Berberine coated biocomposite hemostatic film based alginate as absorbable biomaterial for wound healing

https://doi.org/10.1016/j.ijbiomac.2022.04.132Get rights and content

Highlights

  • Biocomposite hemostatic films (BHFs) based alginate/chitosan/college

  • Bioactivity of BHFs enhanced by calcium/berberine dual-crosslinking system

  • The BHF-6B facilitated wound healing.

  • The BHF-6B possessed excellent bacteriostatic activity with long-term effect.

  • The BHF-6B possessed excellent biocompatibility and biodegradability.

Abstract

In wound treatment, severe bleeding and infection are always primary challenges. Therefore, it is highly desired to develop novel dressing with both hemostatic and antibacterial capability. Herein, a series of biocomposite hemostatic films (BHFs) based alginate/chitosan/collagen-berberine have been prepared and well characterized for further biofunctional study. We have demonstrated that the hemostatic and antibacterial activities were significantly enhanced by calcium/berberine dual-crosslinking system in the film. Through the synergistic effects, BHF-6B exhibited a shorter in vivo clotting and wound healing time than that of commercial dressing in rat tail amputation and full-thickness skin defect models. Additionally, BHF-6B showed excellent bacteriostatic activity with long-term effects. Moreover, hemolysis and cytotoxicity tests in vitro illustrated the prominent biocompatibility of the composite films. Notably, BHF-6B could be degraded quickly and completely in vivo. Overall, the present work indicated that the functionalized BHF-6B has great potential as an absorbable biomaterial for wound treatment.

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.

References (63)

  • G. Franceschini

    Internal surgical use of biodegradable carbohydrate polymers.Warning for a conscious and proper use of oxidized regenerated cellulose

    Carbohydr. Polym.

    (2019)
  • L. Jaiswal et al.

    Carrageenan-based functional hydrogel film reinforced with sulfur nanoparticles and grapefruit seed extract for wound healing application

    Carbohydr. Polym.

    (2019)
  • V. Pawar et al.

    Chitosan nanoparticles and povidone iodine containing alginate gel for prevention and treatment of orthopedic implant associated infections

    Int. J. Biol. Macromol.

    (2018)
  • A. Verlee et al.

    Recent developments in antibacterial and antifungal chitosan and its derivatives

    Carbohydr. Polym.

    (2017)
  • J. Shao et al.

    Antibacterial effect and wound healing ability of silver nanoparticles incorporation into chitosan-based nanofibrous membranes

    Mater. Sci. Eng.

    (2019)
  • L. Jaiswal et al.

    Lignin-mediated green synthesis of AgNPs in carrageenan matrix for wound dressing applications

    Int. J. Biol. Macromol.

    (2020)
  • X. Li et al.

    The combined antibacterial effects of sodium new houttuyfonate and berberine chloride against growing and persistent methicillin-resistant and vancomycin-intermediate Staphylococcus aureus

    BMC Microbiol.

    (2020)
  • J. Jin et al.

    Alginate-based composite microspheres coated by berberine simultaneously improve hemostatic and antibacterial efficacy

    <sb:contribution><sb:title>Colloids Surf.</sb:title> </sb:contribution><sb:host><sb:issue><sb:series><sb:title>B Biointerfaces</sb:title></sb:series></sb:issue></sb:host>

    (2020)
  • Y. Wang et al.

    Highly transparent, highly flexible composite membrane with multiple antimicrobial effects used for promoting wound healing

    Carbohydr. Polym.

    (2019)
  • H. Qiao et al.

    A novel microporous oxidized bacterial cellulose/arginine composite and its effect on behavior of fibroblast/endothelial cell

    Carbohydr. Polym.

    (2018)
  • H.W. Ju et al.

    Wound healing effect of electrospun silk fibroin nanomatrix in burn-model

    Int. J. Biol. Macromol.

    (2016)
  • P.H. Wang et al.

    Wound healing

    J. Chin. Med. Assoc.

    (2018)
  • B.L. Guo et al.

    Haemostatic materials for wound healing applications

    Nat. Rev. Chem.

    (2021)
  • R.N. Dong et al.

    Smart wound dressings for wound healing

    Nano Today

    (2021)
  • D.R. Spahn et al.

    The European guideline on management of major bleeding and coagulopathy following trauma: fifth edition

    Crit. Care

    (2019)
  • D.A. Hickman et al.

    Biomaterials and advanced technologies for hemostatic management of bleeding

    Adv. Mater.

    (2018)
  • M. Caspers et al.

    Current strategies for hemostatic control in acute trauma hemorrhage and trauma-induced coagulopathy

    Expert. Rev. Hematol.

    (2018)
  • K. Quan et al.

    Diaminopropionic acid reinforced graphene sponge and its use for hemostasis

    ACS Appl. Mater. Interfaces

    (2016)
  • W. Wang et al.

    Chitosan derivatives and their application in biomedicine

    Int. J. Mol. Sci.

    (2020)
  • B.S. Kheirabadi et al.

    Comparison of new hemostatic granules/powders with currently deployed hemostatic products in a lethal model of extremity arterial hemorrhage in swine

    J. Trauma

    (2009)
  • A.H. Smith et al.

    Haemostatic dressings in prehospital care

    Emerg.Med.J.

    (2013)
  • Cited by (17)

    View all citing articles on Scopus
    View full text