer chain in the hydrogel matrix. As a result, the viscosity of the hydrogel was increased and the polymer chain was not easily diffused into the surface on the tissue to form intimate contact. Thus, on increasing the concentration of cross-linking agent, decreases the adhesiveness of the hydrogel. Adhesiveness of hydrogel binding with tissues of fly has been visually recorded (Video 1) which supports the adhere property of hydrogel on tissues of the fly. Furthermore, hydrogels are known for their medical importance and have a wide range of applications including protective dressing, drugs carrier, contact lens production, tissue engineering etc. [37]. To investigate the tissue adhesive behaviour of the hydrogel, CS-PAP hydrogel was allowed to spread on wounded flies; and it was found that the hydrogel was easily spreadable over the wounded area, and it displayed a strong adhesion property with the Drosophila's tissues (Fig. 3B–E). Further, flies were monitored for their survival for thirty days after implementation of hydrogel over the injury and found that no-fly died even after 30 days which concludes that hydrogel reduces the risk of wound infection and also shown non-toxic nature of hydrogel. Adhesive nature of the hydrogel over the wound area encourages its application as protective covering agent. No lethality/ death
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
associated with hydrogel on applied flies, the same lifespan was observed as in control flies were observed, suggesting the non-toxic property of the hydrogel. Hence, the hydrogel has medical importance and can be used as a dressing material for protecting wound/cut area. 3.5. Antimicrobial assay 3.5.1. Antibacterial activity Introduction of the antibacterial activity in the biomedical polymers would lead towards reducing infection risk. CS, acryloyl-phenylalanine and CS-PAP hydrogel were tested against E. coli, K. pneumonia, S. aureus, P. aeruginosa, S. boydii, A. hydrophila bacteria by reported method [46–50] and were found active against broad spectrum bacteria mean both gram-positive and gram-negative bacteria. Among all the bacteria CS based hydrogel showed good activity against E. coli and S. aureus bacteria and the inhibition zone ranges were found from 10 mm to 20 mm (Fig. 4). Incorporation of the acryloyl phenylalanine into the chitosan backbone via free radical polymerization makes it more potent and increases its antibacterial activity (Table 1). 3.5.2. Antifungal activity To examine the antifungal activity of synthesized CS based hydrogel on human pathogenic fungi i.e. Microsporum gypseum, T. mentagrophytes and Candida albicans were taken. Fig. 5 reveals that hydrogel showed visible antifungal activity against C. albicans and M. gypseum but it did not show any activity against T. mentagrophytes. The minimum inhibition zone against Candida albicans and Microsporum gypseum were found to be in the range from 9 mm to 17 mm. Modified chitosan hydrogel was found more antifungal than pure chitosan hydrogel, because acryloyl-phenylalanine act as a key factor in the hydrogel which increases the antifungal activity of the synthesized hydrogel while bare chitosan did not show impressive antifungal activity (Table 2). 4. Conclusion We have developed a tissue adhesive, antimicrobial CS based porous hydrogel using various concentration of MBA as a cross-linking agent, and resulted in hydrogel shown enhanced mechanical strength and other biological applications such as tissue adhesive and antimicrobial activity. The robust mechanical strength can be attributed due to the formation of more interconnected 3D hydrogel network on increasing the concentration of cross-linker. The synthesized hydrogel was explored in the biomedical field in which it showed good tissue adhesive property, antibacterial and antifungal activity as well. Therefore, on the basis of the above findings, we conclude that synthesized hydrogel can be used as a promising candidate in the biomedical field. Thus, hydrogel prepared by easy, eco-friendly methods provides numerous opportunities in a biomedical application such as wound dressing and tissue adhesiveness. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2018.12.018. Acknowledgement The authors would like to acknowledge funding from Banaras Hindu University, India under the Scheme DST-PURSE (5050) and UGC-UPE (4204), authors are thankful to Ms. Zurryat Fatima, Aimil Ltd., New Delhi for rheology related studies using Malvern Kinexus Pro Rheometer.
115
References [1] D. Seliktar, Science 336 (2012) 1124–1128. [2] R. Censi, P. Di Martino, T. Vermonden, Hennink, J. Control. Release 161 (2012) 680–692. [3] K.Y. Lee, D.J. Mooney, Chem. Rev. 101 (2001) 1869–1880. [4] A.S. Hoffman, Adv. Drug Deliv. Rev. 54 (2002) 3–12. [5] P. Calvert, Adv. Mater. 21 (2009) 743–756. [6] X. Zhao, Soft Matter 10 (2014) 672–687. [7] J.P. Gong, Soft Matter 6 (2010) 2583–2590. [8] M.A. Haque, T. Kurokawa, J.P. Gong, Polymer 53 (2012) 1805–1822. [9] K. Haraguchi, Polym. J. 43 (2011) 223–241. [10] J.P. Gong, Science 344 (2014) 161–162. [11] J.Y. Sun, X. Zhao, W.R. Illeperuma, O. Chaudhuri, K.H. Oh, D.J. Mooney, J.J. Vlassak, Z. Suo, Nature 489 (2012) 133–136. [12] T.L. Sun, T. Kurokawa, S. Kuroda, A.B. Ihsan, T. Akasaki, K. Sato, M.A. Haque, T. Nakajima, J.P. Gong, Nat. Mater. 12 (2013) 932–937. [13] Y. Yang, M.W. Urban, Chem. Soc. Rev. 42 (2013) 7446–7467. [14] Y. Zhao, T. Nakajima, J.J. Yang, T. Kurokawa, J. Liu, J. Lu, S. Mizumoto, K. Sugahara, N. Kitamura, K. Yasuda, A.U. Daniels, J.P. Gong, Adv. Mater. 26 (2014) 436–442. [15] Y.M. Chen, R. Ogawa, A. Kakugo, Y. Osada, J.P. Gong, Soft Matter 5 (2009) 1804. [16] F. Brandl, F. Sommer, A. Goepferich, Biomaterials 28 (2007) 134–146. [17] J.L. Drury, D.J. Mooney, Biomaterials 24 (2003) 4337–4351. [18] M. Krogsgaard, M.A. Behrens, J.S. Pedersen, H. Birkedal, Biomacromolecules 14 (2013) 297–301. [19] S. Sharma, A. Kumar Deepak, S. Afgan, R. Kumar, ChemistrySelect 2 (2017) 11281–11287. [20] C.E. Brubaker, H. Kissler, L.J. Wang, D.B. Kaufman, P.B. Messersmith, Biomaterials 31 (2010) 420–427. [21] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Science 318 (2007) 426–430. [22] H. Lee, B.P. Lee, P.B. Messersmith, Nature 448 (2007) 338–341. [23] B.K. Ahn, D.W. Lee, J.N. Israelachvili, J.H. Waite, Nat. Mater. 13 (2014) 867–872. [24] N. Holten-Andersen, M.J. Harrington, H. Birkedal, B.P. Lee, P.B. Messersmith, K.Y. Lee, J.H. Waite, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 2651–2655. [25] G. Sudre, L. Olanier, Y. Tran, D. Hourdet, C. Creton, Soft Matter 8 (2012) 8184–8193. [26] N. Annabi, S.M. Mithieux, G. Camci-Unal, M.R. Dokmeci, A.S. Weiss, A. Khademhosseini, Biochem. Eng. J. 77 (2013) 110. [27] J. Fang, A. Mehlich, N. Koga, J.Q. Huang, R. Koga, X.Y. Gao, C.G. Hu, C. Jin, M. Rief, J. Kast, D. Baker, H.B. Li, Nat. Commun. 4 (2013) 2974. [28] M.J. Glassman, B.D. Olsen, Soft Matter 9 (2013) 6814. [29] A.E.X. Brown, R.I. Litvinov, D.E. Discher, P.K. Purohit, J.W. Weisel, Science 325 (2009) 741. [30] K. Nagapudi, W.T. Brinkman, J. Leisen, B.S. Thomas, E.R. Wright, C. Haller, X. Wu, R.P. Apkarian, V.P. Conticello, E.L. Chaikof, Macromolecules 38 (2005) 345. [31] M.D. Golinska, M.K. Wlodarczyk-Biegun, M.W.T. Werten, M.A.C. Stuart, F.A. de Wolf, R. de Vries, Biomacromolecules 15 (2014) 699. [32] B.J. Kim, D.X. Oh, S. Kim, J.H. Seo, D.S. Hwang, A. Masic, D.K. Han, H.J. Cha, Biomacromolecules 15 (2014) 1579. [33] W.A. Petka, J.L. Harden, K.P. McGrath, D. Wirtz, D.A. Tirrell, Science 281 (1998) 389. [34] S. Lv, D.M. Dudek, Y. Cao, M.M. Balamurali, J. Gosline, H.B. Li, Nature 465 (2010) 69. [35] H.B. Li, W.A. Linke, A.F. Oberhauser, M. Carrion-Vazquez, J.G. Kerkviliet, H. Lu, P.E. Marszalek, J.M. Fernandez, Nature 418 (2002) 998. [36] F. Shamekhi, E. Tamjid, K. Khajeh, Int. J. Biol. Macromol. 120 (2018) 460. [37] S. Kumar, V. Deepak, M. Kumari, P.K. Dutta, Int. J. Biol. Macromol. 84 (2016) 349–353. [38] S. Kumar, P. Garg, S. Pandey, M. Kumari, S. Hoon, K.J. Jang, R. Kapavarapu, P.H. Choung, A.J.F.N. Sobrala, J.H. Chung, J. Mater. Chem. B 3 (2015) 3465. [39] S. Sharma, A. Deepak, R. Kumar, N.K. Rana, B. Koch, Int. J. Biol. Macromol. 116 (2018) 37–44. [40] S. Kumar, J. Dutta, P.K. Dutta, Int. J. Biol. Macromol. 45 (2009) 330. [41] S. Kumar, J. Koh, Int. J. Biol. Macromol. 51 (2012) 1167. [42] T. Wu, J. Huang, Y. Jiang, Y. Hu, X. Ye, D. Liu, J. Chen, Food Chem. 240 (2018) 346–369. [43] X. Jing, H.Y. Mi, Y.J. Lin, E. Enriquez, X.F. Peng, L.S. Turng, ACS Appl. Mater. Interfaces 10 (2018) 20897–22090. [44] R. Dong, X. Zhao, B. Guo, P.X. Ma, ACS Appl. Mater. Interfaces 8 (2016) (171381715). [45] A.R. Karimi, A. Khodadadi, ACS Appl. Mater. Interfaces 8 (2016) 27254–27263. [46] L. Yuan, W. Fan, L. Han, C. Guo, Z. Yan, M. Zhu, X. Mo, Front. Mater. Sci. 12 (1) (2018) 95. [47] D.K. Singh, V.K. Sharma, J. Kumar, A. Mishra, A. Singh, P. Kumari, S.K. Verma, R.N. Kharwar, Nanomedicine 13 (2) (2018) 191–207. [48] F. Wahid, Y.N. Zhou, H.S. Wang, T. Wan, C. Zhong, L.Q. Chu, Int. J. Biol. Macromol. 114 (2018) 1233–1239. [49] M. Zhai, Y. Xu, B. Zhou, W. Jing, J. Photochem. Photobiol. B Biol. 180 (2018) 253–258. [50] A. Moeini, A. Cimmino, et al., Carbohydr. Polym. 195 (2018) 631–641.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Mechanically magnified chitosan-based hydrogel as tissue adhesive and antimicrobial candidate Swati Sharma a, Rajesh Kumar a,⁎, Puja Kumari b, Ravindra Nath Kharwar b, Amarish Kumar Yadav c, Srikrishna Saripella c a b c
Organic Polymer Laboratory, Centre of Advanced Studies in Chemistry, Institute of Science, Banaras Hindu University, Varanasi 221005, UP, India Department of Botany, Institute of Science, Banaras Hindu University, Varanasi 221005, UP, India Department of Biochemistry, Institute of Science, Banaras Hindu University, Varanasi 221005, UP, India
a r t i c l e
i n f o
Article history: Received 21 August 2018 Received in revised form 23 November 2018 Accepted 1 December 2018 Available online 3 December 2018 Keywords: Chitosan Mechanical strength Tissue adhesive Antimicrobial activity
a b s t r a c t The present article reports the development of chitosan (CS) based hydrogel series by varying the concentration of cross-linking agent i.e. N,N′-methylenebisacrylamide (MBA) (0.8–1.4 wt%) via free-radical polymerization in aqueous medium. SEM image analysis confirmed the presence of porous 3D-network in the hydrogel. Prepared hydrogel series exhibited good tissue adhesive property along with antimicrobial activity against E. coli, K. pneumonia, S. aureus, C. albicans & M. gypseum bacteria with the good MIC (4–20 mm). The adhesive strength of hydrogel was found 14 kPa, which seems to be quite efficient in tissue adhesiveness applications, which was also validated and tested on Drosophila (Oregon-R) tissues, results were promising. Magnified mechanical strength i.e. storage modulus (G′) and loss modulus (G″) were found 106 Pa and 104 Pa, respectively, which makes the hydrogel a potential candidate in the biomedical field. Moreover, CS hydrogel showed good swelling ratio in aqueous medium up to 390% at room temperature. © 2018 Elsevier B.V. All rights reserved.
1. Introduction Hydrogels have a 3D network which is similar in structure with soft tissues and can be engineered to resemble an extracellular matrix, therefore, hydrogel has various applications in the biomedical field [1–4]. Generally, hydrogels have a tendency to absorb a large amount of water inside the hydrogel matrix due to which it has brittle in nature, and it's the biggest challenge to develop biomimetic hydrogels with efficient mechanical properties [5]. Numerous work had been done to develop tough hydrogels such as interpenetrating network [6], nanocomposite and double network hydrogels [7], but they generally consist of interpenetrated brittle [8] and ductile networks [9], that can effectively dissipate energy. A reported hydrogel showed lack of selfhealing ability of natural tissues after damage because they are unable to reform covalent bond [10]. To overcome this challenge the sacrificial covalent bonds were replaced in the brittle networks with easily reformable non-covalent bonds like ionic-bond in cross-linked alginate [11] and electrostatic cross-linked networks [12]. The incorporation of the non-covalent bond which has the ability to reform also enhances the self-healing ability in the hydrogels [13]. In addition, excellent cell affinity and tissue adhesiveness enable integration with surrounding tissue after implantation. However, it is challenging to integrate these ⁎ Corresponding author. E-mail address: [email protected] (R. Kumar).
https://doi.org/10.1016/j.ijbiomac.2018.12.018 0141-8130/© 2018 Elsevier B.V. All rights reserved.
characteristics into one hydrogel. Hydrogels require for tissue repair must be designed in such a manner where hydrogel should allow tissues and cells to attach for acceleration after implantation [14]. Most of the reported hydrogel have poor cell affinity, therefore, unable to show adhesive property [15–17]. Tough, hydrogel does not have an affinity to adhere to the tissue during surgical operation. Poly-dopamine (PDA) based hydrogel has excellent adhesiveness and cell affinity property along with the self-healable ability due to the presence of catechol groups [18–24]. Most of the reported PDA based hydrogel were produced via metal chelation, therefore, toxicity may be the concern for the biomedical application. In general, the adhesive property of the hydrogel is weakened due to the water uptake property of hydrogel because water molecules interact with the adhesive groups of the hydrogel resulting diminish adhere property [25]. The multipurpose and strong adhesion is more applicable to complicated interfaces like surgical operations, wound dressing and bioglue [25]. Tissue-adhesive materials are clinically used for local hemostasis in surgery, for stopping body fluids and air leaks that may be resistant to conventional suture or stapling techniques. In the past decades, numerous work has been reported regarding the tissue adhesive hydrogels which include collagen sheets with fibrin glues [26], fibrillar collagen [27], collagen with citric acid derivative [28], gelatin with resorcin and formalin (GRF glue™) [29], albumin with glutaraldehyde [30], cyanoacrylate [31], and synthetic polymers [32–35]. But the reported hydrogel is not suitable for surgical operations due to the presence of glutaraldehyde inside the
110
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
matrix because of its toxic nature. Hydrogels having tissue adhesive, self-healing property and the antimicrobial activity plays a significant role in the biomedical field. Hydrogel having antimicrobial activity reduces the risk of infection over the wound dressing, surgical operation and other biological applications. Recently, chitosan attracted attention towards its usage in the food industry as a preserving agent [36]. Bio-films based on chitosan combined with materials such as proteins, polysaccharides and antimicrobial peptides have been successfully probed at an experimental level on food products such as eggs, fruits, vegetables, dairy products and meat [37]. One of the effective biological applications of chitosan is found in the biomedical field for gene delivery because of its ability to interact with anionic DNA [38]. Researchers also developed chitosan coupled with AMP which enhances its solubility in water and facilitates it's binding with DNA, which ultimately enhances the delivery of DNA into the cancer cells [38]. Although, Chitosan (CS) is a naturally occurring stable, safe, bio-adhesive and pH-sensitive biopolymers, which is used in food and pharmaceutical industry [36,37]. It possesses a linear straight-chain, composed of randomly distributed β-(1 → 4)-linked Dglucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit) [39]. Chitosan-based hydrogels were found to be a very potential candidate in the field of medicine because of its unique properties such as non-toxic, biocompatibility, biodegradability, stimuli-responsive etc. [37,39]. Chitosan-based hydrogel found to be very potent because of its optical properties as well as a biomedical application such as antimicrobial behaviour [40,41]. The hydrogels could provide the warm and moist micro-environments, its good biological properties could diminish the risk of bacterial infection, and therefore hydrogels are promising candidates used as soft tissue adhesives [42]. In view of the above shortcomings of above-discussed hydrogel and chitosan, the developed hydrogel series revealed a remarkable responsiveness in our study. Thus, in the present article, we explored the biomedical application such as tissue adhesiveness on tissues of flies of Drosophila and antimicrobial activity of CS-PAP hydrogel in a different environment. In the earlier work, we reported the CS-PAP hydrogel as drug carrier vehicle and also proved that hydrogel was not toxic and it was biocompatible but mechanical strength was not up to expectation [39]. An ideal hydrogel for biomedical application should have similar intrinsic properties of natural tissue, especially moderate mechanical strength and self-healing ability in order to increase the lifespan of the polymeric material. Therefore, herein we developed a series of CS-PAP hydrogel for good adhesiveness along with good antimicrobial activity against E. coli, K. pneumonia, S. aureus, C. albicans & M. gypseum. Further, the study reveals that the synthesized series of hydrogel shown very good viscoelastic properties/mechanical strength.
2. Experimental section
2.2. Instrumentation The shape and morphology of the hydrogel were analyzed by AFM (Make: NT-MDT Model: Solver NEXT) and HR-SEM (HRSEMSUPRA 40, ZEISS (Germany)), stereomicroscope (Magnus MSZBI). The rheology studies were carried out on Malvern Kinexus Pro Rheometer. The adhesive strength of the hydrogel with different concentration of crosslinking agent was measured on the Instron Materials Test system (MTS Criterion 43, MTS Criterion). 2.3. Preparation of tissue adhesive hydrogel A well-developed procedure [39] with some modification was chosen to prepare the hydrogel. We reported the synthesis of chitosan-based multi-responsive biocompatible hydrogel via free radical polymerization [FRP] using ammonium persulfate as initiator and N,N′-methylenebisacrylamide (1 wt%) as a cross-linking agent. In the present article, we developed a series of the CS-based hydrogel by varying the concentration of cross-linking agent i.e. N,N′methylenebisacrylamide from 0.8 to 1.4 wt% using ammonium persulfate as FRP initiator. Briefly, chitosan solution was prepared by dissolving CS (1.5 wt%) in aqueous acetic acid solution, the solution was stirred for 2 h for homogenization and then acryloyl phenylalanine [39] was added to the CS solution. Further, a known amount of ammonium persulfate (0.5 wt%) was added to the above solution and stirred the reaction mixture for an additional few minutes for homogenization. To move ahead, the N,N′-methylenebisacrylamide was added as a cross-linking agent in the reaction mixture and stirred the reaction mixture for 15 min. After 15 min a soft jelly type polymeric hydrogel material was obtained and left it for few hours in a vacuum oven for drying. By following the abovedescribed method, a series of hydrogel with different concentration of N,N′-methylenebisacrylamide was prepared (Scheme 1). The characterization data such as FTIR, NMR, XRD, were same as reported elsewhere [39]. 2.4. Water content measurement Since the developed polymeric material was a hydrogel, so its water uptake property was studied to understand its water holding and releasing behaviour. To investigate this, the hydrogels were vacuumdried to obtain a constant weight and then immersed in distilled water at 37 ± 0.1 °C. The polymeric material was swollen in water at different pH (3 to 10) and swollen gels were removed from the water at a determined interval of time and weighed until equilibrium was reached. The water content of the hydrogels was calculated using the Eq. (1) [37,39]. Swelling Ratio ð%Þ ¼
Ws −Wo 100 Wo
ð1Þ
2.1. Materials Chitosan (CS), the degree of deacetylation was determined as reported elsewhere [39] and it was found 85.8% and molecular weight was 96,950 Da. Phenylalanine and N,N′-methylenebisacrylamide (MBA) were purchased from Sigma-Aldrich Co. Ltd. (Gillingham, Dorset, SP8 4XT, UK.) Beef extract, casein hydrolysate, starch, Dextrose, peptone, cornmeal agar media, agar all were purchased from Himedia (Mumbai, India) and used without any purification. Drosophila (Oregon-R) stock was procured from Bloomington Drosophila Stock Centre, USA. Staphylococcus aureus (IMS/GN7), Escherichia coli (ATCC 25922), Salmonella typhi (MTCC 3216), Aeromonas hydrophila (IMS/GN11), Klebsiella pneumonia, Pseudomonas aeruginosa (clinical isolates), Shigella boydii (IMS/GN2), Microsporum gypseum, Trichophyton mentagrophytes and Candida albicans were obtained from Institute of Medical Sciences Banaras Hindu University, India.
where Ws: The weight of the swollen sample. Wo: The original weight of the sample. 2.5. Rheology experiment The viscoelastic property of CS-PAP hydrogel was 140 times better than the earlier report [39] of CS-PAP hydrogel with a fixed amount of cross-linking agent. In the present study, the effect of N,N′methylenebisacrylamide from 0.8 to 1.4 wt% concentration was studied on storage (G′) as well as loss modulus (G″) values by varying the different parameters like time sweep study, strain amplitude sweep, and frequency sweep and found up to 140 × 103 Pa as storage modulus. All the experiments were carried out at 37 ± 0.1 °C using a 40 mm
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
111
Scheme 1. Cross-linked CS based tissue adhesive, mechanically strong and soft polymeric hydrogel.
parallel plate with plate gap of 1.0 mm. The hydrogel was placed between the parallel plate and the platform with special care to avoid evaporation of water. The storage modulus (G′) and loss modulus (G″) were measured as a function of frequency (ω), time, and strain amplitude.
2.6. Tissue adhesiveness property To examine the adhesive property of CS-PAP hydrogel, adhesion strength test was carried out on Instron Materials Test system (MTS Criterion 43, MTS Criterion) by varying the concentration of cross-linking
Fig. 1. The swelling ratio of hydrogel with different concentration of MBA in distilled water at 37 °C (i), SEM images showing the porous behaviour of hydrogel and more compact network formation on increasing MBA concentration.
112
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
Fig. 2. Rheology analysis of hydrogel, (a) more stability of CS-based hydrogel series as G′ was found higher than G″, (b) mechanical strength of hydrogel was performed by strain amplitude sweep study, (c) frequency sweep study of CS-based hydrogel.
agent (0.8% to 1.4 wt%). Furthermore, the adhesive nature of CS-based hydrogel on the tissue was also investigated by applying a definite/ known amount of hydrogel on Drosophila flies. Drosophila flies were cultured on standard cornmeal agar media and the stock was maintained in BOD with alternative light-dark cycle at 24 ± 1 °C. In each group of the experiment, fifteen virgin female flies of the same age were taken and under the stereomicroscope, with the help of fine sterile surgical blade (Pro-care, No. 11) small cut/wound was made on the abdomen (ventral side) of each anaesthetized fly. The hydrogel was applied immediately over the wound area in the one set of the group.
Experimental group flies were kept in fresh food vials and monitored for their survival till thirty days and adhesive nature of CS-based hydrogel was analyzed. 2.7. Antimicrobial assay 2.7.1. Antibacterial activity The antibacterial activity of chitosan-based hydrogel was tested against six different human pathogenic bacteria named as Staphylococcus aureus (IMS/GN7), (Gram +ve) and Escherichia coli (ATCC 25922),
Fig. 3. Representing the adhesion property of CS-PAP hydrogel, wounded fly and hydrogel applied over the wounded fly. Plot represents percent survival of wounded flies and wounded flies with hydrogel (n = 15).
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
113
Fig. 4. MHA plates showing the antibacterial activity and inhibition zone against E. coli and S. aureus performed by the Well diffusion method. (1) - The synthesized amino acid-based hydrogel, (2) - chitosan, (3) - acryloyl phenylalanine and (4) - cross-linking agent.
Salmonella typhi (MTCC 3216), Aeromonas hydrophila (IMS/GN11), Klebsiella pneumonia (clinical isolates), Shigella boydii (IMS/GN2) and a clinical isolate Pseudomonas aeruginosa (all Gram −ve) taking the pure chitosan as control through well diffusion method on MHA (Mueller Hinton Agar) plates. The MHA plates for antibacterial assay were prepared by dissolving beef extract (2.0 g), casein hydrolysate (17.5 g), starch (1.5 g), agar (17.0 g) in 1000 mL of distilled water. Wells (5 mm) were prepared by borer on media plates and the plate was swabbed separately with different organisms to be tested. The MHA plates were then incubated for 18–24 h at 37 °C and the zone of inhibition was calculated. 2.7.2. Antifungal activity To investigate the antifungal activity of synthesized CS based hydrogel against different human pathogenic fungi viz. M. gypseum, T. mentagrophytes and C. albicans in SDA (Sabouraud Dextrose Agar) plates were screened via a well diffusion method. Sabouraud's Dextrose agar, SDA (dextrose 40 g; peptone 10 g; agar 15 g; distilled water 1 L) were used. Wells (5 mm) were prepared by borer on media plates and the plate was swabbed separately with different organisms to be tested. SDA plates were incubated for 2 to 3 days at 27 °C and the zone of inhibition was measured. 3. Results and discussion 3.1. Preparation and characterization of CS-based adhesive hydrogel A series of CS-based tissue adhesive hydrogel was successfully synthesized via free radical polymerization by varying the concentration of cross-linking agent from 0.8 to 1.4 wt%. Effect of concentration of cross-linking agent on the mechanical strength of hydrogel was carried out on rheometer. Morphology of the synthesized hydrogel was
examined by performing scanning electron microscopy. The prepared hydrogel was further explored as tissue adhesive as well as an antimicrobial agent. 3.2. Swelling ratio and morphology of hydrogel Swelling behaviour of the hydrogel series was carried out by immersing the hydrogels in distilled water having different pH (3 to 10) at 37 ± 0.1 °C (Fig. S1). As shown in Fig. 1, the equilibrium state of swelling was reached after 60 min and hydrogel showed different swelling degree with a different concentration of the cross-linking agent. As the concentration of N,N′-methylenebisacrylamide from 0.8 to 1.4 wt% has increased the rate of swelling was increased from 190 to 390% (Fig. 2i). The swelling property of the hydrogel was might be due to its porous network development in the hydrogel, on increasing the concentration of cross-linking agent from 0.8 to 1.4 wt%, leading towards the formation of more tougher hydrogel network and provided more space for water diffusion [36,39]. On increasing the cross-linker concentration, the matrix of the hydrogel become more compact and more strengthen which reflects its water absorbance capacity i.e. absorbs more amount of water and show the higher swelling rate at maximum cross-linker concentration. When the concentration of the cross-linking agent was 0.8 wt% only 190% swelling ratio was obtained while when the concentration of cross-linking agent was 1.4 wt%, its swelling ratio was 390%, which confirms the vital role of the concentration of the cross-linking agent in the compactness, strength, as well as toughness of the hydrogel. The porous behaviour of the hydrogel was further confirmed by analysis of images taken by a SEM microscope. The films were developed with varying concentration of N,N′-methylenebisacrylamide. The SEM images shown that hydrogel network becomes more and more compact on increasing the concentration of cross-linking agent [43], the porous structure becomes non-porous due to the formation of the
Table 1 Antibacterial activity test of CS-based hydrogel. S. no.
Sample
E. coli
K. pneumonia
S. aureus
++(12 mm) ++(13 mm) − −
++(17 mm) +(7 mm) ++(14 mm) +(4 mm)
P. aeruginosa
S. boydii
A. hydrophila
− − − −
− − − −
− − − −
Inhibition zone (mm) 1. 2. 3. 4.
CS-PAP AP CS Cross-linking agent
++(20 mm) +(10 mm) ++(11 mm) −
Note: Table representing the result of the antibacterial activity test on the basis of the inhibition zone. (+ denotes weak activity (less than or equal to 10 mm), ++ denotes strong activity (more than 11 mm), and − denotes no activity).
114
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
Fig. 5. SDA plates showing the antifungal activity and inhibition zone against C. albicans & M. gypseum performed by the Well diffusion method. (A)-Chitosan, (B)-amino acid-based hydrogel.
compact network (Fig. 1A–D). When the concentration of cross-linking agent was minimum i.e. 0.8 wt% hydrogel showed porous network. Hydrogel with a maximum concentration of crosslinking agent i.e. N,N′methylenebisacrylamide (1.4 wt%) shown more compact type network rather than porous hydrogel matrix but seems to be more strengthen network which was further confirmed by rheology mechanical experiment. 3.3. Rheology behaviour of CS-based adhesive hydrogel Regardless of its porous attributes, the synthesized CS based hydrogel series with different concentration of MBA exhibited tough mechanical properties. To investigate the effect of cross-linker with different ratios on the mechanical properties of CS-based hydrogels, dynamic rheological measurements were carried out. The viscoelastic properties were studied by rheological experiments on Malvern Kinexus Pro instrument. The storage modulus (G′) and loss modulus (G″) were indicated that the amount of energy stored and energy dissipated under the oscillatory stress, respectively. Synthesized soft polymeric material exhibited gel behaviour, which was revealed by the wide linear viscoelastic region in the dynamic frequency sweep study and confirmed from the storage modulus (G′) i.e. 72 × 103 Pa which was higher than loss modulus (G″) i.e. 48 × 103 Pa (Fig. 2) when cross-linker concentration was minimum (0.8 wt%). On increasing the concentration of crosslinking agent from 0.8 to 1.4 wt% (Fig. 2a), both the storage and loss modulus was increased up to 132 × 103 Pa and 85 × 103 Pa, respectively, and found G′ was 61% higher than G″, proved the gel behaviour of hydrogel [44,45]. Stability of the hydrogel series was checked by time sweep experiment at constant strain amplitude % i.e. 50%. Fig. 2b reveals the more stability of hydrogel as storage and loss modulus was found almost constant i.e. 138 × 103 Pa in the time range 20 to 400 s, when hydrogel had maximum cross-linker concentration (1.4%wt) storage modulus(G′) was 138 × 103 Pa which was found to be 49% higher than loss modulus (G″) i.e. 65 × 103 Pa. On increasing the concentration of MBA, same results were obtained i.e. storage modulus was found to be more than loss modulus in each case, shown that stability was
Table 2 Antifungal activity test of CS-based hydrogel against human pathogenic fungi. Sample name
C. albicans
M. gypseum
T. mentagrophytes
CS-PAP CS AP
+17 mm +15 mm −
14 mm 9 mm −
− − −
“+” represents the positive response of pathogen against the test. While “−” represents the negative response against the test.
increased on increasing the MBA concentration in hydrogel matrix [43]. The high mechanical strength of hydrogel was investigated by performing strain amplitude% study (Fig. 2b) from where it was found that CS-PAP hydrogel exhibited 850% strain value at storage modulus 140 × 103 Pa and loss modulus 80 × 103 Pa value, respectively. It was confirmed that CS based hydrogel exhibited more G′ & G″ than the previous report [39], therefore, it was observed that on increasing the concentration of cross-linker from 0.8 to 1.4 wt%, storage as well as loss modulus both were increased appreciably (Fig. 2c). Therefore, this study supports that hydrogel exhibited excellent mechanical properties. This enhances mechanical strength which was due to more crosslinking taken place between the polymeric hydrogel networks and resulted in the stiffer network on increasing concentration of crosslinking agent [44,45]. Improved mechanical strength in hydrogel was obtained by varying the concentration of MBA. 3.4. Adhesive property of hydrogel To know the strength of adhesion of the synthesized hydrogel series, the adhesion test was carried out on the Instron Materials Test system (MTS Criterion 43, MTS Criterion). From the observation, it was found that on increasing the concentration of cross-linking agent i.e. MBA from 0.8 to 1.4 wt%, the adhesion strength of hydrogel decreases (Fig. 3A), while pure chitosan solution did not show any adhesion strength, the reason behind it might be due to the formation of compact hydrogel network, which restricted the mobility of polymer chain in the hydrogel matrix. As a result, the viscosity of the hydrogel was increased and the polymer chain was not easily diffused into the surface on the tissue to form intimate contact. Thus, on increasing the concentration of cross-linking agent, decreases the adhesiveness of the hydrogel. Adhesiveness of hydrogel binding with tissues of fly has been visually recorded (Video 1) which supports the adhere property of hydrogel on tissues of the fly. Furthermore, hydrogels are known for their medical importance and have a wide range of applications including protective dressing, drugs carrier, contact lens production, tissue engineering etc. [37]. To investigate the tissue adhesive behaviour of the hydrogel, CS-PAP hydrogel was allowed to spread on wounded flies; and it was found that the hydrogel was easily spreadable over the wounded area, and it displayed a strong adhesion property with the Drosophila's tissues (Fig. 3B–E). Further, flies were monitored for their survival for thirty days after implementation of hydrogel over the injury and found that no-fly died even after 30 days which concludes that hydrogel reduces the risk of wound infection and also shown non-toxic nature of hydrogel. Adhesive nature of the hydrogel over the wound area encourages its application as protective covering agent. No lethality/ death
S. Sharma et al. / International Journal of Biological Macromolecules 125 (2019) 109–115
associated with hydrogel on applied flies, the same lifespan was observed as in control flies were observed, suggesting the non-toxic property of the hydrogel. Hence, the hydrogel has medical importance and can be used as a dressing material for protecting wound/cut area. 3.5. Antimicrobial assay 3.5.1. Antibacterial activity Introduction of the antibacterial activity in the biomedical polymers would lead towards reducing infection risk. CS, acryloyl-phenylalanine and CS-PAP hydrogel were tested against E. coli, K. pneumonia, S. aureus, P. aeruginosa, S. boydii, A. hydrophila bacteria by reported method [46–50] and were found active against broad spectrum bacteria mean both gram-positive and gram-negative bacteria. Among all the bacteria CS based hydrogel showed good activity against E. coli and S. aureus bacteria and the inhibition zone ranges were found from 10 mm to 20 mm (Fig. 4). Incorporation of the acryloyl phenylalanine into the chitosan backbone via free radical polymerization makes it more potent and increases its antibacterial activity (Table 1). 3.5.2. Antifungal activity To examine the antifungal activity of synthesized CS based hydrogel on human pathogenic fungi i.e. Microsporum gypseum, T. mentagrophytes and Candida albicans were taken. Fig. 5 reveals that hydrogel showed visible antifungal activity against C. albicans and M. gypseum but it did not show any activity against T. mentagrophytes. The minimum inhibition zone against Candida albicans and Microsporum gypseum were found to be in the range from 9 mm to 17 mm. Modified chitosan hydrogel was found more antifungal than pure chitosan hydrogel, because acryloyl-phenylalanine act as a key factor in the hydrogel which increases the antifungal activity of the synthesized hydrogel while bare chitosan did not show impressive antifungal activity (Table 2). 4. Conclusion We have developed a tissue adhesive, antimicrobial CS based porous hydrogel using various concentration of MBA as a cross-linking agent, and resulted in hydrogel shown enhanced mechanical strength and other biological applications such as tissue adhesive and antimicrobial activity. The robust mechanical strength can be attributed due to the formation of more interconnected 3D hydrogel network on increasing the concentration of cross-linker. The synthesized hydrogel was explored in the biomedical field in which it showed good tissue adhesive property, antibacterial and antifungal activity as well. Therefore, on the basis of the above findings, we conclude that synthesized hydrogel can be used as a promising candidate in the biomedical field. Thus, hydrogel prepared by easy, eco-friendly methods provides numerous opportunities in a biomedical application such as wound dressing and tissue adhesiveness. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2018.12.018. Acknowledgement The authors would like to acknowledge funding from Banaras Hindu University, India under the Scheme DST-PURSE (5050) and UGC-UPE (4204), authors are thankful to Ms. Zurryat Fatima, Aimil Ltd., New Delhi for rheology related studies using Malvern Kinexus Pro Rheometer.
115
References [1] D. Seliktar, Science 336 (2012) 1124–1128. [2] R. Censi, P. Di Martino, T. Vermonden, Hennink, J. Control. Release 161 (2012) 680–692. [3] K.Y. Lee, D.J. Mooney, Chem. Rev. 101 (2001) 1869–1880. [4] A.S. Hoffman, Adv. Drug Deliv. Rev. 54 (2002) 3–12. [5] P. Calvert, Adv. Mater. 21 (2009) 743–756. [6] X. Zhao, Soft Matter 10 (2014) 672–687. [7] J.P. Gong, Soft Matter 6 (2010) 2583–2590. [8] M.A. Haque, T. Kurokawa, J.P. Gong, Polymer 53 (2012) 1805–1822. [9] K. Haraguchi, Polym. J. 43 (2011) 223–241. [10] J.P. Gong, Science 344 (2014) 161–162. [11] J.Y. Sun, X. Zhao, W.R. Illeperuma, O. Chaudhuri, K.H. Oh, D.J. Mooney, J.J. Vlassak, Z. Suo, Nature 489 (2012) 133–136. [12] T.L. Sun, T. Kurokawa, S. Kuroda, A.B. Ihsan, T. Akasaki, K. Sato, M.A. Haque, T. Nakajima, J.P. Gong, Nat. Mater. 12 (2013) 932–937. [13] Y. Yang, M.W. Urban, Chem. Soc. Rev. 42 (2013) 7446–7467. [14] Y. Zhao, T. Nakajima, J.J. Yang, T. Kurokawa, J. Liu, J. Lu, S. Mizumoto, K. Sugahara, N. Kitamura, K. Yasuda, A.U. Daniels, J.P. Gong, Adv. Mater. 26 (2014) 436–442. [15] Y.M. Chen, R. Ogawa, A. Kakugo, Y. Osada, J.P. Gong, Soft Matter 5 (2009) 1804. [16] F. Brandl, F. Sommer, A. Goepferich, Biomaterials 28 (2007) 134–146. [17] J.L. Drury, D.J. Mooney, Biomaterials 24 (2003) 4337–4351. [18] M. Krogsgaard, M.A. Behrens, J.S. Pedersen, H. Birkedal, Biomacromolecules 14 (2013) 297–301. [19] S. Sharma, A. Kumar Deepak, S. Afgan, R. Kumar, ChemistrySelect 2 (2017) 11281–11287. [20] C.E. Brubaker, H. Kissler, L.J. Wang, D.B. Kaufman, P.B. Messersmith, Biomaterials 31 (2010) 420–427. [21] H. Lee, S.M. Dellatore, W.M. Miller, P.B. Messersmith, Science 318 (2007) 426–430. [22] H. Lee, B.P. Lee, P.B. Messersmith, Nature 448 (2007) 338–341. [23] B.K. Ahn, D.W. Lee, J.N. Israelachvili, J.H. Waite, Nat. Mater. 13 (2014) 867–872. [24] N. Holten-Andersen, M.J. Harrington, H. Birkedal, B.P. Lee, P.B. Messersmith, K.Y. Lee, J.H. Waite, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 2651–2655. [25] G. Sudre, L. Olanier, Y. Tran, D. Hourdet, C. Creton, Soft Matter 8 (2012) 8184–8193. [26] N. Annabi, S.M. Mithieux, G. Camci-Unal, M.R. Dokmeci, A.S. Weiss, A. Khademhosseini, Biochem. Eng. J. 77 (2013) 110. [27] J. Fang, A. Mehlich, N. Koga, J.Q. Huang, R. Koga, X.Y. Gao, C.G. Hu, C. Jin, M. Rief, J. Kast, D. Baker, H.B. Li, Nat. Commun. 4 (2013) 2974. [28] M.J. Glassman, B.D. Olsen, Soft Matter 9 (2013) 6814. [29] A.E.X. Brown, R.I. Litvinov, D.E. Discher, P.K. Purohit, J.W. Weisel, Science 325 (2009) 741. [30] K. Nagapudi, W.T. Brinkman, J. Leisen, B.S. Thomas, E.R. Wright, C. Haller, X. Wu, R.P. Apkarian, V.P. Conticello, E.L. Chaikof, Macromolecules 38 (2005) 345. [31] M.D. Golinska, M.K. Wlodarczyk-Biegun, M.W.T. Werten, M.A.C. Stuart, F.A. de Wolf, R. de Vries, Biomacromolecules 15 (2014) 699. [32] B.J. Kim, D.X. Oh, S. Kim, J.H. Seo, D.S. Hwang, A. Masic, D.K. Han, H.J. Cha, Biomacromolecules 15 (2014) 1579. [33] W.A. Petka, J.L. Harden, K.P. McGrath, D. Wirtz, D.A. Tirrell, Science 281 (1998) 389. [34] S. Lv, D.M. Dudek, Y. Cao, M.M. Balamurali, J. Gosline, H.B. Li, Nature 465 (2010) 69. [35] H.B. Li, W.A. Linke, A.F. Oberhauser, M. Carrion-Vazquez, J.G. Kerkviliet, H. Lu, P.E. Marszalek, J.M. Fernandez, Nature 418 (2002) 998. [36] F. Shamekhi, E. Tamjid, K. Khajeh, Int. J. Biol. Macromol. 120 (2018) 460. [37] S. Kumar, V. Deepak, M. Kumari, P.K. Dutta, Int. J. Biol. Macromol. 84 (2016) 349–353. [38] S. Kumar, P. Garg, S. Pandey, M. Kumari, S. Hoon, K.J. Jang, R. Kapavarapu, P.H. Choung, A.J.F.N. Sobrala, J.H. Chung, J. Mater. Chem. B 3 (2015) 3465. [39] S. Sharma, A. Deepak, R. Kumar, N.K. Rana, B. Koch, Int. J. Biol. Macromol. 116 (2018) 37–44. [40] S. Kumar, J. Dutta, P.K. Dutta, Int. J. Biol. Macromol. 45 (2009) 330. [41] S. Kumar, J. Koh, Int. J. Biol. Macromol. 51 (2012) 1167. [42] T. Wu, J. Huang, Y. Jiang, Y. Hu, X. Ye, D. Liu, J. Chen, Food Chem. 240 (2018) 346–369. [43] X. Jing, H.Y. Mi, Y.J. Lin, E. Enriquez, X.F. Peng, L.S. Turng, ACS Appl. Mater. Interfaces 10 (2018) 20897–22090. [44] R. Dong, X. Zhao, B. Guo, P.X. Ma, ACS Appl. Mater. Interfaces 8 (2016) (171381715). [45] A.R. Karimi, A. Khodadadi, ACS Appl. Mater. Interfaces 8 (2016) 27254–27263. [46] L. Yuan, W. Fan, L. Han, C. Guo, Z. Yan, M. Zhu, X. Mo, Front. Mater. Sci. 12 (1) (2018) 95. [47] D.K. Singh, V.K. Sharma, J. Kumar, A. Mishra, A. Singh, P. Kumari, S.K. Verma, R.N. Kharwar, Nanomedicine 13 (2) (2018) 191–207. [48] F. Wahid, Y.N. Zhou, H.S. Wang, T. Wan, C. Zhong, L.Q. Chu, Int. J. Biol. Macromol. 114 (2018) 1233–1239. [49] M. Zhai, Y. Xu, B. Zhou, W. Jing, J. Photochem. Photobiol. B Biol. 180 (2018) 253–258. [50] A. Moeini, A. Cimmino, et al., Carbohydr. Polym. 195 (2018) 631–641.