Team:Nankai/Design
1. Pudding Health Kit
γ-PGA we produced through synthetic biology method was made into pudding health kit, γ-PGA based hydrogels containing SOD. It's a prototype for chronic wound healing dressing, which proved to be very helpful on speeding up wound healing process on diabetic rats in our experiments. SOD was loaded into hydrogels to scavenge the superoxide and γ-PGA based hydrogels had high water absorption properties delivering the important moist environment. Results showed this type of SOD-PGA hydrogels could promote collagen deposition, epithelialization, and accelerate the healing of moderately exuding wounds. Therefore we believe that our pudding health kit would be a good candidate for wound healing applications in the future.
2. Backgroud
Wound healing is a complex biological process aimed at reconstructing damaged tissue, and it requires precise coordination of connective tissue repair, re-epithelialization, and angiogenesis. During the process, a prolonged inflammation is detrimental to the outcome of healing. Chronic inflammation is characterized by abundant neutrophil infiltration with the associated release of inflammatory mediators including reactive oxygen species (ROS), reactive nitrogen species (RNS), and their derivatives.[1-3]The excessive production of these radicals results in oxidative stress, which is one of the major factors causing non-healing ulcers, such as diabetic wound healing.The remedy for these harms would be the use of wound dressings with antioxidant properties.[4]
Superoxide dismutase (SOD), a non-scavenger enzyme capable of catalyzing the dismutation of superoxide into the less toxic hydrogen peroxide and molecular oxygen, has been widely used as an antioxidant for decreasing reactive oxygen species (ROS) content in injured tissues.
An ideal wound dressing should protect the wound from bacterial infection, control evaporative water loss, remove wound exudates, and promote the establishment of the best milieu for natural healing. At present, high-quality wound dressings are designed to create a moist environment to promote healing.[5]The hydrogel-based wound dressing has recently attracted considerable interest, as they can maintain a moist environment at the wound interface, allow gaseous exchange, absorb wound exudates, and supply a barrier to microorganisms. Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer that is water soluble, non-toxic, edible, and biodegradable,[6-8]which has shown to promote cell migration and enhance cell adhesion.[9-10]Besides, γ-PGA has been reported to prevent postsurgical tissue adhesion and the γ-PGA drug-loaded hydrogel to promote wound healing.[11]
Figure 1. SOD-PGA-hydrogel helps wound repairing.
In this contribution, we developed two types of SOD-loaded γ-PGA hydrogels to promote wound healing. Those γ-PGA based hydrogels could protect the wounded skin and sustained release of SOD promotes the wound repairing process.
3. γ-PGAS/γ-PGA hydrogels
First type of hydrogel here is γ-PGAS/γ-PGA hydrogels. SOD was loaded into hydrogels to scavenge the superoxide anion and γ-PGA was modified with taurine to load more SOD. PGAS/PGA-H had high water absorption properties delivering the important moist environment. SOD released from the hydrogel maintained high enzyme activity and SOD-PGAS/PGA-H could scavenge the superoxide anion effectively. In vivo results showed that SOD-PGAS/PGA-H could promote collagen deposition, epithelialization, and accelerate the healing of moderately exuding wounds. Therefore, SOD-PGAS/PGA-H would be a good candidate for wound healing applications.
3.1 Preparation of SOD-PGAS/PGA-H
Taurine was grafted to the side chain of γ-PGA to obtain sulfonated γ-PGA (γ-PGAS). The interaction between the sulfonic acid groups in γ-PGAS and the amino groups in SOD can increase the load of SOD. Then γ-PGAS and pure γ-PGA was mixed in deionized water at different molar ratios. At last γ-PGAS and γ-PGA was crosslinked with the help of EGDGE to form γ-PGAS/γ-PGA hydrogels.
Dry PGAS/PGA-H samples were dipped in 0.2% SOD/PBS solution at 4℃ for 24 h to load SOD. The hydrogels were then rinsed three times with PBS to remove excess SOD solution.
Figure 2. Synthetic route to c-PGAS/c-PGA hydrogel (PGAS/PGA-H).
3.2 Characteristics of PGAS/PGA-H and SOD-PGAS/PGA-H
The swelling behavior is one of the most important properties of hydrogels for wound dressing. The different swelling ratios of the hydrogels in PBS (pH7.4) are shown in [Figure 3(A)]. The increase of c-PGAS decreased the cross-linking density, which increased the swelling ratio.
The therapeutic efficiency of a drug-loaded hydrogel primarily depends on the dose and release of the drug at the wound site. We observed a sustained release of the entrapped SOD from hydrogels of different formulations, which could provide very accurate dosing and more availability of the drug at the wound site [Figure 3(B)]. The interaction between c-PGAS and SOD imitate the heparin–protein interaction, which increased the amount of SOD loaded in the hydrogel.
To determine the antioxidative property of SOD released from the hydrogels, we studied the accumulated scavenging effect on superoxide radicals [Figure 3(C)]. Forty-eight hours later, the scavenging effect of SOD-PGAS/PGA-H 0 : 1, 0.5 : 1, and 1 : 1 was 49.37%, 56.74%, and 61.97%, respectively. The scavenging effect of the hydrogels increased with SOD release. This was consistent with the results of Figure 2(B). The amount of SOD released from the hydrogel increased with increase of c-PGAS thus scavenged more superoxide radicals.
To test the activity of the released SOD, hydrogels of different ratios were immersed in PBS, and the activity was tested using a modified pyrogallol autoxidation-Vc method.25Figure 3(D) depicted the activity of SOD released from the hydrogels remained at the same level between each time point. Forty-eight hours later, SOD activity retention rate was 93.1%. There was no statistically significant difference between each group. This indicated that the hydrogel system in our study could protect the structure of SOD, and the method used to load SOD was mild enough to guarantee a high activity of SOD.
Figure 3. Characteristics of PGAS/PGA-H and SOD-PGAS/PGA-H (A) Swelling behavior of hydrogels in PBS (pH57.4). (B) The kinetics of SOD released from hydrogels in PBS. (C) Accumulated scavenging effect on superoxide radicals of hydrogels for 12 h. (D) Activity of released SOD.
3.3 Hydrogel Cytotoxicity
Low cytotoxicity is one of the most important properties for biomaterials. Figure 4 showed the cytotoxicity of PGAS/PGA-H evaluated via MTT assay. The cell viability of the specimens was not statistically different from that of the control group (TCPS, tissue-culture polystyrene surfaces) at days 1, 3, and 5 and thus could be considered non-toxic. The excellent cyto-compatibility of the hydrogels was partly ascribed to the biocompatibility of γ-PGA and taurine. γ-PGA is a biocompatible material for various types of cells,[6,12,13] and γ-PGAS has proven biocompatibility.[14]Thus, according to our results, taurine linked to the side chain of γ-PGA had little adverse effect on cellular compatibility.
Figure 4. Cytotoxicity of hydrogels.
3.4 Wound Healing Effects of the Hydrogels
To test the healing effects of hydrogels on chronic wounds, animal model with diabetic rat trauma was used. In our study, SD rats were induced by STZ to develop Type I diabetes, and the full-thickness circular wounds were formed.
Figure 5 showed representative animals from each group (control, PGAS/PGA-H [1:1], SOD-PGAS/PGA-H [1:1]) at days 0, 7, 14, and 21. The results from each group showed that the wound areas treated by SOD-PGAS/PGA-H (1 : 1) were smaller than those of other applications at days 7 and 14 after wounding. These results indicated that SOD encapsulated in hydrogel can promote wound healing. Furthermore, the subcutaneous aspect appeared grossly normal for the test groups (PGAS/PGA-H [1:1], SOD-PGAS/PGA-H [1:1]), and the wound site appeared uninfected. It is known that epithelialization is accelerated if the wound is kept moist.[15]In our experiments, the wound areas treated by hydrogels were smaller than those treated by 3M wound dressing and gauze only. This was probably because keratinocytes migrated more easily over a moist wound surface than underneath a scab.
Figure 5. Photographs of wound treated with control group (A1–A4), PGAS/PGA-H 1 : 1 (B1–B4), SOD-PGAS/PGA-H 1 : 1 (C1–C4) at days 0, 7, 14,and 21, respectively; All wounds were covered with 3M wound dressing and gauze.
Figure 6 depicted the wound closure rates of wounds treated with control, PGAS/PGA-H and SOD-PGAS/PGA-H. After treatments for 7, 14, and 21 days, the rates of wound closure were significantly higher in the SOD-PGAS/PGA-H group than in the control group: the wound closure rate in the control group was 60% after 21 days, while in the PGAS/PGA-H group was 70% and in the SOD-PGAS/PGA-H group was 90%. Compared with other groups, the wounds treated with SOD-PGAS/PGA-H exhibited the fastest wound closure.
Figure 6. Percentages of wound closure after distinct time intervals.
3.5 Conclusions
We prepared SOD-PGAS/PGA-H for wound healing. SOD was loaded into hydrogels to scavenge the superoxide anion and γ-PGA was modified with taurine to load more SOD. PGAS/PGA-H had high water absorption properties delivering the important moist environment. SOD released from the hydrogel maintained high enzyme activity and SOD-PGAS/PGA-H could scavenge the superoxide anion effectively. In vivo results showed that SOD-PGAS/PGA-H could promote collagen deposition, epithelialization, and accelerate the healing of moderately exuding wounds. Therefore, SOD-PGAS/PGA-H would be a good candidate for wound healing applications.
4. PNIPAM/γ-PGA hydrogels
PNIPAM/γ-PGA hydrogel is thermosensitive hydrogels. They are liquid at room temperature but they can transform into hydrogels when they are dropped on skin. The solidified hydrogel can perfectly cover different shape of wounds, leaving no exposed spot on the wound. In this way, SOD loaded hydrogels could have full contact with the damaged tissue and promote the wound repairing process.
Figure 7. PNIPAM/γ-PGA hydrogel at room temperature (D), above 32℃(E, F).
4.1 Characteristics PNIPAM/γ-PGA hydrogels
PNIPAM is a thermosensitive polymeride. It’s liquid at room temperature and could transform into hydrogel when temperature reaches 32℃. PNIPAM has abundant amidogen groups on it’s sidechain which interact well with carboxyl groups on γ-PGA’s sidechain. And thus by directly mixing PNIPAM and γ-PGA solutions at different molar ratios we can get hydrogels solidified at different temperatures. We try several different mixtures of PNIPAM and γ-PGA. Their ability to form hydrogel and the characteristic of the hydrogel was tested at 37℃.
Through the test, we find that hydrogel formed of 75% PNIPAM and 25% γ-PGA has better mechanical capacity.
Figure 8. PNIPAM/γ-PGA 75/25 mixture in different state. a. solution at room temperature. b. hydrogel formed at 37℃. c. hydrogel contains water. d. dry hydrogel.
4.2 γ-PGA’s effect on PNIPAM/γ-PGA hydrogels
Figure 9 shows that when γ-PGA concentration reach 50%, the contact angle of the material drop from 79.6o to 55.3o. Increased γ-PGA concentration in the mixture improved the material’s affinity to water, which can create moister environment and avoid dressing’s adhesion to the wound.[16] Therefore high water content hydrogels can better protect the wound, and in the main time could be removed easily without hurting the tissue underneath.
Figure 9. Contact angle of hydrogels with different γ-PGA concentration. A. PNIPAM/γ-PGA 50/50 B. PNIPAM/γ-PGA 75/25 C. PNIPAM/γ-PGA 100/0.
Figure 10 shows that 50:50, 75:25 and 100:0 PNIPAM/γ-PGA hydrogel’s swelling ratio was 1470%, 936% and 400% respectively. Due to abundant hydrophilic carboxyl groups on γ-PGA’s sidechain, the water absorbing ability of the material is greatly improved. Figure 11 shows that 50:50, 75:25 and 100:0 PNIPAM/γ-PGA hydrogel could still contain 30%,73%, and 61% of its water after 40 hours, which indicates that PNIPAM/γ-PGA hydrogel is good at preserving water.
Figure 10. Swelling ratio of PNIPAN/γ-PGA hydrogel.
Figure 11. Water containing of different PNIPAN/γ-PGA hydrogels through time.
4.3 Hydrogel Cytotoxicity
PNIPAM is a polymer consists of NIPAM monomers, while NIPAM monomers do have cytotoxicity. And thus how PNIPAM is purified and whether it contains redundant NIPAM monomers was one our major concerns. Figure 12 showed the cytotoxicity of PNIPAN/γ-PGA hydrogel evaluated via MTT assay. After 24 hours and 48 hours culture, 3T3 cell’s survival rates in both situations were all above 88% (survival rate above 75% means the material is nontoxic).[17,18]The result shows that PNIPAN/γ-PGA hydrogel had little adverse effect on cellular compatibility.
Figure 12. Cytotoxicity test of PNIPAM/γ-PGA extract liquid at different ratio.
4.4 Wound Healing Effects of the PNIPAN/γ-PGA hydrogel
The wound repair effect of PNIPAN/γ-PGA hydrogel was also tested on diabetic rats. Figure 13 shows the picture of during the recovery of different diabetic rat groups. Figure 14 shows after 21 days, the wound closure rate of control group, PNIPAN/γ-PGA hydrogel group and SOD- PNIPAN/γ-PGA hydrogel was 66%, 79% and 89% respectively. It indicates PNIPAN/γ-PGA hydrogel can promote the healing process of wounded skin and PNIPAN/γ-PGA hydrogel loaded with SOD has even better wound repairing effect.
Figure 13. Photographs of wound treated with control group (A), PNIPAN/γ-PGA hydrogel (B), SOD- PNIPAN/γ-PGA hydrogel (C) at days 0, 7, 14, and 21, respectively.
Figure 14. Percentages of wound closure after distinct time intervals.
4.5 CONCLUSIONS
We prepared SOD-PNIPAN/γ-PGA hydrogel for wound healing. PNIPAN/γ-PGA hydrogel can transform from liquid to hydrogel when the temperature is suitable for the change. This kind of hydrogel can perfectly cover the wound and make full contact to the wound, which promotes the healing of the damaged tissues. When SOD was loaded into hydrogels, it improved the material’s wound repairing effect as the released SOD scavenges the superoxide anion. In vivo results showed that SOD-PNIPAN/γ-PGA and PNIPAN/γ-PGA hydrogel could promote collagen deposition, epithelialization, and accelerate the healing of moderately exuding wounds. Therefore, SOD-PNIPAN/γ-PGA hydrogel would be a good candidate for wound healing applications.
Swollen γ-PGA (crosslinked by γ-irradiation).
Hydrogel appearance and microstructure.
A Bacillus amyloliquefaciens strain.
These are all bioreactors used for fermentation.
References
1. Clark, R. A. J. Am. Acad. Dermatol. 1985, 13, 701. 2. Gopinath, D.; Ahmed, M. R.; Gomathi, K.; Chitra, K.; Sehqal, P. K.; Jayakumar, R. Biomaterials 2004, 25, 1911. 3. Lai, J. J.; Lai, K. P.; Chuang, K. H.; Chang, P.; Yu, I. C.; Lin, W. J.; Chang, C. J. Clin. Invest. 2009, 119, 3739. 4. Moseley, R.; Walker, M.; Waddington, R. J.; Chen, W. Y. J. Biomaterials 2003, 24, 1549. 5. Gong, C. Y.; Wu, Q. J.; Wang, Y. J.; Zhang, D. D.; Luo, F.; Zhao, X.; Wei, Y. Q.; Qian, Z. Y. Biomaterials 2013, 34, 6377. 6. Huang, M. H.; Yang, M. C. Polym. Adv. Technol. 2010, 21, 561. 7. Lee, Y. H.; Chang, J. J.; Yang, M. C.; Chien, C. T.; Lai, W. F.Carbohyd. Polym. 2012, 88, 809. 8. Shih, I. L.; Wu, P. J.; Shieh, C. J. Process Biochem. 2005, 40,2827. 9. Matsusaki, M.; Yoshida, H.; Akashi, M. Biomaterials 2007, 28, 2729. 10. Guan, H.; McGuire, M. J.; Li, S.; Brown, K. C. Bioconjug. Chem. 2008, 19, 1813. 11. Izumi, Y.; Yamamoto, M.; Kawamura, M.; Adachi, T.;Kobayashi, K. Surgery 2007, 141, 678. 12. Lin, Y. H.; Lin, J. H.; Peng, S. F.; Yeh, C. L.; Chen, W. C.;Chang, T. L.; Liu, M. J.; Lai, C. H. J. Appl. Polym. Sci. 2011,120, 1057. 13. Liang, H. F.; Yang, T. F.; Huang, C. T.; Chen, M. C.; Sung,H. W. J. Control. Release 2005, 105, 213. 14. Matsusaki, M.; Serizawa, T.; Kishida, A.; Akashi, M. J. Biomed. Mater. Res. Part A 2005, 73A, 485. 15. Winter, G. D. Nature 1962, 193, 293. 16. Miguel SP, Ribeiro MP, Brancal H, et al. Thermoresponsive chitosan–agarose hydrogel for skin regeneration[J]. Carbohydrate Polymers,2014,111:366-373. 17. Cooperstein MA, Canavan HE. Assessment of cytotoxicity of (N-isopropyl acrylamide) and Poly(N-isopropyl acrylamide)-coated surfaces[J]. Biointerphases,2013,8(1):19-31. 18. International Organization for Standardization. Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity. 2009.