Berberine coated biocomposite hemostatic film based alginate as absorbable biomaterial for wound healing
Graphical abstract
Introduction
Wound issue is the most common challenge to health in the world. Serious trauma, which is susceptible to microbial infection, leads to retard wound healing and even various life-threatening conditions [1]. Therefore, there is an urgent clinical need to develop novel and smart biomaterial which could control bleeding, prevent wound infection and promote wound healing [2], [3].
In recent years, various hemostatic materials have been developed according to the ability of liquid absorption, platelet activation and tissue adhesion [4], [5], [6], such as montmorillonite [7], graphene [8], chitosan [9], alginate [10], collagen [11], etc. However, they have inherent limitations. Montmorillonite, as small particles, presents strong water absorption and excellent hemostatic effect [12], but poor biocompatibility and related inflammation, which limit its application in vivo [13]. Graphene hemostatic material, which has high mechanical strength, is suitable for small wounds rather than extensive wounds [14]. Notably, all these rapid hemostatic materials, such as montmorillonite and graphene, are not conducive to wound healing [15], [16]. Hence, the development of natural polymers, such as chitosan, alginate, collagen, gelatin and hyalurate, has begun to raise concerns [17], [18], [19], [20]. Chitosan, one of the most commonly used natural polymer, shows good hemostatic properties and improves wound healing [21], [22] through shorting the inflammation stage [23], promoting granulation tissue regeneration, and exhibiting rapid hemostasis [24]. However, chitosan is insoluble in polar solvents, thus its further applications are severely limited [25]. Consequently, carboxymethyl chitosan, a carboxymethyl modified chitosan, is developed as a kind of water-soluble polymer [26]. Alginate is another commonly used natural polymer, displays good biocompatibility and water-absorbing ability [27], but the alginate-based dressing performs low mechanical strength [28]. Collagen could be easily obtained as a kind of resorbable ampholyte protein with platelet activation in hemostasis and cell growth in wound healing [11], [29]. However, facile degradation and poor mechanical performance forced it to be combined with other polysaccharides to stop bleeding and facilitate wound healing [27].
To overcome shortcomings of single-component material, researchers have tried combination of multiple materials to meet the complicated application requirements [30], [31]. In recent years, numerous complex polysaccharide hemostatic materials have been developed to promote wound healing and to prevent trauma infection [32]. Guo et al. developed tissue-adhesive cryogels based on polydopamine cross-linked quaternized chitosan as multifunctional wound dressings [33]. Additionally, Pawar et al. prepared the alginate/chitosan/povidone‑iodine composite gel, which performed good anti-Gram positive bacteria activity through povidone‑iodine [34], [35]. Since the excellent antibacterial activity of silver ion was discovered by Cooper [36], silver nanoparticles were added to hemostatic materials for improving wound healing [37], [38], [39]. However, the sedimentary of AgNPs in organs or tissues may induce necrocytosis, cell apoptosis or genetic mutations [40], [41], [42]. Berberine, a natural product and a conventional drug, exhibits inherent antibacterial activity and good safety for clinical use [43], [44], [45]. Previously, we developed SCC-B microspheres coating berberine, which could achieve rapid hemostasis and antibacterial effects [46]. However, microspheres are reported to be more suitable for bleeding control than wound healing [27]. Accordingly, we aim to further develop novel biomaterial that are able to simultaneously control bleeding, prevent infection and accelerate wound healing.
In this work, we combined carboxymethyl chitosan (CMC), sodium alginate (SA), and collagen by adding calcium chloride as crosslinker to prepare a biocomposite hemostatic film (BHF). To improve the antibacterial and mechanical properties of BHF, we further added berberine as the main antibacterial component and cross-linking regulator to prepare a series of novel biocomposite antibacterial hemostatic films (BHF-B series). Subsequently, scanning electron microscope (SEM), fourier infrared spectroscopy (FTIR), atomic force microscope (AFM), contact angle and rotational rheometer were used to characterize the physical and chemical properties of the films. Furthermore, hemostatic and bacteriostatic capacity, biocompatibility as well as biodegradability of BHF series were examined by in vitro and in vivo experiments, such as swelling, degradation, hemostasis, platelet aggregation, bacteriostasis, hemolysis, cytotoxicity, rat-tail amputation and wound healing. A schematic illustration of the characteristic structure and surface properties for the BHF and BHF-6B, and their application as hemostatic wound dressing are shown in Fig. 1.
Section snippets
Materials
Sodium alginate (CAS: 9005-38-3, pharmaceutical degree, M/G = 2/1, MW: 10000–600000), carboxymethyl chitosan (CAS: 83512–85-0, 240Kda, deacetylation degree >90%), were obtained from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Glycerin (CAS: 56–81-5, USP99.7%, MW: 92.09) and berberine hydrochloride (CAS: 633–65-8, 98%, MW: 371.81) were purchased from Shanghai Aladdin Industrial Co., Ltd. (Shanghai, China). Collagen (CAS: 9007-34-5, porcine, type I) was obtained from Shanghai Chiwei
Preparation
BHF series were prepared via a thin-film hydration approach, coating berberine into the composite film for antibacterial application. For in vivo application, the raw materials and cross-linker used in the preparation process were all none-cytotoxic. As expected, the color of the prepared films changed from transparent to dark yellow, indicating the ratio of berberine in films increased from BHF to BHF-9B (Fig. S1). Based on our previous researches, we speculated that the Calcium/Berberine dual
Conclusion
In summary, a series of biocomposite hemostatic films (BHFs) was successfully fabricated. We speculated that berberine was used as a cross-linking agent with alginate, carboxymethyl chitosan and collagen through a co-crosslinking process with Ca2+. The functionalized films not only presented excellent long-term bacteriostasis, but also enhanced hemostasis and adjustable mechanical properties. More importantly, in vivo wound healing assay showed that wound closure ratio was higher in mice
CRediT authorship contribution statement
Haofeng Hu: Investigation, Validation, Data Curation, Writing – Original Draft. Fulin Luo: Investigation, Data Curation, Writing – Original Draft. Qian Zhang: Formal analysis, Validation, Data Curation. Ming Xu: Investigation, Validation, Data Curation. Xin Chen: Data Curation. Zhihao Liu: Investigation. Haodong Xu: Investigation. Lei Wang: Supervision, Writing – Review & Editing. Fei Ye: Supervision, Writing – Review & Editing. Kui Zhang: Conceptualization, Supervision. Bin Chen: Resources,
Declaration of competing interest
We declare that we have no financial and personal relationship with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled.
Acknowledgements
This study was financially supported by the National Natural Science Foundation of China (81803339), Zhejiang Provincial Top Key Discipline of Biology. The authors also sincerely thank the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine and the Electron Microscopy Center of East China Normal University.
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