Uv 2600i

A DPPH assay was employed to evaluate the antioxidant activity of the samples, with certain modifications [39 (link)]. Briefly, DPPH (100 µM) was dissolved in methanol (99.8%). Subsequently, 1 mL of RCs or Cs (1 mg mL⁻¹) was incubated with a DPPH solution (1 mL) for 30 min in the dark. The absorbance of the samples was measured using ultraviolet–visible spectroscopy (UV–vis UV-2600i, Shimadzu, Kyoto, Japan) at a wavelength of 517 nm [40 (link),41 (link)]. The DPPH scavenging activity (%) was calculated using the following equation: $$\mathrm{DPPH}\mathrm{scavenging}\mathrm{activity}(\%)=\left(1-A_{sample}/A_{control}\right)\times 100$$
where A_control and A_sample are the absorbance values of the control (without sample) and sample (with DPPH solution), respectively. Each test was performed at least three times.
Ferric reducing power assay was done as per a previous study [2 (link),42 (link)]. One milligram of sample was added to a mixture of PBS (2.5 mL) and 1 wt% of aqueous potassium ferricyanide (2.5 mL). The above mixture was heated at 50 °C for 20 min. After cooling to room temperature, 10 wt% of aqueous tricholoroacetic acid (2.5 mL) was added and the mixture was centrifuged at 3000 rpm for 10 min. The upper layer of the mixture (2.5 mL) was mixed with DI water and 0.1 wt% aqueous ferric chloride solution and kept for 10 min. Then, the absorbance at 700 nm was measured. Each experiment was done at least three times.

For each formulation of freeze-dried c-CNF hydrogel with PHMB, three random samples were placed into vials containing 20 mL of deionized water. These vials were allowed to stand for 24 h, ensuring the complete dissolution of the hydrogels. The dissolution process was facilitated by a magnetic stirrer set at 100 rpm, maintaining room temperature. After dissolution, the solution samples were filtered using a 0.45 μm membrane filter to exclude minor particles. Subsequently, the filtered samples were subjected to dilution. The average PHMB content was determined using a UV spectrophotometer (UV2600i, Shimadzu Corporation, Kyoto, Japan) operating at a wavelength of 236 nm. The quantification of PHMB content was calculated using a standard PHMB solution with a concentration range from 2.5 μg/mL to 17.5 μg/mL, which displayed high linearity (r² = 0.996). Equation (8) was used to calculate the drug content of the PHMB-incorporated hydrogel.
$$\mathrm{PHMB}\mathrm{loading}\mathrm{content}(\%)=\frac{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{l}}{\mathrm{T}\mathrm{h}\mathrm{e}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}\times 100$$

c-CNF hydrogels containing PHMB, shaped as squares measuring 1 cm × 1 cm, were immersed in 20 mL of PBS buffer with a pH of 7.4 at 37 ± 0.5 °C. The medium was continuously stirred using a magnetic bar at 50 rpm. At predetermined intervals (0.5, 1, 2, 3, 6, 12, 24, 36, 48, 72, and 84 h), 2 mL of the dissolution media were collected, and an equal volume of PBS (2 mL) was replaced. The collected samples were analyzed for PHMB release using a UV-Vis spectrophotometer (UV2600i, Shimadzu, Kyoto, Japan) at 236 nm. All dissolution experiments were performed in triplicate. The amount of released drug was calculated using the following Equation (9).
$$\mathrm{Amount}\mathrm{of}\mathrm{released}\mathrm{drug}(\%)=\frac{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{d}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{e}\mathrm{c}\mathrm{i}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}}{\mathrm{A}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{l}}\times 100$$

The morphology of nanoparticles was verified by TEM (Talos F200X). The dynamic light scattering (DLS) and zeta potential was carried out by Zetasizer Pro (Malvern Panalytical). The UV–vis spectra were characterized by UV–vis spectrophotometer (UV-2600i, Shimadzu Corporation). The Mn content was detected by ICP-OES (Optima 2100, PerkinElmer). XPS analysis was conducted by an Axis Ultra DLD instrument. The dissolved oxygen was performed on an oxygen probe (ST300 D, OHAUS).

Three random MN patches for each formulation were dissolved in DI water (10 mL) in a beaker under magnetic stirring. After that, the average amount of lidocaine HCl was examined using a UV-spectrophotometer (UV 2600i, Shimadzu Corporation, Kyoto, Japan) at 263 nm. The contents of lidocaine HCl were calculated from the standard curve of lidocaine HCl (0.125–0.625 μg/mL) with a high linear regression (r² = 0.998) according to Equation (3).
$$\% \mathrm{Drug} \mathrm{loading} \mathrm{content}=\frac{\mathrm{T}\mathrm{h}\mathrm{e} \mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{l}\mathrm{i}\mathrm{d}\mathrm{o}\mathrm{c}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{e} \mathrm{H}\mathrm{C}\mathrm{l} \mathrm{i}\mathrm{n} \mathrm{M}\mathrm{N} \mathrm{p}\mathrm{a}\mathrm{t}\mathrm{c}\mathrm{h}}{\mathrm{T}\mathrm{h}\mathrm{e}\mathrm{o}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{a}\mathrm{m}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{l}\mathrm{i}\mathrm{d}\mathrm{o}\mathrm{c}\mathrm{a}\mathrm{i}\mathrm{n}\mathrm{e} \mathrm{H}\mathrm{C}\mathrm{l} \mathrm{i}\mathrm{n} \mathrm{d}\mathrm{r}\mathrm{y} \mathrm{p}\mathrm{o}\mathrm{l}\mathrm{y}\mathrm{m}\mathrm{e}\mathrm{r}}\times 100$$