Characterization and evaluation of antibacterial and wound healing activity of naringenin-loaded polyethylene glycol/polycaprolactone electrospun nanofibers

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Abstract

Antibiotic resistance is one of the main reasons for the prolonged wound-healing process in the world. Therefore, natural compounds that possess excellent antioxidant, anti-inflammatory, and antimicrobial properties, can promote the high performance of therapeutic avenues in wound treatment. We report the preparation and evaluation of Naringenin (Nrg), a poorly water-soluble model ingredient, loaded into Polycaprolactone (PCL)/Polyethylene glycol (PEG) nanofibers (NFs) as a novel antibacterial wound dressing. The electrospinning method was used to produce a smooth nanofiber. The structure, morphology, components interactions, contact angle, swelling ratios, tensile strength, antibacterial activity, and cell biocompatibility have been investigated. The SEM images revealed that linear morphology, homogeneous, and smooth surface nanofibers had been prepared. The electrospun mats show sustained Nrg-releasing properties at a high concentration of Nrg. It supplemented the FT-IR results that exhibit the cross-link between the –OH functional group of Nrg and C=O of PCL. The results illustrated significantly enhanced antibacterial performance of mats when minimal Nrg was applied in nanofibers, compared to the higher Nrg amounts in NFs. The non-toxic nature of mats was confirmed by increased survival of human fibroblast cells in MTT assay. In in vivo wound injury model in rat, the Nrg-loaded mats shows comparable re-epithelialization and wound closure to commercially available phenytoin ointment. Altogether, the study addresses the Nrg-loaded PCL/PEG NFs mats that can serve as the prototype biodegradable and antibacterial wound dressing to treat cutaneous wound infection.

Introduction

Bacterial infections are the leading cause of prolonged wound-healing processes in the world [1]. Acute and chronic wounds healing rates in burns and diabetic ulcers are slower than other ones in that about two-thirds of these wounds are vulnerable to developing an infection [2]. Although there are different clinical methods for wound care management, the treatment of bacterial infections and wounds is still a public health issue due to the life losses and economic burden [3]. This challenge represented the need for wound dressings that will simultaneously hinder and eliminate infection, induce a high healing rate, provide a moist environment, pass oxygen, and finally reduce costs [4].

The various strategies to enhance wound healing process efficiency include negative pressure wound therapy, hyperbaric oxygen therapy, and skin substitutes (porcine xenograft and porcine collagen) [5,6]. The major factors limiting the applicability of the methods mentioned above are cost and the possibility of treatment failure. In recent years, nanofiber mats spring up a new class of material with high surface area and extracellular matrix (ECM) like structure, which could hold novel therapeutic promise in managing wound treatment conditions [7].

Increased antibiotic resistance is one of the main reasons bounded the systematic use of antibiotics. Antibiotic resistance causes kidney and liver toxicity, loss of normal body flora, allergic reactions, prolonged hospital stays, and increased mortality [8]. In acute and chronic wounds, blood circulation at the damage site is poor and topical use of antibiotics, in addition to overcoming the downside above, leads to selective delivery of antibiotics to the target site with a lower concentration in a shorter time. In light of these issues, antibacterial dressings have been paid much attention to because they can physically protect the wound against microorganisms. By hindered the biofilm formation can be effective against a wide range of bacteria [9]. Many documents have demonstrated that dressings comprising antimicrobial agents such as antibiotics [10,11], active secondary metabolites [12], and metal nanoparticles [13,14] have shown incredible results to wound healing.

Nrg is a predominant flavanone found in citrus fruits, mainly grapefruit and bitter orange. Pharmacologically, it exhibited immunomodulatory, anticancer, antibacterial, antimutagenic, antioxidant, anti-inflammatory, antiproliferative, antiestrogenic, hepato-protective, and antiatherogenic activities [[15], [16], [17], [18]]. As shown in Fig. 1, It possesses two benzene rings linked together via a pyrone ring which gives it antibacterial properties [19,20]; nevertheless, due to the few reports, the mode of action and mechanism of Nrg antibacterial activity is unknown. However, its lipophilic nature (log P value 2.6) and poor water-solubility limited its therapeutic application. Many efforts have been made to increase the solubility of Nrg, such as biotransformation modification [21,22], formulated into various dosage forms [[23], [24], [25]], and physical or chemical cross-linking [26,27].

Therefore, the hypothesis of Nrg incorporation in PCL/PEG NFs could provide a novel strategy to enhance its antibacterial activity by increasing the available surface area and effective delivery of the ingredient to the target site. We first investigated the nanofibers mats characterization and in vitro release profile. The effect of nanofibers on human fibroblasts’ cell survival was accessed. The Nrg-loaded mats were further challenged for their antibacterial activity against Gram-positive and negative bacteria. The ability of prepared NFs as a dressing to promote rat skin wound healing was studied to test the applicability of PCL/PEG NFs as a carrier of Nrg.

Section snippets

General

Dimethyl Sulfoxide (DMSO), Nrg, Formalin, Hematoxylin-eosin, and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Polycaprolactone (80 000 Da), Polyethylene glycol (400 Da), Dichloromethane, Dimethylformamide (DMF), Diethyl ether, Xylenol, Xylazine hydrochloride, Ketamine KH2PO4, and NaOH were obtained from Merck. Müller-Hinton agar and Müller Hinton Broth culture medium and Dialysis bag (12 KDa) were purchased from Himedia laboratories.

Characterizations

Electrospinning of polymers with a short-chain length such as PEG is challenging, and thus, the use of co-spinning polymer seems to be an effective solution [28]. PCL as an approved polymer by US-FDA is widely used in biomedical applications, though, its hydrophobicity cause decreased cell attachment [29]. Given that a hydrophilic polymer is required to increase cell recognition sites and biocompatibility of PCL, and since PEG provides this property in the mixture, it was necessary to adjust

Conclusion

In this work, we developed the antibacterial dressing via a simple electrospinning method based on Nrg-embedded PCL/PEG NFs to accelerate the wound healing process. From FT-IR spectra, it can be deduced that the nature of the interaction between PCL/PEG and the Nrg was a hydrogen bonding, and a Nrg can cross-linked nanofibers which results in sustained antimicrobial wound dressings. The mats have great antibacterial activity and minimize the chances of bacterial resistance due to Nrg's natural

Authorship statement

All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in the Journal of Drug Delivery

Declaration of competing interest

The authors declare no competing financial interest.

Acknowledgments

This study was supported by Kermanshah University of Medical Sciences, Kermanshah, Iran (Grant number: 980524).

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