2.4.2. Crystal Violet Assay, Laser Microscopy Scanning, Viability Staining and ImageJ Analysis

A 0.5% crystal violet solution was used to determine the biofilm formation on the sample's surface. The PBS washed samples were placed into a 12-well plate filled with 500 µL crystal violet solution and incubated at room temperature for 20 min on shaker. The samples were then transferred to a new 6-well plate and washed four times gently with 5 mL PBS to excrete excess crystal violet. The plates were gently shaken to completely remove the residual dye. Then, the samples were measured using a laser microscope to measure the bacterial attachment and biofilm formation on the sample surface. The laser micrographs are then analyzed with ImageJ 1.50, to measure the area filled by bacteria. The analysis of biofilm density was measured by conversion of confocal images into red, green and blue colors. The background subtraction was applied to remove background noise. The images were then assembled into color channels and the integrated density of pixels was calculated. Correspondingly, the same samples were used to measure the OD of the samples with crystal violet stained which represent the amount of biofilm. The samples were transferred into a new 12-well plate filled with 95% ethanol and the plate was incubated at room temperature on a shaker at 270 rpm for 15 min. Then, 100 µL of the ethanol solution containing the crystal violet stained by the biofilms was transferred into the 96-well plate. The optical density at 595 nm was determined for total biomass quantification.

#### *2.5. Statistical Analysis 2.5. Statistical Analysis 2.5. Statistical Analysis*

The experimental data were statistically analyzed with Student's t-test (t-test) where *p* < 0.05 was considered statistically significant and marked with an asterisk (\*) and not statistically significant data marked with (*ns*). The experimental data were statistically analyzed with Student's t-test (t-test) where *p* < 0.05 was considered statistically significant and marked with an asterisk (\*) and not statistically significant data marked with (*ns)*. The experimental data were statistically analyzed with Student's t-test (t-test) where *p* < 0.05 was considered statistically significant and marked with an asterisk (\*) and not statistically significant data marked with (*ns)*.

the 96-well plate. The optical density at 595 nm was determined for total biomass quanti-

the 96-well plate. The optical density at 595 nm was determined for total biomass quanti-

#### **3. Results 3. Results 3. Results**

fication.

fication.

#### *3.1. Surface Characterization 3.1. Surface Characterization 3.1. Surface Characterization*

#### 3.1.1. Laser Microscopy 3.1.1. Laser Microscopy

Figure 2 shows the laser micrographs images of five samples with different concentration of AlN ranging from 0 to 20 wt. %. The dispersion of AlN on the surface could be clearly observed as a function of concentration. 3.1.1. Laser Microscopy Figure 2 shows the laser micrographs images of five samples with different concentration of AlN ranging from 0 to 20 wt. %. The dispersion of AlN on the surface could be clearly observed as a function of concentration. Figure 2 shows the laser micrographs images of five samples with different concentration of AlN ranging from 0 to 20 wt. %. The dispersion of AlN on the surface could be clearly observed as a function of concentration.

**Figure 2.** Laser microscope images of CA/AlN composites. **Figure 2.** Laser microscope images of CA/AlN composites. **Figure 2.** Laser microscope images of CA/AlN composites.

*Antibiotics* **2021**, *10*, x FOR PEER REVIEW 5 of 17

*Antibiotics* **2021**, *10*, x FOR PEER REVIEW 5 of 17

The white particles observed are the AlN. The surface roughness Ra increases as the amount of AlN increases. Figure 3 shows the quantification of surface roughness where a clear increasing trend with AlN concentration was observed. As shown, 20 wt. % shows the highest surface roughness and the whitish particle observed in image is AlN which increases the surface roughness of the material. The white particles observed are the AlN. The surface roughness R<sup>a</sup> increases as the amount of AlN increases. Figure 3 shows the quantification of surface roughness where a clear increasing trend with AlN concentration was observed. As shown, 20 wt. % shows the highest surface roughness and the whitish particle observed in image is AlN which increases the surface roughness of the material. The white particles observed are the AlN. The surface roughness Ra increases as the amount of AlN increases. Figure 3 shows the quantification of surface roughness where a clear increasing trend with AlN concentration was observed. As shown, 20 wt. % shows the highest surface roughness and the whitish particle observed in image is AlN which increases the surface roughness of the material.

**Figure 3.** Surface microscopic roughness of CA/AlN composites. \* = *p* < 0.05 **Figure 3.** Surface microscopic roughness of CA/AlN composites. \* = *p* < 0.05 **Figure 3.** Surface microscopic roughness of CA/AlN composites. \* = *p* < 0.05.

#### 3.1.2. FTIR Spectroscopy

*Antibiotics* **2021**, *10*, x FOR PEER REVIEW 6 of 17

Figure 4 shows the FTIR spectra of the pure aluminum nitride, and the CA/AlN composites ranging from 0 wt. % to 20 wt. %. The composite material has a wide range spectrum from 500 to 3750 cm−<sup>1</sup> . The FTIR spectra do not shows the presence of AlN as it can be clearly observed that none of the CA/AlN composites has the strong peak of AlN at 672 cm−<sup>1</sup> (peak 1) which is assigned to E1(TO) [11–13]. Peak 2 to 10 represents cellulose acetate functional groups. The band at 1750 cm−<sup>1</sup> (peak 7) was attributed to C=O from cellulose acetate and the band at 1428 cm−<sup>1</sup> (peak 6) was assigned to CH<sup>2</sup> vibrations. The sharp absorption peaks at 1041 cm−<sup>1</sup> (peak 3) and 1250 cm−<sup>1</sup> (peak 4) were due to presence of C-O stretching [14,15]. The band at 912 cm−<sup>1</sup> (peak 2) can be attributed to C-O stretching and CH<sup>2</sup> rocking vibrations. In addition, 1366 cm−<sup>1</sup> (peak 5) was assigned to CH3. The broad peak at and 2942 cm−<sup>1</sup> (peak 9) and the peak at 3487 cm−<sup>1</sup> (peak 10) attributed to C-H aromatic vibrations and O-H stretching of cellulose acetate [16,17], respectively. FTIR do not show any functional groups of AlN which are present in the CA/AlN composites. This might be because the strong signal of CA blocks the weak signals of AlN in the composite. 3.1.2. FTIR Spectroscopy Figure 4 shows the FTIR spectra of the pure aluminum nitride, and the CA/AlN composites ranging from 0 wt. % to 20 wt. %. The composite material has a wide range spectrum from 500 to 3750 cm−1. The FTIR spectra do not shows the presence of AlN as it can be clearly observed that none of the CA/AlN composites has the strong peak of AlN at 672 cm−1 (peak 1) which is assigned to E1(TO) [11–13]. Peak 2 to 10 represents cellulose acetate functional groups. The band at 1750 cm−1 (peak 7) was attributed to C=O from cellulose acetate and the band at 1428 cm−1 (peak 6) was assigned to CH2 vibrations. The sharp absorption peaks at 1041 cm−1 (peak 3) and 1250 cm−1 (peak 4) were due to presence of C-O stretching [14,15]. The band at 912 cm−1 (peak 2) can be attributed to C-O stretching and CH2 rocking vibrations. In addition, 1366 cm−1 (peak 5) was assigned to CH3. The broad peak at and 2942 cm−1 (peak 9) and the peak at 3487 cm−1 (peak 10) attributed to C-H aromatic vibrations and O-H stretching of cellulose acetate [16,17], respectively. FTIR do not show any functional groups of AlN which are present in the CA/AlN composites. This might be because the strong signal of CA blocks the weak signals of AlN in the composite.

**Figure 4.** FTIR spectra of the CA/AlN composites. **Figure 4.** FTIR spectra of the CA/AlN composites.

#### 3.1.3. Raman Spectroscopy 3.1.3. Raman Spectroscopy

The functional groups of CA could by clearly observed by FTIR, while Raman spectroscopy was used to further investigate both the CA/AlN composites and AlN. The various spectra acquired were illustrated in Figure 5. The characteristics of Raman signal for cellulose was clearly observed at 2934 cm−1 (peak 6) which is assigned to C-H stretching and asymmetric stretching vibrations of the C-O-C glycosidic linkage. In addition, Raman signals at 1380 (peak 3), 1435 (peak 4) and 1754 cm−1 (peak 5) are attributed to C=O vibrations of the carbonyl group and asymmetric and symmetric vibrations of C-H bond which exist in the acetyl group from CA [17–19]. Besides, the presence of AlN could be clearly observed at 612 (peak 1) and 656 cm−1 (peak 2) which are associated with A1 (TO) and E2 (high) [20–22]. As AlN concentration increases the peak intensity also increases. The red spots are a marker for the presence of the AlN in the CA matrix, which is green. The intensity of the red spot increases in conjunction with the peak intensity, which can be explained with the increased presence of AlN. The AlN signals, which cannot be detected The functional groups of CA could by clearly observed by FTIR, while Raman spectroscopy was used to further investigate both the CA/AlN composites and AlN. The various spectra acquired were illustrated in Figure 5. The characteristics of Raman signal for cellulose was clearly observed at 2934 cm−<sup>1</sup> (peak 6) which is assigned to C-H stretching and asymmetric stretching vibrations of the C-O-C glycosidic linkage. In addition, Raman signals at 1380 (peak 3), 1435 (peak 4) and 1754 cm−<sup>1</sup> (peak 5) are attributed to C=O vibrations of the carbonyl group and asymmetric and symmetric vibrations of C-H bond which exist in the acetyl group from CA [17–19]. Besides, the presence of AlN could be clearly observed at 612 (peak 1) and 656 cm−<sup>1</sup> (peak 2) which are associated with A<sup>1</sup> (TO) and E<sup>2</sup> (high) [20–22]. As AlN concentration increases the peak intensity also increases. The red spots are a marker for the presence of the AlN in the CA matrix, which is green. The intensity of the red spot increases in conjunction with the peak intensity, which can be explained with the increased presence of AlN. The AlN signals, which cannot be

detected by FTIR, were clearly seen in the Raman spectra which proves the presence of AlN particles in the composite. by FTIR, were clearly seen in the Raman spectra which proves the presence of AlN particles in the composite.

**Figure 5.** Raman average spectra of the CA/AlN composites. **Figure 5.** Raman average spectra of the CA/AlN composites.

#### *3.2. Mechanical Property 3.2. Mechanical Property*

Figure 6a–b illustrates the stress versus strain curves for the various CA/AlN composites. It provides the information on how the composite materials deforms with increasing force. The composites containing 5 wt. % of AlN has similar strength to the 0 wt. %. Mechanical strength was then reduced in the samples containing 10 wt. % to 20 wt. %. This proves that 5 wt. % reinforcement concentration has the highest strength among the composite samples. Figure 6a,b illustrates the stress versus strain curves for the various CA/AlN composites. It provides the information on how the composite materials deforms with increasing force. The composites containing 5 wt. % of AlN has similar strength to the 0 wt. %. Mechanical strength was then reduced in the samples containing 10 wt. % to 20 wt. %. This proves that 5 wt. % reinforcement concentration has the highest strength among the composite samples.

Figure 6c shows the young modulus of the CA/AlN composites which were calculated using gradient of the stress versus strain curves. The Young's modulus of the samples containing 5 wt. % and 10 wt. % of AlN increases up to 1100 and 1221 MPa, respectively. However, when the further AlN were added, the Young's modulus of the 15 wt. % and 20 wt. % samples decrease to 989 and 892 MPa, respectively Therefore, it can be deduced that the stiffness of the samples increases with low AlN concentration (5 wt. % and 10 wt. %) and then reduce when the AlN reached to 15 and 20 wt. %. Figure 6c shows the young modulus of the CA/AlN composites which were calculated using gradient of the stress versus strain curves. The Young's modulus of the samples containing 5 wt. % and 10 wt. % of AlN increases up to 1100 and 1221 MPa, respectively. However, when the further AlN were added, the Young's modulus of the 15 wt. % and 20 wt. % samples decrease to 989 and 892 MPa, respectively Therefore, it can be deduced that the stiffness of the samples increases with low AlN concentration (5 wt. % and 10 wt. %) and then reduce when the AlN reached to 15 and 20 wt. %.

In addition, the changes in toughness of the material were calculated by area un-der the graph as shown in Figure 6d. The toughness of the 0 wt. % sample was 2.7 MPa. When 5 wt. % of AlN was added to the composite, the toughness significantly increased, up to 4.0 MPa. which shows it is toughest sample. However, the toughness started to reduce when higher AlN concentrations were added to the composites. The calculations analysis was made as stated in the literature [18,23,24].

HDPE, PVC, and PVDC are the commercially available material which is used to produce cling film for food packaging, tablecloths and other plastic materials. Theoretically, the commercial HDPE, PVC and PVDC clings film has the tensile strength of ≥10, ≥15 and ≥60 MPa respectively. Therefore, the CA/AlN composites can be used as the food wrap or tablecloths since it has the tensile strength in the range of 57 to 41 MPa which meets the range of tensile strength of commercial product. As a summary, though the tensile strength reduces at 20 wt. % but it has the enough mechanical strength to be an alternative

**Figure 6.** (**a**). Tensile stress-strain curves of the CA/AlN composites, (**b**) Max strength, (**c**) Young's modulus and (**d**) Toughness. \* = *p* < 0.05, *ns* = *p* > 0.05 **Figure 6.** (**a**). Tensile stress-strain curves of the CA/AlN composites, (**b**) Max strength, (**c**) Young's modulus and (**d**) Toughness. \* = *p* < 0.05, *ns* = *p* > 0.05.

#### In addition, the changes in toughness of the material were calculated by area un-der *3.3. Thermal Properties*

the graph as shown in Figure 6d). The toughness of the 0 wt. % sample was 2.7 MPa. When 5 wt. % of AlN was added to the composite, the toughness significantly increased, up to 4.0 MPa. which shows it is toughest sample. However, the toughness started to reduce when higher AlN concentrations were added to the composites. The calculations analysis was made as stated in the literature [18,23,24]. HDPE, PVC, and PVDC are the commercially available material which is used to produce cling film for food packaging, tablecloths and other plastic materials. Theoretically, the commercial HDPE, PVC and PVDC clings film has the tensile strength of ≥10, ≥15 and **≥**60 MPa respectively. Therefore, the CA/AlN composites can be used as the food The thermal property of the CA/AlN composites was analyzed by differential scanning calorimetry, DSC. The desorption temperature (Td), glass transition temperature (Tg), melting temperature (Tm), and degree of crystallinity χ*<sup>c</sup>* were measured. Figure 7 shows the first heating curve of DSC of cellulose acetate and CA/AlN composites. The first endothermic peak was clearly seen in all the samples. This peak appears due to the desorption of water. This phenomenon occurs due to the existence of residual moisture or low boiling point of solvents. T<sup>d</sup> varies from control (0 wt. %) to 20 wt. % in the range from 71.82 ◦C to 79.97 ◦C. The differences of T<sup>d</sup> values are due to their different ability of holding water of the polymeric matrices [14].

wrap or tablecloths since it has the tensile strength in the range of 57 to 41 MPa which meets the range of tensile strength of commercial product. As a summary, though the tensile strength reduces at 20 wt. % but it has the enough mechanical strength to be an alternative material when compared with the theoretical values of the commercial prod-Figure 8a shows the second heating curve where the T<sup>g</sup> and T<sup>m</sup> could be identified. The T<sup>g</sup> value were calculated by the maximum Tan delta peak [25–29]. The T<sup>g</sup> values increases as the concentration of AlN increases as illustrated in Figure 8b. The T<sup>g</sup> value of the CA improved with increasing the amount of AlN reinforcement.

ucts. However, 20 wt. % showed high thermal and antibacterial property which enhance the composite material's efficiency. *3.3. Thermal Properties*  The thermal property of the CA/AlN composites was analyzed by differential scanning calorimetry, DSC. The desorption temperature (Td), glass transition temperature (Tg), melting temperature (Tm), and degree of crystallinity χ*<sup>c</sup>* were measured. Figure 7 shows the first heating curve of DSC of cellulose acetate and CA/AlN composites. The first en-The T<sup>m</sup> temperature also shows similar trend where it increases as the amount of AlN increases. However, the increment seems to be small (about 7.35 ◦C from 0 wt. % to 20 wt. %). From Figure 8c, an endothermic peak appears in the first cycle close to the melting peak, but according to literature [26,29,30], the first heating curve is influenced by the thermal and mechanical history, and the second heating curve is conventionally used for the determination of material properties, such as meting point or glass transition temperature, under a given dynamic condition. For this reason, the melting point and glass transition temperature were investigated using the second heating curve [25–32].

dothermic peak was clearly seen in all the samples. This peak appears due to the desorption of water. This phenomenon occurs due to the existence of residual moisture or low boiling point of solvents. Td varies from control (0 wt. %) to 20 wt. % in the range from 71.82 °C to 79.97 °C. The differences of Td values are due to their different ability of holding

water of the polymeric matrices [14].

**Figure 7.** DSC first heating curves for the CA/AlN composites. **Figure 7.** DSC first heating curves for the CA/AlN composites.

**Figure 8.** DSC curves for the CA/AlN composites (**a**), 2nd heating curve (**b**), Glass transition temperature, Tg, (**c**), Melting temperature and (**d**), crystallinity degree. \* = *p* < 0.05, *ns* = *p* > 0.05 **Figure 8.** DSC curves for the CA/AlN composites (**a**), 2nd heating curve (**b**), Glass transition temperature, Tg, (**c**), Melting temperature and (**d**), crystallinity degree. \* = *p* < 0.05, *ns* = *p* > 0.05.

enthalpy of (Δܪ (and the respective value for the totally crystalline material (∆ܪ

߯ ൌ Δܪ ܪ∆ω

Another parameter extrapolated from the DSC curves is the degree of crystallinity χ*<sup>c</sup>* which is a fundamental property for properties of plastics. A higher degree of crystalliza-

Based on Figure 8d the degree of crystallinity reduces upon addition of AlN. However, the concentration of AlN does not affect the crystallinity degree much as the value

Figure 9 demonstrates the antibacterial test conducted against the CA/AlN composites material with *S. epidermidis* using WST method. It is clearly observed that absorbance level increases until 15 wt. % and start to reduce at 20 wt. % for 12 h. Besides, the same samples were tested with the laser microscope, where the biofilm production was clearly seen on the surface as shown in Figure S1. Almost similar results were obtained for 12 h, the biofilm formation reduces slightly from the control sample and the antibacterial effect was not clearly observed. Whereas, for 24 h, the absorption shows the similar trend as 12 h, but at 20 wt. % shows a very lower absorption than control. The biofilm formation shows a very clear trend at 24 h where, at control it was observed the full area covered by

) mul-

ൈ 100 (1)

was almost similar from 5 wt. % to 20 wt. %

3.4.1. Microbial Viability Assay (WST)

ܪ∆ where

*3.4. In Vitro Testing* 

tiply with weight fraction of CA using the following formula:

= 58.8 J g−1 as stated by Cerquiera et al. [27].

Another parameter extrapolated from the DSC curves is the degree of crystallinity χ*<sup>c</sup>* which is a fundamental property for properties of plastics. A higher degree of crystallization makes the material stiffer and stronger but also increases brittleness [26–31]. The crystallinity degree of CA/AlN composites was calculated as the ratio between the melting enthalpy of (∆*Hm*) and the respective value for the totally crystalline material (∆*H*<sup>0</sup> *<sup>m</sup>*) multiply with weight fraction of CA using the following formula:

$$\chi\_{\mathcal{L}} = \frac{\Delta H\_m}{\omega \Delta H\_m^0} \times 100 \tag{1}$$

where ∆*H*<sup>0</sup> *<sup>m</sup>* = 58.8 J g−<sup>1</sup> as stated by Cerquiera et al. [27].

Based on Figure 8d the degree of crystallinity reduces upon addition of AlN. However, the concentration of AlN does not affect the crystallinity degree much as the value was almost similar from 5 wt. % to 20 wt. %
