X-ray fluorescence spectroscopy: The XRF spectrum confirms the presence of Zn in the CMC@ZnAsp hydrogel (Figure 1). The zinc content was determined to be 3.3%. By assuming that the dry hydrogel contains only the added components without any residual water, the incorporation of 0.00732 g of ZnAsp (containing 14.2% Zn) to 0.02 g of CMC yields a theoretical zinc content of 3.8% in CMC@ZnAsp.

Crystal and molecular structure of ZnAsp: The crystal and molecular structure of ZnAsp is shown in Figure 2. While the structure of ZnAsp has been previously reported, in this work we provide a more precise refinement and verify its chemical formulation (Hartmann and Vahrenkamp, 1994; Lemoine et al., 2004; Marsh, 2004).

¹H-NMR spectroscopy: The ¹H-NMR spectrum of CMC@ZnAsp was recorded in D₂O and compared with that of ZnAsp (Figure 3). The singlet signal at 1.905 ppm of CMC@ZnAsp is assigned to the methyl protons of aspirin which is in accordance with the corresponding -CH₃ group of ZnAsp which also appeared at 1.951 ppm. The resonance signals of CMC@ZnAsp at 6.936–6.868 (t, H^b aromatic), 7.413–7.361 (t, H^d,e aromatic) and 7.771–7.739 (d, H^f aromatic) are in agreement with those dominated in initial compound ZnAsp (6.913–6.844 (t, aromatic H^b), 7.406–7.344 (t, aromatic H^d,e) and 7.754–7.723 (d, aromatic H^f)) (Figure 3). Moreover, the resonance signals of CMC are observed at 4.246–3.161 ppm (Kono et al., 2016; Stathopoulou et al., 2018) (Figure 3).

Stability of ZnAsp or CMC@ZnAsp in double distilled water (ddw) solutions: Stability of ZnAsp or CMC@ZnAsp in ddw solutions was assessed using UV-Vis spectroscopy. Figure 4 presents the absorption spectra of ZnAsp, CMC@ZnAsp, Aspirin and Salicylic acid. These data indicate that the solid-state structure of ZnAsp is preserved in solution, whether as the free complex or when dispersed within CMC (CMC@ZnAsp). A similar conclusion was drawn when the corresponding spectra were recorded in phosphate-buffered saline (PBS, pH 7), representative of cell culture conditions. Stability was also evaluated under mildly acidic conditions, mimicking healthy skin (pH 4.5–5.5), using a sodium acetate buffer at pH 5.5 (Figure 4). The results confirmed that the structural integrity of the complex was retained in all tested media—double-distilled water (ddw), PBS, and sodium acetate buffer solution (SABS). Chiaverini et al. (2024) showed that the Mo₂(Asp)₄ complex maintains its structural stability longer than other Mo₂(NSAID)₄ analogues, while still undergoing controlled disassembly in biological environments to release active components. This dual behavior supports its potential as a therapeutic candidate, especially in applications requiring targeted and sustained release of NSAIDs. Moreover, the synthesis of the CMC@ZnAsp hydrogel involves dissolving ZnAsp in water with the simultaneous addition of the appropriate amount of CMC. The process lasts approximately 3 h. The UV-Vis spectrum of the hydrogel containing CMC@ZnAsp, recorded after this period, is identical to that of the free ZnAsp complex recorded immediately after its preparation (Figure 4).

Ultraviolet Protection Factor (UPF) of ZnAsp and CMC@ZnAsp : The UV-protective properties of ZnAsp and CMC@ZnAsp were evaluated according to standard techniques aligned with ISO 23675 principles. A solution of ZnAsp (5 × 10^-4 M) or CMC@ZnAsp (5 × 10^-4 M in respect of ZnAsp) was prepared, and its absorbance was measured across the UV range of 290–400 nm (UVA and UVB radiation) using a UV-Vis spectrophotometer. The absorbance values were converted to % transmittance (T%), and a transmittance spectrum (T% vs. wavelength) was generated (Figure 5).

To quantify the protective effect, the surface area under the T% vs. wavelength curve was calculated. Ultraviolet Protection Factor (UPF) determination was performed by calculating the quotient of the surface integrals of transmittance multiplied by wavelength, over the 290–400 nm range, without and with the Zn-aspirinate solution: U P F = ∫ 290 400 T r e f % · d λ ∫ 290 400 T Z n A s p % · d λ where T_ref is the transmittance without ZnAsp or CMC@ZnAsp and T_ZnAsp or T_CMC@ZnAsp is the transmittance of the ZnAsp or CMC@ZnAsp solution.

This value was subsequently normalized to a standard application density of 2 mg/cm², as required for consistent comparison with standard protective formulations.

The resulting UPF values of the ZnAsp and CMC@ZnAsp solutions were 9.85 and 11.05, respectively, indicating moderate UV protection in both cases. These results demonstrate that the CMC@ZnAsp hydrogel exhibits a higher UPF compared to its ZnAsp component alone. Furthermore, the hydrogel’s enhanced ability to absorb and attenuate UV radiation across both the UVA and UVB radiations highlights its potential for use in formulations designed for effective UV shielding.

Continuous photolysis: Photostability under sun exposure or ultraviolet (UVB) radiation is essential to ensure the effectiveness and safety of commercial sunscreens (Gonzalez et al., 2007). While the average daily outdoor exposure is typically 1–2 h, this can increase substantially to 5–6 h during holidays (Diffey, 2011). In light of this, we evaluated the photostability of both CMC@ZnAsp and ZnAsp under UVB radiation (λ_max = 280 nm) in D₂O solution for a continuous 6-h period. The reaction was monitored using ¹H-NMR spectroscopy (Supplementary Figure S1). No changes were detected between the initial spectra of CMC@ZnAsp or ZnAsp and those recorded after UVB exposure, indicating that both the hydrogel CMC@ZnAsp and its ZnAsp component remain stable without undergoing decomposition. This photostability supports their potential use in photoprotective hydrogel formulations.

In Vitro Toxicity against Immortalized Human Keratinocytes (HaCaT) Cells: To assess the toxicity of CMC@ZnAsp and ZnAsp, HaCaT cells were chosen as a representative model for studying the cellular pharmacokinetics of compounds in keratinocytes (Pessina, et al., 2001). HaCaT cells are useful for testing preventive and therapeutic strategies for skin-related conditions (Tyagi et al., 2015). The cells screening performed using the sulforhodamine B (SRB) assay. HaCaT cells were incubated with CMC@ZnAsp, and ZnAsp for 48 h. The IC₅₀ value for CMC@ZnAsp is 80.0 ± 2.4 μΜ, while the corresponding value for ΖnAsp is 83.3 ± 2.4 μΜ. Moreover, CMC@Znasp and ZnAsp were irradiated with UVB light for a period of 6 h and the HaCaT cells were incubated with the radiated, CMC@Znasp and ZnAsp. The IC₅₀ values of the radiated CMC@Znasp and ZnAsp are 72.6 ± 0.8 and 84.4 ± 2.1 μΜ, respectively. Thus, both CMC@ZnAsp and ZnAsp, along with their UV-irradiated counterparts, exhibited similar levels of toxicity towards HaCaT cells, indicating good photostability and low cytotoxicity for the hydrogel CMC@ZnAsp and its active component ZnAsp.

In vivo toxicity evaluation by brine shrimp A. salina: The zooplanktonic crustacean A. salina is used as a model organism for in vivo toxicological testing by the U.S. Environmental Protection Agency (EPA) (Banti and Hadjikakou, 2021). The A. salina assay is closely correlated with toxicity data from rodent and human studies and is commonly used to assess the in vivo toxicity of the hydrogel, its active ingredient, and its photoproducts (Banti and Hadjikakou, 2021).

The survival rates (%) of A. salina larvae were assessed at a concentration of 50 μM, which corresponds to the concentration used for evaluating keratinocyte cell migration in the in vitro scratch wound assay (see below). Additionally, higher concentrations of 90, 180, and 360 μM were tested after a 24-h incubation period. No toxic effects on the survival of A. salina were observed for the CMC@ZnAsp hydrogel and ZnAsp, or their corresponding irradiated CMC@ZnAsp and ZnAsp, indicating that both the CMC@ZnAsp and ZnAsp and their irradiated counterparts are non-toxic.

Anti-inflammation activity by albumin denaturation assay: The anti-inflammatory activity of CMC@ZnAsp and ZnAsp was initially assessed in vitro using the albumin denaturation assay. This assay measures the inhibition of protein denaturation, specifically the disruption of its secondary and tertiary structures (Chick, et al., 2025). Generally, protein denaturation is a critical pathological process associated with inflammation and conditions such as rheumatoid arthritis (Rajamohan et al., 2025). The non steroidal anti-inflammatory drug, diclofenac was used for a standard positive control (Chick, et al., 2025). The IC₅₀ value of diclofenac is 2.45 ± 0.50 mM. ZnAsp demonstrated a stronger effect, with a calculated IC₅₀ value of 1.96 ± 0.26 mM, indicating significant anti-inflammatory activity by effectively inhibiting protein denaturation—surpassing the activity of diclofenac sodium.

Migration of keratinocytes in wound margin by in vitro scratch wound assay: Self-healing properties are essential to the effectiveness of the formulated hydrogel sunscreens (Heydari et al., 2025). The re-epithelialization process is a critical phase of wound healing, during which cells migrate from the wound margins toward the center to facilitate ulcer closure (Stathopoulou et al., 2018).

The in vitro scratch wound assay is based on creating an artificial gap, or “scratch,” on a confluent cell monolayer. The cells migrate toward the gap, healing the wound by combining migration and proliferation (Stathopoulou et al., 2018).

The effects of the hydrogel, its ingredients, and their photoproducts on cell migration were evaluated after 48 h of incubation with HaCaT cells (Figures 6, 7). The tested concentration of 50 μM can be considered non-cytotoxic, as the cells’ viability at this level exceeds 70%. According to FDA ISO 10993-5 that regulates the “Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity,” if the percent of viability is higher than 70% upon treatment with an agent, this agent is considered as noncytotoxic. in accordance with ISO 10993-5, which regulates the Biological evaluation of medical devices–Part 5: Tests for in vitro cytotoxicity (Banti et al., 2023).

The recovered wound area (RWA %) is defined as: R W A % = 100 * i n i t i a l   w o u n d   a r e a − f i n a l   w o u n d   a r e a i n i t i a l   w o u n d   a r e a

Upon 48 h incubation, the RWA % was 59.1% in the untreated keratinocytes, on the contrary of 66.7% of CMC@ZnAsp, and 76.5% of the irradiated CMC@ZnAsp, 58.6% of the ZnAsp, and 73.8% of the irradiated of ZnAsp, suggesting that CMC@ZnAsp and ZnAsp as well as their counterparts show wound healing properties towards HaCaT cells after 48 h incubation at the concentration of 50 μΜ.

Solutions of CMC@ZnAsp and ZnAsp for biological assays were freshly prepared by dissolving the hydrogel material or complex directly in ddw or cell culture medium (DMEM) at the desired low concentrations. Under these dilute conditions, no gelation occurred, and the material behaved as a solution suitable for biological testing. The scratch test and the cell viability assessment by SRB assay were carried out using H₂O/DMEM for CMC@ZnAsp and ZnAsp.

SRB assay: Solutions of CMC@ZnAsp and ZnAsp (0.01 M) in ddH₂O were freshly prepared and diluted with the cell culture medium to the desired concentration (5–150 μM). This study was performed as previously reported (Banti et al., 2023).

In vitro scratch wound healing assay: HaCaT cells were grown at a density of 80,000 cells/well for 24 h on a 24-well plate. The next day, shaped wounds were made using a sterile 10 μL pipette tip to scrape across each well, creating a cell free-area. Then, the cells were washed with PBS. The cells were exposed to CMC@Znasp, Znasp and their irradiated counterparts for 6 hours-at 50 μΜ in culture medium. Cells for the negative control were also scratched, washed. The scratch closure was monitored for 48 h using phase-contrast microscopy. The scratch area was measured using the ImageJ software (Stathopoulou et al., 2018).

In vivo toxicity evaluation by brine shrimp A. salina: The brine shrimp assay was performed as previously reported (Banti et al., 2023).

Anti-inflammation activity by albumin denaturation assay: The samples were screened for anti-inflammatory properties using the inhibition of albumin denaturation assay, as previously described (Chaiya et al., 2022; Chavan and Hosamani, 2018; Chick et al., 2025). The mixture (2.5 mL) consisted of 0.1 mL of egg albumin (from fresh chicken’s egg), 1.4 mL of phosphate buffered saline (PBS, pH 6.4) and 1 mL of varying concentrations of diclofenac sodium or ZnAsp (0.4–4.0 mM) in ddH₂O. The double-distilled water served as negative control. Then the mixtures were incubated at 37 °C in an incubator for 15 min and then heated at 70 °C for 5 min. After cooling for 10 min, the absorbance of the samples was measured at 660 nm. The anti-inflammatory activity was estimated as the percentage inhibition or clearance of protein denaturation and calculated according A _sample is the absorbance of the test sample (albumin-mixed PBS solution with tested agent) towards A _control is the absorbance of the control (albumin-mixed PBS solution without tested compound). The mean values and confidence limits are determined by independent experimental replicates.