*4.9. Evaluation of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of CuI NPs*

The antibacterial activity of gallic acid–CuI NPs against Gram-negative *E. coli* (ATCC 25922) and Gram-positive methicillin-resistant *Staphylococcus aureus* (MRSA, ATCC 33591) was tested by broth dilution method to evaluate minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Gram-negative *E. coli* and Gram-positive MRSA were separately dispersed in saline solution; the optical density was measured to be 0.5 using a densitometer, resulting in 1.5 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/mL of bacteria. Test tubes containing 1.5 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/mL were diluted by pipetting 100 <sup>µ</sup>L into 3 mL of saline solution; then, <sup>5</sup> <sup>×</sup> <sup>10</sup><sup>6</sup> CFU/mL of *E. coli* and of MRSA was obtained. After the bacterial suspension was prepared, serial dilution of the copper iodide solution was conducted. An amount of 512 mg of copper iodide was dissolved in 2 mL of DMSO, and 2 mL of MHB was added to this test tube. Then, serial dilution was conducted 10 times in 24-well plate. Bacterial suspension of 5 <sup>×</sup> <sup>10</sup><sup>6</sup> was transferred to a 24-well plate from column 1 to column 10 and was incubated in a shaking incubator with overnight cultures of bacteria.

#### *4.10. Physical Properties of PVA Liquid Bandage Formulation*

To evaluate the drying time of the PVA formulations on human skin, a test was applied on the inner side of the forearm. Different PVA formulations were spread on human forearm and let dry for a period of 1, 2 and 3 min. If there was no liquid visible on the skin, the PVA film was considered to be dry. The PVA liquid bandage with minimum drying time was chosen to create a liquid bandage.

Additionally, the water vapor transmission rate describes the amount of water passed through a unit area of film in a unit time. The water vapor transmission rate was evaluated to study the water permeability of the films, as this has an effect on skin temperature and the hydration of the wound. Following Zurdo Schroeder et al. [40], PVA liquid bandages loaded with different amounts of CuI suspension were cast on glass plates to evaporate the solvent, forming PVA films. These films were cut into circular samples with diameters of 0.03 m and covered on centrifuge tubes containing 30 mL of distilled water. The tubes were placed into an incubator with a temperature of 37 ◦C and relative humidity of 52%. Then, the tubes were weighed at each time point—0, 3, 6 and 24 h—to calculate the water vapor transmission rate. The weight loss of the glass tubes was determined as ∆m (g), m0 h–m24 h. The water vapor transmission rate is calculated by the amount of water transmitted through in PVA film related to surface area A (m<sup>2</sup> ) and time t (day), following the equation below:

$$\text{Water vapor transmission rate (WVTR)} = \frac{\text{\textdegree (m)}}{\text{A \textdegree (m}^2\text{)} \times \text{time (day)}}$$

The swelling ratio is usually measured for polymer hydrogel or polymer films to determine water resistance. The study of swelling behavior was performed referring to J. Liu et al. [36]. Pure PVA, PVA/CuI (0.095%) and PVA/CuI (0.190%) were cast in glass Petri dishes to form films. Then, each film was cut into a circular film with a diameter of 12 mm, and the average thickness was measured. After that, PBS buffer solution was prepared with sodium chloride, potassium chloride, sodium phosphate dibasic and monopotassium phosphate to make a buffer solution with pH 7.2 ± 0.1. Each film was weighed before the swelling test; the weight was determined as m0. Then, the samples were incubated at 37 ◦C. After the specified time, the samples were removed from PBS, then quickly cleaned with Kimwipes paper to remove water and weighed with an electronic balance. At time t, the weight was recorded as m<sup>t</sup> . All samples were continuously incubated in PBS solution and weighed at each time point up to 48 h to ensure that equilibrium was reached. The swelling ratio was calculated by the equation below:

$$\text{Swelling ratio} = \frac{\text{m}\_{\text{t}} - \text{m}\_{0}}{\text{m}\_{0}} \times 100$$

#### *4.11. In Vitro Indirect Cytotoxic Test of PVA Liquid Bandage Formulations*

The indirect cytotoxicity of PVA loaded with CuI suspension was tested following the ISO10993-5 (biological evaluation of medical devices) standard test using adult human dermal fibroblasts (GIBCO, Grand Island, NY, USA). Firstly, the culture medium was prepared and collected by exposing samples to UV radiation for 30 min for sterilization. Then, the samples were immersed in DMEM in a 96-well tissue culture polystyrene (TCPS) plate at 0.5, 5 and 10 mg/mL for 1 and 3 days to produce different concentrations of sample extractions. After, the samples were placed into a 24-well culture plate containing of Dulbecco's phosphate-buffered saline modified with Eagle's medium (Hyclone, Logan, UT, USA), with the addition of 10% fetal bovine serum (Gibco), 1% L-glutamine (Gibco), 100 µg/mL of streptomycin, 100 units/mL of penicillin (Gibco) and 5 µg/mL of amphotericin B (Gibco) for 24 h. Then, 10,000 cells of the human dermal fibroblast (HDFa) cell line were cultured in the prepared medium in a 96-well plate and incubated overnight in a humidity chamber with 5% CO<sup>2</sup> at 37 ◦C. The number of viable cells were counted using (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (USB Corporation, Cleveland, OH, USA). An absorbance microplate reader (BiotekELx800; Biotek Instruments Inc., Winooski, VT, USA) was used to determine the optical density (O.D.) at 570 nm of cells. The control group was 2D cell culture in normal conditions.

#### *4.12. Antibacterial Test by Time–Kill Assay*

The antibacterial activities of PVA liquid bandage with different concentration of CuI suspension were characterized through time–kill assay. It was necessary to determine the ability of the liquid bandage to kill bacteria in a fixed time. Briefly, the antibacterial activity of PVA liquid solutions was tested against *E. coli* (Gram-negative) and MRSA (Gram-positive) bacteria. The samples included pure PVA solution and PVA liquid bandages with different concentrations of CuI suspension. Firstly, both the *E. coli* and MRSA microorganisms were cultured in Mueller Hinton Broth (MHB) at 37 ◦C for 18 h. After, the bacterial solutions of *E. coli* and MRSA were adjusted to 0.5 McFarland standard solution using a densitometer, determined as 1.5 <sup>×</sup> <sup>10</sup><sup>8</sup> CFU/mL. Then, the bacterial solutions were diluted with MHB to 5 <sup>×</sup> <sup>10</sup><sup>5</sup> CFU/mL. Each sample contained 3 mL of <sup>5</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> CFU/mL diluted inoculum with continual turbulence at 37 ◦C for 0, 1, 3, 6, 12 and 24 h. At each time, 20 µL of each inoculum was brought out and replaced with normal saline solution. Eventually, 5 drops of 10 µL of each serial dilution were dropped on prepared Mueller–Hinton agar (MHA) in a Petri dish and incubated overnight in an incubator with controlled relative humidity and temperature of 37 ◦C. The number of growing bacterial colonies at each time point was counted, averaged and compared with the number of the control culture by estimating the CFU/mL values. The percentage of bacterial reduction was calculated following this equation:

$$\text{Bacteria reduction } (\%) = \frac{\text{N}\_{\text{control}} - \text{N}\_{\text{specimen}}}{\text{N}\_{\text{control}}} \times 100$$

where Ncontrol is the number of bacterial colonies in the control (CFU/mL); Nspecimen is the number of bacterial colonies in the specimen (CFU/mL).

**Author Contributions:** Conceptualization, supervision and funding acquisition, P.S. (Pitt Supaphol); data curation, formal analysis, investigation and methodology, P.P., C.C., S.C., P.S. (Pakakrong Sangsanoh), O.S. and P.C.; data curation, validation and review of the manuscript, O.S. and P.C.; visualization, P.P., C.C. and S.C.; writing—original draft, P.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the 90th Anniversary of Chulalongkorn University Scholarship Batch 50: GCUGR1125643048M No. 2–14 and Fundamental Fund 2565: FF65, Chulalongkorn University, Thailand.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors appreciate the full scholarship from the Petroleum and Petrochemical College (PPC), Chulalongkorn University. This work was performed by the support of Herbal Extract-Infused Advanced Wound Dressing Research Unit, Ratchadaphisekphot Endowment Fund, Chulalongkorn University, Thailand.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Declaration of Competing Interest:** The authors declare that they have no known competing financial interests or personal relationships that could appear to influence the work reported in this paper.

#### **References**


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