Synthesis of Antibacterial Gelatin/Sodium Alginate Sponges and Their Antibacterial Activity

In the present study, sponges with the antibiotic tetracycline hydrochloride (TCH) loaded into alginate incorporated with gelatin (G/SA) were fabricated. The G/SA sponges were characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric (TG) analysis. G/SA sponges show a three-dimensional network structure with high porosity. An excellent swelling behavior and a controlled TCH release performance are observed from G/SA sponges. Moreover, they exhibit good antibacterial activity against both Gram-positive and Gram-negative bacteria.

Polymers 2020, 12, 1926 3 of 11 Akron, OH, USA). TG analysis was carried out using a model Q5000 TGA (TA Instruments, New Castle, DE, USA) with a heating rate of 10 • C/min under nitrogen atmosphere.

Swelling Assays and Porosity Determination
The swelling behaviors of the sponges were determined through a gravimetric method. First, the sponges were cut into round pieces with the diameter of 10 mm and their dry weights (W 0 ) were accurately measured. Each sample was immersed in DI water at room temperature. The swollen sponges were withdrawn from the water and their wet weights of the swollen sponges (W 1 ) was measured after the removal of excess surface water by gently blotting with a filter paper. The swelling ratio of the sponges was calculated as follows: The porosity was determined using an immersing method in the ethanol until saturation. The samples with the diameter of 10 mm were weighed before and after immersion into the ethanol. All testing was proceeded in triplicate. The porosity was calculated according to Equation (2): where W 1 and W 2 are the weights of tested sponges before and after immersion in the ethanol, ρ is the density of ethanol and V is the volume of the sponge.

TCH Loading Calculation
TCH loadings were calculated according to the original concentration of TCH (C 0 ) and the concentration of unloaded TCH (C 1 ) determined by a Spectrum Two Spectrometer (Perkin Elmer, Akron, OH, USA) at the monitoring wavelength of 356 nm. TCH loadings (W) in the G/SA sponges were calculated using the following equation: where V is the volume of total TCH solution, A is the area of the G/SA sponge.

TCH Release Behaviors
The TCH release behaviors were studied in 0.1 M phosphate buffer saline (PBS) buffers with pH 7.4. The sponges were cut into round pieces with the diameter of 10 mm. The tested samples were fully immersed in a beaker containing 50 mL PBS at 37 • C and placed in a dark place. At specific time points, an aliquot of 3.5 mL was collected from each solution and the absorbance was then measured at 356 nm on a Spectrum Two Spectrometer (Perkin Elmer, Akron, OH, USA). An equivalent volume of fresh PBS buffer was replaced into the system after each sampling to maintain constant medium volume. Thus, the concentrations of released TCH obtained at different times can be calculated. As a result, the cumulative released TCH amounts can be calculated accordingly. The experiments were performed in triplicate.

Antibacterial Activity
The antibacterial activities of G/SA sponges were investigated against Gram-negative E. coli ATCC 25,922 and B. subtilis ATCC 9372, Gram-positive S. aureus ATCC 6538 and P. aeruginosa ATCC 27,853 by disk diffusion method. G/SA sponges were cut into round shapes with 10 mm diameter and sterilized by ultraviolet lamp for 60 min. Lawns of test bacteria (about 1 × 10 5 CFU/plate) were prepared on TSA. The sterilized samples were then carefully placed upon the lawns. The plates were placed in a

Surface Morphology
The exudate absorption capacity is directly affected by the surface morphology and porosity of polymer-based wound dressings, so the morphologies of G/SA and TCH-loaded G/SA (G/SA5) sponges were studied using SEM ( Figure 1). Figures 1A,B show the cross-sectional morphologies of G/SA sponge with different magnifications. It can be clearly seen that G/SA sponge exhibits a highly porous three-dimensional network structure with interconnected pores throughout the hybrid sponge. After TCH was loaded into the sponge, small difference in pores shape and size can be found although G/SA5 sponge still keeps its 3D structure, as shown in Figures Figure 2 shows the porosities of G/SA and TCH loaded G/SA sponges. The G/SA sponge shows a relatively high porosity of 90.2%. After TCH was loaded into G/SA sponges, a slight decrease in porosity to a range of 87.1%-88.5% can be observed. TCH loadings of G/SA composite sponges are listed in Table 1. It can be clearly found that TCH loading increases with the increase of initial TCH concentration. These high porosities benefit for exudates absorbing, making the fabricated sponges show great potentials in wound dressings application.   Figure 2 shows the porosities of G/SA and TCH loaded G/SA sponges. The G/SA sponge shows a relatively high porosity of 90.2%. After TCH was loaded into G/SA sponges, a slight decrease in porosity to a range of 87.1-88.5% can be observed. TCH loadings of G/SA composite sponges are listed in Table 1. It can be clearly found that TCH loading increases with the increase of initial TCH concentration. These high porosities benefit for exudates absorbing, making the fabricated sponges show great potentials in wound dressings application.

Thermal Properties
The TG and derivative thermogravimetric (DTG) curves of SA, gelatin and G/SA5 are shown in Figure 3. Three weight loss stages were found in the TG curve of SA (curve a in Figure 3A). The first weight loss stage occurs below 120 °C, which is attributed to the evaporation of absorbed water. The second weight loss occurred in the range of 200 °C-350 °C involving the degradation of main backbone of SA and the formation of sodium carbonate. The third weight loss stage starting from 350 °C to 730 °C is the decomposition of both SA and sodium carbonate [24,25]. The residue of SA is 30.8%, corresponding to the formed carbon dioxide and calcium oxide [26]. Thermal degradation of gelatin occurs in two stages (curve b in Figure 3A). The first stage occurs from room temperature up to 180 °C and the second stage occurs between 180 °C and 370 °C corresponding to the degradation of protein [27].

Thermal Properties
The TG and derivative thermogravimetric (DTG) curves of SA, gelatin and G/SA5 are shown in Figure 3. Three weight loss stages were found in the TG curve of SA (curve a in Figure 3A). The first weight loss stage occurs below 120 • C, which is attributed to the evaporation of absorbed water. The second weight loss occurred in the range of 200-350 • C involving the degradation of main backbone of SA and the formation of sodium carbonate. The third weight loss stage starting from 350 • C to 730 • C is the decomposition of both SA and sodium carbonate [24,25]. The residue of SA is 30.8%, corresponding to the formed carbon dioxide and calcium oxide [26]. Thermal degradation of gelatin occurs in two stages (curve b in Figure 3A). The first stage occurs from room temperature up to 180 • C and the second stage occurs between 180 • C and 370 • C corresponding to the degradation of protein [27].

Thermal Properties
The TG and derivative thermogravimetric (DTG) curves of SA, gelatin and G/SA5 are shown in Figure 3. Three weight loss stages were found in the TG curve of SA (curve a in Figure 3A). The first weight loss stage occurs below 120 °C, which is attributed to the evaporation of absorbed water. The second weight loss occurred in the range of 200 °C-350 °C involving the degradation of main backbone of SA and the formation of sodium carbonate. The third weight loss stage starting from 350 °C to 730 °C is the decomposition of both SA and sodium carbonate [24,25]. The residue of SA is 30.8%, corresponding to the formed carbon dioxide and calcium oxide [26]. Thermal degradation of gelatin occurs in two stages (curve b in Figure 3A). The first stage occurs from room temperature up to 180 °C and the second stage occurs between 180 °C and 370 °C corresponding to the degradation of protein [27]. In the case of G/SA5 sponge (curve c in Figure 3A), two weight loss stages were shown. The first one is the loss of adsorbed water which occurs below 200 • C. The second one is the complex decomposition Polymers 2020, 12,1926 6 of 11 of SA, gelatin, sodium carbonate and TCH in the range of 200-780 • C. The residue of G/SA5 is 31.2%, which is higher than those of SA and gelatin (21.1%) due to the existence of TCH. Therefore, TCH are demonstrated successfully to be incorporated into G/SA sponges.

FTIR Analysis
FTIR spectra of G/SA, G/SA1, G/SA2, G/SA3, G/SA4, G/SA5 and TCH are given in Figure 4. In the case of G/SA (curve a), characteristic bands located at 3328 cm −1 , 1559 cm −1 correspond to the stretching of -NH and -OH groups, and the N-H deformation (amide II) of gelatin [28]. Moreover, the peak at 1624 cm −1 is assigned to the C=O stretching (amide I) of gelatin and the asymmetric COO stretching vibration of SA. The absorption band at 1419 cm −1 corresponds to the symmetric COO stretching vibration of SA [13,29]. The asymmetric and symmetric COO stretching vibrations of the reported SA without any cross-linking are located at 1612 and 1417 cm −1 , respectively [30]. They show a blue shift due to the formation of ionic bonding between -COOand Ca 2+ in this study. In the FTIR spectrum of TCH (curve g), the peaks located at 1665 cm −1 , 1617 cm −1 , 1590 cm −1 , 1545 cm −1 and 1440 cm −1 are assigned to the C=O vibration of amide I, the C=O vibration of A-ring, the C=O vibration of C-ring, the N-H deformation of amide II and the C=C vibration of aromatic ring, respectively [31]. For TCH-loaded G/SA sponges (curves b-f), the intensity of peak at 1545 cm −1 increases with TCH loading raising although most characteristic peaks of TCH overlaps with the peaks of G/SA. Thus, the existence of TCH in the G/SA sponges is verified.
Polymers 2020, 12, x FOR PEER REVIEW 6 of 11 of G/SA5 is 31.2%, which is higher than those of SA and gelatin (21.1%) due to the existence of TCH. Therefore, TCH are demonstrated successfully to be incorporated into G/SA sponges.

FTIR Analysis
FTIR spectra of G/SA, G/SA1, G/SA2, G/SA3, G/SA4, G/SA5 and TCH are given in Figure 4. In the case of G/SA (curve a), characteristic bands located at 3328 cm −1 , 1559 cm −1 correspond to the stretching of -NH and -OH groups, and the N-H deformation (amide II) of gelatin [28]. Moreover, the peak at 1624 cm −1 is assigned to the C=O stretching (amide I) of gelatin and the asymmetric COO stretching vibration of SA. The absorption band at 1419 cm −1 corresponds to the symmetric COO stretching vibration of SA [13,29]. The asymmetric and symmetric COO stretching vibrations of the reported SA without any cross-linking are located at 1612 and 1417 cm −1, respectively [30]. They show a blue shift due to the formation of ionic bonding between -COOand Ca 2+ in this study. In the FTIR spectrum of TCH (curve g), the peaks located at 1665 cm −1 , 1617 cm −1 , 1590 cm −1 , 1545 cm −1 and 1440 cm −1 are assigned to the C=O vibration of amide I, the C=O vibration of A-ring, the C=O vibration of C-ring, the N-H deformation of amide II and the C=C vibration of aromatic ring, respectively [31]. For TCH-loaded G/SA sponges (curves b-f), the intensity of peak at 1545 cm −1 increases with TCH loading raising although most characteristic peaks of TCH overlaps with the peaks of G/SA. Thus, the existence of TCH in the G/SA sponges is verified.

Swelling Behaviors In Vitro
The water absorbing and holding abilities are essential characteristics for polymer-based wound dressings which facilitate the adsorption of exudates, body fluids or metabolites. Figure 5 shows the dynamic swelling performance of G/SA composite sponges. The swelling ratio of G/SA sponge is 2190% after 24 h, which is the highest swelling ratio among the tested sponges. SA has many hydrophilic groups including carboxylic and hydroxyl groups, and gelatin also contains carboxylic and hydroxyl groups as well as amino groups, leading to a high swelling performance. Meanwhile, TCH loaded G/SA sponges show slightly reduced swelling performance with the swelling ratios ranging from 1780 to 1960% after 24 h. This phenomenon could be due to the fact that the porosities of sponges were slightly decreased after being loaded with TCH.

Swelling Behaviors In Vitro
The water absorbing and holding abilities are essential characteristics for polymer-based wound dressings which facilitate the adsorption of exudates, body fluids or metabolites. Figure 5 shows the dynamic swelling performance of G/SA composite sponges. The swelling ratio of G/SA sponge is 2190% after 24 h, which is the highest swelling ratio among the tested sponges. SA has many hydrophilic groups including carboxylic and hydroxyl groups, and gelatin also contains carboxylic and hydroxyl groups as well as amino groups, leading to a high swelling performance. Meanwhile, TCH loaded G/SA sponges show slightly reduced swelling performance with the swelling ratios ranging from 1780 to 1960% after 24 h. This phenomenon could be due to the fact that the porosities of sponges were slightly decreased after being loaded with TCH.

TCH Release Behavior
The TCH release behaviors were determined in 0.1 M PBS buffer at pH 7.4 and the result is shown in Figure 6A. A burst release behavior was displayed in the first 1 h for the TCH-loaded G/SA sponges and this behavior could be probably due to the accumulation of TCH on the surface of G/SA sponges. Then a gradual increased release behavior for the G/SA composite sponges was shown and the release behavior lasts for 12 h. Moreover, the released amount is positively related to the TCH loadings in the G/SA sponges.

TCH Release Behavior
The TCH release behaviors were determined in 0.1 M PBS buffer at pH 7.4 and the result is shown in Figure 6A. A burst release behavior was displayed in the first 1 h for the TCH-loaded G/SA sponges and this behavior could be probably due to the accumulation of TCH on the surface of G/SA sponges. Then a gradual increased release behavior for the G/SA composite sponges was shown and the release behavior lasts for 12 h. Moreover, the released amount is positively related to the TCH loadings in the G/SA sponges.
Polymers 2020, 12, x FOR PEER REVIEW 7 of 11 Figure 5. Swelling behaviors of G/SA composite sponges.

TCH Release Behavior
The TCH release behaviors were determined in 0.1 M PBS buffer at pH 7.4 and the result is shown in Figure 6A. A burst release behavior was displayed in the first 1 h for the TCH-loaded G/SA sponges and this behavior could be probably due to the accumulation of TCH on the surface of G/SA sponges. Then a gradual increased release behavior for the G/SA composite sponges was shown and the release behavior lasts for 12 h. Moreover, the released amount is positively related to the TCH loadings in the G/SA sponges.  Thus, G/SA composite sponge is considered to be a good TCH carrier with controlled release performance, demonstrating its potential wound dressing application in the treatment of infections and inflammation.
Four mathematical models, including zero order, first order, Higuchi and Korsmeyer-Peppas models were used to fit the TCH release profiles. The obtained results are shown in Table 2. The best fitting mathematical model for the TCH release mechanism was determined by the calculated correlation coefficient (R 2 ). It can be found that the Korsmeyer-Peppas model is the best fitting one, which is a semi-experimental exponential function equation considering diffusion of drug and erosion of polymer matrix [16]. The plots of ln(Mt/M) versus ln(t) using Korsmeyer-Peppas model are displayed in Figure 6B. The diffusional constant n indicates the drug release mechanism. In this study, n values lie between 0.45 and 0.89, suggesting the TCH release behavior follows non-Fickian diffusion, which corresponds to the combined effects of the drug releases by dissolution, diffusion and erosion of matrix.

Antibacterial Activity
The antibacterial activities of G/SA composite sponges towards Gram-negative E. coli ATCC 25,922 and B. subtilis ATCC 9372, Gram-positive S. aureus ATCC 6538 and P. aeruginosa ATCC 27,853 were investigated by the disc diffusion method. The antibacterial activity is determined by measuring the clear zone of inhibition around the samples after 24 h incubation (Figure 7). For G/SA without any TCH loading, no inhibition zones were observed around the tested four strains, implying that G/SA sponge does not have any antibacterial activities. While, G/SA composite sponges have different levels of antibacterial activities. The measured average diameters of inhibition zones are shown in Figure 8. It can be clearly seen that the inhibition zone increases with the increase of TCH loading in the G/SA sponge. Moreover, G/SA composite sponges show a good antibacterial activity against E. coli, S. aureus and B. subtilis. However, little antibacterial activity against P. aeruginosa was shown. The differences in the antibacterial activity could be due to the susceptibility difference against different strains of the loaded TCH. Therefore, G/SA composite sponges show good antibacterial activities, which makes them have great potentials in the antibacterial wound dressing materials applications.
Polymers 2020, 12, x FOR PEER REVIEW 9 of 11 antibacterial activities, which makes them have great potentials in the antibacterial wound dressing materials applications.

Conclusions
In summary, TCH-loaded G/SA sponges were successfully synthesized and they display good swelling behavior and high porosity. G/SA composite sponges exhibit three-dimensional network structures, which help the G/SA sponges to facilitate TCH loading. The TCH-loaded G/SA sponges show controlled release performance and good antibacterial effects against E. coli ATCC 25922 and B. subtilis ATCC 9372 and S. aureus ATCC 6538. Thus, the fabricated G/SA sponges show potential as promising substrates for drug delivery applications in wound dressing and other biomedical applications.

Conclusions
In summary, TCH-loaded G/SA sponges were successfully synthesized and they display good swelling behavior and high porosity. G/SA composite sponges exhibit three-dimensional network structures, which help the G/SA sponges to facilitate TCH loading. The TCH-loaded G/SA sponges show controlled release performance and good antibacterial effects against E. coli ATCC 25922 and B. subtilis ATCC 9372 and S. aureus ATCC 6538. Thus, the fabricated G/SA sponges show potential as promising substrates for drug delivery applications in wound dressing and other biomedical applications.

Conclusions
In summary, TCH-loaded G/SA sponges were successfully synthesized and they display good swelling behavior and high porosity. G/SA composite sponges exhibit three-dimensional network structures, which help the G/SA sponges to facilitate TCH loading. The TCH-loaded G/SA sponges show controlled release performance and good antibacterial effects against E. coli ATCC 25,922 and B. subtilis ATCC 9372 and S. aureus ATCC 6538. Thus, the fabricated G/SA sponges show potential as promising substrates for drug delivery applications in wound dressing and other biomedical applications.