640 Mussel-inspired Polydopamine-assisted Bromelain Immobilization onto Electrospun Fibrous Membrane for Potential Application as Wound Dressing
Authors: Chen Xinxin , Yu Jiaao , Zhang Xiu-Hang 
Published: March 2020

Abstract

Introduction
Skin damage as a result of injury, be it acute or chronic, can adversely impact the quality of life of affected individuals, with serious wounds being life-threatening in certain cases, leading to a high clinical demand for effective wound dressing materials. Effective wound dressings are those which maintain moisture in the wound site, maintaining hemostasis while preventing the development of any infections, thereby allowing air and water to more effectively facilitate epithelization. At present the bioactivity of most wound dressings is relatively poor, with more effective dressings being very costly and difficult to produce, necessitating the development of novel wound healing materials. Recent research has highlighted the potential of electrospun fibrous membranes as a material for use in wound dressing design. Membranes designed using electrospinning technology have desirable features including a pore size that is readily tunable, as well as substantial air permeability and a high surface-to-volume ratio. These membranes also have a 3D structure that is similar to the structure of the extracellular matrix (ECM), which is essential for mediating effective cellular adhesion and proliferation. These advantageous properties make such electrospun fibers potentially ideal for use in wound dressings. Studies have further sought to functionalize these fibers so as to enhance their ability to promote wound healing via imbuing them with growth factors, antibacterial compounds, or other bioactive materials. Current ongoing efforts have to functionalize electrospun fibers offer great potential for efforts to regulate wound healing.
Given their safety and relatively low cost, bioactive plant extracts have been the subject of particular interest in the context of wound healing. One such extract is bromelain, which is a crude extract isolated from the pineapple fruit that contains a number of therapeutically valuable proteolytic enzymes. Previous work has shown bromelain to be capable of mediating anti-inflammatory and anti-edematous properties in many contexts including sinusitis, thrombophlebitis, angina pectoris, bronchitis, surgical trauma, and pyelonephritis. Bromelain is also known to be capable of hydrolyzing devitalized tissues so as to enhance rates of wound healing. Efforts to maintain the stability of bromelain are essential for its therapeutic utilization, with previous groups having demonstrated that bromelain can be effectively incorporated into electrospun or other polymer matrices. For example, Bayat et al. were able to use a blending electrospinning process to generate chitosan nanofibers loaded with bromelain, while Korrapati et al. used coaxial electrospinning in order to incorporate bromelain into electrospun fibers. In addition to direct incorporation, bromelain can also be immobilized directly onto the surface of electrospun fibers, allowing for more direct interaction between bromelain and wound tissue. Unfortunately, electrospun fibers tend to interact poorly with enzymes, resulting in relatively poor enzymatic activity and stability. Therefore there is a need to develop better techniques for immobilizing bromelain onto electrospun fibers.
Previous studies have highlighted the potential of mussel-inspired polydopamine (PDA), which is produced via oxidatively polymerizing dopamine, as a substrate with the potential for utilization in the development of advanced biomaterials. PDA is readily deposited onto a wide range of surfaces without the need of expensive or complex instruments and procedures in order to achieve this deposition. Importantly, PDA is highly versatile owing to the abundance of available functional groups, allowing it to be developed into a platform well-suited to use in myriad applications. PDA has been shown to ameliorate enzymatic immobilization via both Schiff base reactions and Michael-type addition. However, at present there are no reports regarding the potential for PDA to facilitate bromelain immobilization.
In the present report we describe the successful use of PDA to mediate bromelain immobilization on electrospun fibers, with the resultant fibers being effective when used for dressing wounds. The synthetic polymer poly(e-caprolactone) (PCL), which exhibits good biocompatibility, was selected for use as a matrix that was used to generate a fibrous membrane onto which bromelain could be immobilized. The FDA has approved the biomedical use of PCL, and it has previously been shown to exhibit both a high degree of mechanical stability while also remaining biodegradable. In this report, we first immobilized bromelain onto electrospun PCL fibers via use of PDA and then explored the physical and chemical properties of the resultant membranes and of the bromelain deposited thereupon. We additionally assessed the utility of these membranes as mediators of cellular adhesion, cell proliferation, and antibacterial activity in vitro, and as drivers of enhanced wound healing in vivo. Together our findings highlight these bromelain-immobilized electrospun PCL fiber membranes as having great promise for use in wound healing applications.

Methods
PCL fiber were prepared via electrospinning, then bromelain-immobilized PCL fiber were generated. The surface morphology of fibers was obtained by scanning electron microscopy. Swelling test, water vapor transmission rates (WVTR), in vitro bromelain release and loading, enzyme activity and stability were tested to demonstrate the fiber scaffold characteristic. The prepared composite scaffolds had satisfactory antibacterial activity via Kirby–Bauer (K-B) disc method. The cell studies were performed to reveal that the scaffolds were biocompatible and safe for cell attachment. Histological and immunohistochemical examinations were performed in rats. In Vivo wound healing model were chosen to verify the PCL functionalization using PDA and bromelain can accelerate the wound healing process.

Results
We found that bromelain activity could be better stabilized when via its immobilization on electrospun fibers. The resultant BrPDA-PCL fibers exhibited promising properties including optimal mechanical stability, wettability, and rates of water vapor transmission. In addition, these BrPDA-PCL fibers were biocompatible, allowing for effective cellular adhesion and proliferation. The results of zone of inhibition testing further confirmed that these fibers achieved effective antibacterial activity against Escherichia coli and Staphylococcus aureus. When used in vivo, as compared with PCL fibers or control animals the BrPDA-PCL fibers enhanced wound healing rates while reducing associated inflammation.

Conclusions
Herein we found that using PDA to immobilize bromelain onto electrospun PCL fibers resulted in the production of membranes that were highly effective wound dressings. Bromelain activity was markedly stabilized via the immobilization process, and the resultant BrPDA-PCL fibers were capable of supporting both cellular adhesion and proliferation. Given the observed synergy between PDA and bromelain, these BrPDA-PCL fibers also exhibited antibacterial activity. When utilized in a rat model of wound healing, inflammation was reduced and healing rates were improved in rats treated using BrPDA-PCL fibers relative to untreated controls. We therefore propose that future studies further examine the value of bromelain-immobilized electrospun PCL fibers as a means of enhancing epithelial regeneration.

Applicability of Research to Practice
The electrospun BrPDA-PCL fibers were capable of loading and delivering drugs, and could be potentially used as novel antibacterial burn wound dressings. These scaffolds have promising potential applications in infection control at the early phase of burn injury and accelerate wound healing process.