**1. Introduction**

Dura mater is the outermost membrane of meninges which surrounds the brain and spinal cord to retain the cerebrospinal fluid (CSF) inside. During neurosurgical procedures, intracranial lesions might lead to partial dura removal or dural perforation and the restoration of the dura mater to regain a watertight dural closure should be performed. The solitary use of native dura for such reconstruction could struggle to achieve an adequate primary closure in certain situations and might result in post-operative complications, morbidity and even loss of life [1,2]. Several dural substitutes and dural sealants are commercially available and used in place of native dura mater for dural repair and closure restoration. Recently, a new bilayered knitted fabric reinforced composite (ORC/PCL bilayered composite) was developed for potential use as a dural substitute [3–5]. This composite membrane contained two different morphologies including a composite layer which incorporated a relatively faster resorption ORC that could in situ generate pores for tissue ingrowth and a dense nonporous layer of relatively slower resorption PCL on the opposite side which helped provide the load bearing and liquid leakage resistance [6–8]. This type of dural substitute also offered several advantages including low cost, simple production as well as similar mechanical properties to human dura mater.

**Citation:** Sanpakitwattana, A.; Suvannapruk, W.; Chumnanvej, S.; Hemstapat, R.; Suwanprateeb, J. Cefazolin Loaded Oxidized Regenerated Cellulose/Polycaprolactone Bilayered Composite for Use as Potential Antibacterial Dural Substitute. *Polymers* **2022**, *14*, 4449. https://doi.org/10.3390/ polym14204449

Academic Editors: Ariana Hudita and Bianca Galˇ a¸ˇteanu

Received: 27 August 2022 Accepted: 20 September 2022 Published: 21 October 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In general, post-operative CNS infection such as meningitis and post-operative surgical site infections (SSIs) have been reported following craniotomy and neurosurgical procedures [8,9]. Several risk factors associated with these post-operative infections have been identified, including CSF leakage, prolonged operation procedure (>3 h), diabetes, implantation of prosthetic devices such as shunt and cranioplasty materials [9,10]. The predominant pathogens causing these infections are Gram-positive bacteria, particularly *Staphylococcus aureus* (*S. aureus*) and *coagulase*-*negative staphylococci* (*CoNS*). The incidence rates of post-operative surgical site infections (SSIs) have been reported to decrease when antibiotic prophylaxis strategies were implemented [11,12]. The ideal antimicrobial drugs for prophylaxis should be effective against the most common organisms found at the surgical sites without eliminating other causative organisms [9,13]. Cephalosporins are the most commonly used prophylactic antibiotics in different types of surgical procedure [14]. In particular, cefazolin, the first generation cephalosporin, has been considered as the prophylactic drug of choice for post-neurosurgical infections [11]. This is because it is most effective against Gram-positive bacteria such as *S. aureus* and *CoNS*, which are the major causes of the post-operative SSIs and post-craniotomy infections. Moreover, several advantages of cefazolin have been reported including a low toxicity and low cost [12,15]. Cefazolin is given parenterally via intramuscular or intravenous administration since its absorption from the GI tract is insufficient.

Localized drug delivery is one of the most effective strategies for delivering drugs to the specific targeted site, which led to more preferable therapeutic effects [16]. It has been reported that a localized delivery system showed superiority over the conventional systemic antibiotic delivery [17]. A higher dose reaching directly to the desired site, decreased systemic toxicity, reduced the bacterial resistance as well as improved patient compliance [17–19]. The ideal localized system should exhibit a burst release of a large amount of drug in the initial period and be followed by a continual therapeutic dose release to prevent latent infection [16,19]. Cefazolin has been experimentally loaded into several carriers including a PCL sponge pad or fibers [20,21], PCL/sodium-alginate [22], mesoporous silicon microparticles [23], hydroxyapatite [24] and gelatin nanofiber mats [25,26] for use as localized drug release systems. Initial burst release of cefazolin was typically seen and followed by a sustained release from 24 h to 32 days depending on the type of carrier.

Preventing the incidence of post-neurosurgical infection is clinically crucial during neurosurgical procedures. Since an implant was reported to be one of the risk factors for SSIs, the use of an antibacterial dural substitute with the localized antibiotics delivery would probably help in decreasing this risk. This would be used as an adjuvant or add-on treatment and not as a replacement for systemic antibiotics prophylaxis via an intravenous route. Previously, commercial collagen artificial dura mater was experimentally mixed with cefuroxime sodium, ceftriaxone sodium or norvancomycin aimed to provide sustained drug release directly to the brain surface and it overcame the problem of the blood–brain barrier that lowered drug concentrations in the cerebrospinal fluid compared to the venous blood [27]. However, no antibacterial activity was carried out and only a short release study for up to 72 h was performed, although the release time of 6–7 days was designed. Vancomycin soaked commercial crosslinked collagen based dural substitute was also investigated for possible use in infected and contaminated wounds. However, the study was brief and only colony counting and antibacterial activity at 24 h were measured [28]. Therefore, data of an antibiotic loaded dural substitute that can perform dual functions for dural restoration and local release of antibiotics in the prevention of surgical site infection are still limited and not exhaustively studied.

It was hypothesized that ORC/PCL bilayered composite which was developed and already passed in vitro and in vivo studies [3–5] could be further modified to act as an antibiotic dural substitute. The characteristics and properties of the carriers are important factors which could influence the drug releasing behavior of the material and the incorporation of drug in the carriers would in turn affect the property of the materials. This study was carried out to gain the understanding and knowledge to further develop

an antibacterial dural substitute, which has rarely been reported. In this study, cefazolin was chosen as a model antibiotic for investigation. Various concentrations of cefazolin were loaded into previously selected ORC/PCL bilayered composite (P20 formulation) and their effects on physical properties, mechanical properties, drug loading and releasing characteristics and antibacterial activity were investigated for potentially being used as an antibacterial dural substitute in dural closure application.

#### **2. Materials and Methods**

#### *2.1. Raw Materials*

All raw materials including polycaprolactone (PCL, Mw ~ 80,000, Sigma-Aldrich, St. Louis, MI, USA), oxidized regenerated cellulose (ORC, Surgicel®®, Nu-Knit®®, Ethicon Inc., Raritan, NJ, USA) and N-methyl-2-pyrrolidone (NMP, PharmasolveTM, Ashland Inc., Wilmington, DE, USA) were purchased and used in the as-received form. The antibiotic used was cefazolin sodium (Fazolin®®, Siam Bheasach Co., Ltd., Bangkok, Thailand) and was supplied in the powder form.

#### *2.2. Sample Preparation*

NMP was equally divided into two parts. Cefazolin powders were dissolved in the first part of NMP at the concentrations of 0, 25, 50, 75 and 100 mg/mL and stirred at room temperature until clear solution was obtained while 20 g of PCL was dissolved in the second part of NMP by heating at 60 ◦C using a hotplate until obtaining a clear viscous PCL solution. After cooling down to a temperature of approximately 40 ◦C, both parts were then mixed well together, producing 20% *w*/*v* cefazolin loaded PCL solutions. PCL solutions was used to evenly infiltrate both sides of ORC knitted woven fabric (30 × <sup>70</sup> × 1.0 mm3) and was then additionally recoated on one side of the loaded fabric as described previously [3,4]. The infiltrated and coated fabrics were then submerged in deionized water for 30 s to solidify the PCL and to leach out the solvent and they were then further dried in the oven at 40 ◦C for 24 h to obtain cefazolin loaded ORC/PCL bilayered composite (designated P20, P20\_25, P20\_50, P20\_75 and P20\_100, respectively). Cefazolin loaded PCL was also fabricated for use as comparative samples. The cefazolin loaded PCL solutions, which were prepared in the same manner as described previously, were poured into a cavity of a mold (30 × <sup>70</sup> × 1.0 mm3). The mold was then submerged in deionized water for 30 s and dried in the oven at 40 ◦C for 24 h to obtain cefazolin loaded PCL (designated PCL, PCL\_25, PCL\_50, PCL\_75 and PCL\_100, respectively). The concentration of cefazolin solution used to infiltrate the ORC/PCL bilayered composite or mix in PCL in this study was selected based on the concentration of cefazolin solution used for typical intravenous or intramuscular injection which ranges from about 20 mg/mL to 250 mg/mL. Table 1 shows the formulations that were employed to prepare the samples.


**Table 1.** Formulations of the prepared samples.
