Antibacterial wound dressing nanobrous mats composed of cellulose acetate loaded with chitosan/erythromycin nanoparticles

This study attempts to fabricate the antibacterial nanobrous mats composed of cellulose acetate (CA) nanobers loaded with erythromycin-chitosan nanoparticles (Ery-CS NPs) intended for infected wound dressing. The Ery-loaded CS NPs were prepared by ionic gelation process and then incorporated into the CA electrospun nanobers (NFs). The characterization of the NPs and mats were investigated by using dynamic light scattering (DLS), scanning electron microscopy (SEM), Attenuated Total Reection Fourier transform infrared (ATR-FTIR) to show the size distribution, morphology and surface chemistry along with other physiochemical properties including hydrophilicity property, porosity, and water absorption capacity. The antimicrobial activity and cell viability of broblast cells were also evaluated. The results indicated that Ery was loaded into CS NPs with high encapsulation eciency (95%). The CA NFs (17%) incorporated with the Ery-CS NPs (12 wt%) displayed smooth homogenous morphology with 141.7 ± 91.7 nm average diameter. The relevant analyses revealed that the NPs incorporated in NFs and provided high water holding capacity with high porosity. Finally, Ery-CS NPs/CA mats were able to inhibit the growth of both Gram-positive and Gram-negative bacteria as well as showed no cytotoxic effect on the human dermal broblast cells. Overall, our ndings concluded that the proposed system could be potentially applied as the proper antibacterial mats for infected wound dressing applications.

diverse therapeutics drugs or natural agents [9,22,23]. It seems that by blending the natural polymers with CA, these electrospun NFs can be provided the better properties for wound dressing applications [9].
The treatment by using natural polymers comprising chitosan (CS), propolis and starch have been exclusively considered for the wide variety of illnesses [24][25][26][27]. Chitosan, (1-4)-2-amino-2-deoxy-β-dglucan (Scheme 1b), is a polysaccharide gained from alkaline hydrolysis of chitin with that possesses plentiful proper features for wound dressing applications [25,26,28]. Moreover, the non-toxic products of CS, after degradation, are oligosaccharides which can be then extruded or combined to glycosaminoglycans and glycoproteins [26]. CS is a natural water-insoluble polymer that extensively applied for wound healing owing to several advantages including, excellent water absorption capacity, biodegradability, biocompatibility and antibacterial activity [28,29]. Thanks to mentioned-properties, chitosan can be exclusively used for the preparation of new products for wound dressing applications [26].
Erythromycin (Ery), is a medium-spectrum antibiotic, achieved by Streptomyces erythreus that contains a 13-carbon ring with two sugars involved by glycosidic linkages (Scheme 1c) [30,31]. It is very soluble in organic solvents and poorly soluble in water; it is valuable in the remedy of pneumonia, diphtheria, whooping cough, and acne handlings [30][31][32]. The common dosage for topical administration is about 20 mg/g [33].
The combination of Ery and natural polymers into electrospun NFs is an attractive issue if wound healing and drug delivery are desired. These systems can provide a novel and effective therapy to protecting the wound site from bacterial infection [30]. It is hypothesized that a proper electrospun nano ber containing both antibacterial agents and antibacterial NPs could be result in a perfect antibacterial wound dressing material. Therefore, we tried to integrate the advantages of CA electrospun NFs with Ery-loaded CS NPs to improve antibacterial activity for wound healing applications. In this regard, fabrication of polyvinyl alcohol (PVA)/carboxymethyl cellulose (CMC)-ZnO NFs containing Ery was investigated to employ for wound dressing. The results obtained from this study revealed that the proposed system provided excellent antibacterial activity against S. aureus and E. coli. In vitro study displayed that Ery release from PVA-CMC/ZnO-EM NFs was gradually increased [34].
The major aim of this study was to fabricate an ideal nano brous wound dressing mats based on CA loaded with CS/Ery NPs through the electrospinning process. For this purpose, The Ery-loaded CS NPs were made and then incorporated into the CA electrospun nano bers. Physicochemical properties of nanoparticles and CA NFs were measured by using dynamic light scattering (DLS), scanning electron microscopy (SEM), Attenuated Total Re ection Fourier transform infrared (ATR-FTIR) and contact angle measurement. Finally, the antibacterial activity and biocompatibility of this proposed system were assessed.

Synthesize Ery-loaded CS NPs
Ery loaded CS NPs were synthesized by ionic gelation method previously described [35]. First, CS was dissolved in an aqueous solution of acetic acid (1% (v/v)) to form 2 mg/ml of CS solution under overnight stirring at room temperature. Then, Ery (10-30 mg) was mixed with the CS solution and stirred for 1 hour. Next, TPP in water solution (0.2 w/v) was injected drop wisely ( ow rate 1 mL/min) to the CS solution (TPP to CS: 20% (v/v)) under moderate stirring for 1 hour. After that, the Ery-CS NPs were collected after centrifugation at 10,000 rpm for 1 h at 4 °C, redispersed in deionized water and freeze-dried.At last, the supernatant was gathered for the quanti cation of loading and encapsulation e ciency.

Fabrication of CA NFs loaded with Ery-CS NPs
CA NFs loaded with Ery-CS NPs were fabricated under optimized conditions using the electrospinning process. Electrospinning the solutions was carried out using an electrospinning apparatus set at an 18 kV applied voltage, ow rate 1 mL/h, and a 15 cm tip-to-collector distance. The NFs were collected on a metallic rotating drum wrapped with a metal foil. According to the given conditions, CA solutions (17% w/v) were prepared with a mixture of acetic acid/water at different proportions 80:20, 90:10 and 100:0 v/v. Then, the obtained NFs were evaluated which the ratio 90:10 v/v was selected as the proper proportion. Following, CA solution (17% w/v) in acetic acid-water (90:10 v/v) and Ery-CS NPs at different concentration (5 ,8 ,12 ,15 wt%) were blended and was produced the electrospun bers. Finally, the NFs removed from the metal foil and stored for further characterizations and applications.

Dynamic light scattering and Zeta potential analyses
The size and size distribution of Ery-CS NPs were measured by scatteroscope I (K-ONE, Korea) after the preparation of CS NPs. Moreover, the zeta potential of the NPs was determined by electrophoretic light scattering (Zeta sizer, Malvern, UK) at 25 °C, 150 V. For this purpose, the NPs were dispersed in phosphate buffer saline (pH 7.4), and the zeta potential was measured in triplicate for each sample.

SEM analysis of the NPs
The size and morphology of the Ery-CS NPs were also studied by SEM (XL30 Philips, The Netherlands) at an accelerating voltage of 20.0 kV after sputtering gold.

Encapsulation e ciency and drug loading measurements
The percentage of encapsulation e cacy (EE %) of Ery in CS NPs and drug loading (DL%) were analyzed by UV Vis spectrophotometer (Cecil Instruments Ltd., UK) at 205 nm. The concentration of free Ery in the supernatant was measured based on a previously created calibration curve for Ery and then EE and DL were calculated by using the following equation: Where A is the total amount of Ery, B is the amount of free Ery available in the supernatant, and C is the weight of NPs after freeze-drying.

Morphology and Size assessment
The morphology and diameter of the NFs were observed by using a SEM (DSM 960A, Zeiss, Germany) applied at an acceleration voltage of 15 kV and the NFs coated with gold plasma sputter. Image-J software was also used to analyze the NFs diameter distributions.

Attenuated total re ection FTIR
The analysis of the functional groups and bonding con gurations of the samples were characterized using ATR-FTIRspectroscopy (Bruker 125HR), which was recorded between 600-4000 cm − 1 at room temperature.

Water absorption
The water absorption capacity of free CA NFs and Ery-CS/CA NFs mats was determined by immersion in water (pH 7.4) at 37 °C [36]. The piece of mats precisely weighed and then were immersed for 1, 3, 6, 12, 24 and 72 hours in the water. After immersion for respective periods, the samples were taken out from the medium, and excess uid on the surface of each specimen was carefully drained off by lter paper. Afterward, the mats were weighed as wet weight. The water absorption percentage (W) of the mats was calculated using the following equations: The W0 and W1 de ne the weight of dried and wet mat, respectively. The mean value was obtained from three parallel samples.

Contact-angle measurement
The hydrophilicity of free CA NFs and Ery-CS/CA NFs mats was determined by water contact angle measurement. The sessile drop technique was performed using a contact angle measuring system (G10, KRUSS, and Germany). For this objective, 4 µL distilled water droplet was located on the surface of each mat. The values were reported as the average of three samples for each test.

Porosity evaluation
The determination of the porosity of free CA NFs and Ery-CS/CA NFs was measured using the liquid displacement method [37]. The mats immersed in absolute ethanol for 1 hour and then the porosity was measured by the following equation: where V1, V2, and V3 represent the initial volume of ethanol, the volume after immersing and the volume of the ethanol after the mat removal (after 1 h), respectively.

Antibacterial activity assessment
The antibacterial experiments of samples were performed using disc-diffusion and broth microdilution methods against Gram-positive (S. aureus ATCC 25923) and Gram-negative (E. coli ATCC 2592 and P. aeruginosa ATCC 12228) bacteria. These micro-organisms commonly found on infectious skin and wounds. In disc-diffusion method, the nano brous mats including CA, Ery/CA, CS NPs/CA and Ery-CS NPs/CA, were cut into circular discs and were placed on the surface of nutrient agar plates. The discscontaining plates incubated for 24 h at 37 °C. Then, the clearance zones were measured in diameter. The broth microdilution method was also applied to determine the minimal inhibitory concentration (MIC) of Ery, CS NPs and Ery-CS NPs [38,39]. All strains were grown in nutrient broth media adjusted to 10 6 colony-forming units (CFU)/mL, then was added to each tube. After UV sterilization, using the serial dilution method, 10 different concentrations of each sample were added to the bacterial suspensions. All tubes were incubated at 35 °C for 24 h in an orbital shaker, and then their turbidity was examined against light. Bacterial suspension without treatment and nutrient broth media were used as a positive and negative control, respectively. The lowest concentration at which inhibit the visible growth was considered as the MIC.

Cell viability assay
The viability of cultured human dermal broblast cells (Pasteur Institute of Iran, Tehran, Iran) on CA NFs, Ery/CA NFs, CS/CA NFs and Ery-CS NPs/CA NFs was evaluated using MTT colorimetric assay [40]. The mats were rst to cut into proper sizes and placed in each well of the 96-well plate. After sterilizing the mats by UV radiation, the cells were seeded onto the mats at a density of 1 × 104 cells/well, followed by incubation for 24 h, 48 h, and 72 h. After each incubation time, the culture medium was removed from each well and the wells were washed with PBS three times. Then fresh medium with the MTT (5 mg/mL) reagent was added to each well followed by incubation for 3 h at 37 °C. The optical density of each well at 570 nm was measured by an ELISA reader (Perkin-Elmer, USA,). The cell viability was identi ed by comparing the absorbance of cells cultured on the mats to that of control well having cells. The experiments carried out for each sample in triplicate.

Statistical analysis
All values were reported as the mean ± standard deviation (SD). Data were analyzed by one-or two-way analysis of variance (ANOVA) by GraphPad Prism 8 software. P < 0.05 was considered statistically signi cant (*: P < 0.05).

Results And Discussion
The fabrication of antibacterial wound dressing/matrices is necessary for amelioration of lesions and the prevention of infection in the skin and soft tissues. In this regard, CA electrospun NFs have indicated striking properties to dermal/transdermal or wound dressing applications [9,23]. Moreover, CS NPs have brought great bene ts for the loading/delivery of therapeutics agents and wound dressing materials due to its unique physicochemical and inherent antibacterial properties [41][42][43]. Herein, we attempt to present the bene ts of CA NFs concurrent with the therapeutic effects of CS NPs to delivery of Ery for enhancing antibacterial wound healing applications.

Particle size and zeta potential characterization of Ery loaded CS NPs
The unloaded CS particles and loaded with Ery were analyzed by dynamic light scattering (DLS) technique to obtain the particle size distribution. The resultant plot showed that the mean particle size of unloaded particles was 115 nm (Fig. 1a) and loaded particles with Ery was 251 nm (Fig. 1b). The observed difference in the average size of the prepared NPs is probably due to the high drug loading in Ery-CS NPs. Also, different concentration of Ery (10 to 30 mg) was used to preparation of Ery-CS NPs, which does not signi cant effect on the size distribution of Ery-CS NPs.
The morphology of loaded CS NPs with the formulation having 30 mg Ery in aqueous phase was observed by SEM, which is shown in Fig. 1c and d. The SEM micrograph was con rmed a spherical shape with smooth surfaces and uniform size of the prepared NPs with around 50 nm > in size. Furthermore, the zeta potential of CS NPs and Ery-CS NPs was about + 14.7 mV (Fig. 1e) and + 14.9 mV (Fig. 1f), respectively, which is considered a reasonable surface charge for CS NPs due to positive charge of functional groups in CS chains. Besides, positively charged NPs indicated that can be adsorbed on the bacterial surface and closely connected with bacteria [44].

Loading and encapsulation e ciency
The high concentration of Ery (30 mg) resulted in higher EE and DL, as a result, this preparation condition was chosen as the optimal formula and used for all further experiments including EE, DL and electrospinning. EE and DL of the optimized Ery loaded CS NPs were measured to be 95.3 ± 3.1% and 41.14 ± 0.05%, respectively. Hence, Ery was well encapsulated into CS NPs, and indicated a high amount of Ery could incorporate in CS NPs.

The optimum condition for electrospun ber mats of CA and Ery-CS NPs/CA
Prior to the addition of Ery-CS NPs to CA solution for electrospinning, CA solution (17% w/v) with the different ratios of acetic acid/water solvents were prepared and the diameter, morphology and their berforming abilities were tested through electrospinning. Figure 2a-c shows the SEM micrograph images of CA electrospun bers at different ratio of the solvents as follows: 80:20 v/v (a), 90:10 v/v (b) and 100:0 v/v (c); the diameter distribution of the prepared NFs was 1133.8 ± 446.7 nm, 269 ± 74.6 nm, and 336.3 ± 133.9 nm, respectively. Moreover, the SEM micrographs revealed that the electrospinning of CA (17% w/v) produced the smooth, cylindrical, ribbon-shaped and bead-free NFs. Whereas increasing the acetic acid concentration from 90 to 100% v/v led to the formation of non-uniform CA NFs in diameter and morphology (Fig. 2c). The reason why the morphology of the electrospun bers was changed is rooted in the fact that changing acetic acid concentration is associated with the rheological behavior of the CA solution as well as with the stretching of the electrospinning jet [45,46]. Subsequently, the prepared NFs with 90:10 v/v acetic acid/water was used for further work due to the lower diameter, proper morphology and uniform structure.
In the following, different concentration of Ery-CS NPs (5-8-12-15 wt%) was incorporated in the optimized CA solution and then their ber-forming abilities by electrospinning as well as the diameter distribution and morphology were evaluated. The obtained SEM micrographs along with their diameter distributions are illustrated in Fig. 3a-d. The morphological observation revealed that the CA solution containing 5 to 15 wt% of Ery-CS NPs generated bers with different morphologies. Electrospinning of CA having 5 wt% of the NPs produced relatively uniform and bead-free NFs with the diameter distribution of 204.6 ± 67.7 nm (Fig. 3a). The more uniform and cylindrical bead-free bers with diameter distribution of 210 ± 72.6 nm were formed while the NPs concentration was augmented to 8 wt% (Fig. 3b). Increasing concentration of the NPs to 12 wt% resulted in a blend of cylindrical and at bers with negligible beads of diameter distribution 141.7 ± 91.7 nm as depicted in Fig. 3c. As the concentration of the NPs increased to 15 wt% generated non-continuous, ribbon or at-shaped bers with elongated beads (Fig. 3d), which could reduce the active surface area of bers [45] and is not suitable for our purposes. The morphology of the prepared bers was affected by the incorporated amount of the NPs. Besides, the amounts of the NPs higher than 12 wt% not produced the proper bers. This is probably due to the escalated viscoelasticity of the solution and the lack of CA chains entanglement in the solution which is crucial for electrospinning. Finally, the mats containing 12 wt% of the NPs due to the lowest diameter, unique morphology, and maximum loading in the NFs were selected and employed to further use. Also, under the same condition, 12 wt% of Ery was incorporated in CA NFs for use in relevant tests.

ATR-FTIR Characterization
ATR-FTIR spectroscopy analysis was conducted to characterize the surface functional groups and the interaction between the constituted components of the Ery-CS NPs/CA NFs. The spectra are shown in Fig. 4a. The spectrum of CA NFs (Fig. 4a 1)  The spectrum of CS NPs (Fig. 4a 3) presents characteristic absorption peaks located at 1040 cm − 1 and 1234 cm − 1 correspond to the stretch vibration of C-O bond, 1639 cm − 1 related to the N-H bending vibration, 2926 cm − 1 corresponds to CH 2 stretching vibration, and a peak at 3500 to 3200 cm − 1 corresponds to -OH stretching of alcoholic and phenolic groups, which are in agreement with the literature [51][52][53]. In the FTIR spectra of Ery-CS NPs (Fig. 4a 4), it is clear that the characteristic peaks of CS NPs and Ery are both presented in the Ery-CS NPs, which demonstrated the CS NPs were loaded with Ery. The spectrum of the CA NFs incorporated with 12 wt% Ery-CS NPs showed both distinctive peaks of CA NFs and the characteristic bands attributed to Ery-CS NPs (Fig. 4a 5), indicating the NPs were e ciently loaded on the CA NFs. Besides, the widening of absorption peaks was detected in the region of 3100-3600 cm −1 related to the stretching of intermolecular hydrogen bonds of hydroxyl groups (-OH) as well as the peak related to the stretching of acetyl groups is weakened. These properties are generally caused by intermolecular interactions, as well as due to the presence of physical interactions between CA and Ery-CS NPs.

Determination of Porosity
The proposed NFs with higher surface area and higher porosity was exclusively applied for antibacterial wound dressing. The porosity measurements revealed that the CA NFs and Ery-CS NPs/CA NFs possess 90.03 ± 1.5% and 91.3 ± 0.9% porosity of the total mat volume, respectively. The resultant NFs mats exhibit a remarkably high porosity, as well as the addition of Ery-CS NPs, caused a slight increment in porosity, however, the changes between porosity values are not statistically signi cant. The presence of large volume of porosity would be helpful for the exchange of oxygen and nutrients to the interior of the wound dressing [54].

Contact angle measurement
An appropriate wound dressing should be hydrophilic to be capable of absorbing the wound exudate and maintaining the moisture levels at the wound bed [55]. The contact angle of a water droplet on the CA NF mats and Ery-CS NPs/CA NF mats are shown in Fig. 4b during the period of 1 to 10 s. The measured contact-angle of CA mats (Fig. 4b 1) was found to be 116.5°, 77.8°, 63.3° and 0°, and also for Ery-CS NPs/CA mats (Fig. 4b 2) was found to be 100.7°, 63.7°,46.5° and 0° in 10, 5, 2 and 1 s, respectively.
Although CA is hydrophobic, water drops rapidly penetrate into the CA mats because of its highly porous structure [27]. Moreover, the accumulation of the NPs into CA NFs led to a decrease in the contact angle values. This could be due to the presence of Ery-CS NPs that increased surface hydrophilicity of the mats.

Water absorption ability
As the other valuable characteristics of a wound dressing, water absorption was measured and the results were shown in Fig. 4c. The water holding capacity of CA mats reached to about 206% and 301% in the 1 and 48 h soaking time, and Ery-CA NPs/CA mats reached to about 276% and 491% in the 1 and 48 h soaking time, respectively. After 48 h immersion, the Ery-CA NPs/CA mats exhibited a high water absorption capacity of about 500% and about 200% more than CA NFs, which is due to the high wateruptake ability of CS NPs [56]. The Ery-CA NPs/CA mats provided a signi cant water-holding capacity, which can be applied as a wound dressing to absorb the wound uids\exudates.

Antibacterial activity
The antibacterial activity of CA, Ery/CA, CS NPs/CA and Ery-CS NPs/CA ber mats against the both positive and negative bacteria was tested by using the disk diffusion method. No zone of inhibition was observed near the disk made only CA nano brous mats in the three strains of bacteria (Fig. 5a-c 1). Nevertheless, such nano brous mats can create perfect blocks and pores that can prevent bacterial penetration and exogenous infections. The diameter of inhibition ring was around 20 mm, 18 mm and 15 mm for Ery/CA mats (Fig. 5a-c 2) and was around 25 mm, 22 mm and 18 mm for the CA mats loaded CS NPs (Fig. 5a-c 3) against S. aureus, E. coli and P. aeruginosa respectively. In case of Ery-CS NPs/CA mats, the diameter of the inhibition zones reached 30 mm, 25 mm and 20 mm, as shown in Fig. 5a-c 4. These results demonstrated the superior antibacterial activity of the Ery-CS NPs/CA mats in comparison to Ery/CA and CS NPs/CA nano brous mats that is probably due to the merged antibacterial activity of Ery and CS NPs.
Subsequently, the antibacterial activity of the constituents of Ery-CS NPs/CA mats were also investigated by MIC testing that the results are shown in Table 1. The applied strains possessed MIC values ≥ 100 µg/mL and presented highly resistant to Ery. CS NPs showed high bactericidal activity against both positive/negative strains, however, indicated the lower antibacterial effect on the negative strains than the positive strain. The antibacterial activity of CS NPs is resulted from the alteration of membrane penetrability leading to membrane disruption and leakage of cytoplasm [57]. Furthermore, the MIC value of Ery-CS NPs was about 5 µg/mL against S. aureus, and also 20 and 80 µg/mL against E. coli and P. aeruginosa (Table 2), which is lower than Ery and CS NPs. This could be due to the combinational antibacterial effects of the CS NPs and Ery. As a result, the ndings con rmed that the developed nano brous mat provides a high antibacterial e ciency and could be operative to treat infected wounds. This could be owing to the greater hydrophilic nature of the CS NPs (Fig. 6). Besides, in case of Ery/CA mats, cell proliferation decreased which was probably due to adverse effects of Ery on the cell growth on the broblasts. There was no signi cant distinction in cell viability between the different groups compared to the control group, indicating the biocompatibility of the proposed nano brous mats. The results showed that Ery-CS NPs/CA NFs can be potentially applied as biocompatible and biodegradable mat for wound dressing applications.

Conclusions
This study seeks to integrate the potential antibacterial activity of CS NPs and Ery incorporated into CA electrospun NFs to develop a suitable antibacterial wound dressing. The optimized solvent ratio of acetic acid: water (90:10 v/v) was established to fabricate CA electrospun NFs mat. Ery loaded CS NPs was synthesized by ionic gelation method that exhibited the nano-scale size with reasonable zeta potential and high encapsulation e ciency and drug loading. Afterward, different concentrations of Ery-CS NPs were incorporated into the CA NFs that the 12 wt% of NPs produced the more appropriate size distribution and morphology speci cations. Moreover, the physicochemical characteristics of the fabricated mats were con rmed by ATR-FTIR, porosity, contact angle and water absorption analyses. Ery-CS NPs/CA mats showed the effective antibacterial activity toward both Gram-positive and Gram-negative bacteria. The cell viability assay showed non-cytotoxic effects on human broblasts, con rming biocompatibility of the nano brous mats. All these ndings indicate that these Ery-CS NPs/ CA mats can be employed as an e cient nano ber-based antibacterial wound dressing material.    MTT cell growth measurement assay of the nano brous mats after 48 and 72 h incubation. The viability of control cells was set at 100%.