In Vitro Antioxidant and Cellular Activities of Functionalized Spermidine by Conjugating with Ascorbic Acid in Human Skin Cells

TL;DR

This study shows that combining spermidine with ascorbic acid improves antioxidant effects and skin cell functions, making it promising for biomedical and cosmetic use.

Contribution

The novel conjugation of spermidine with ascorbic acid enhances antioxidant activity and skin cell benefits.

Findings

AA-SPMD showed high stability and strong antioxidant activity under rigorous conditions.

AA-SPMD increased cell proliferation and migration in skin cells without cytotoxic effects.

AA-SPMD boosted collagen I production in fibroblasts, suggesting potential biomedical and cosmetic applications.

Abstract

Spermidine (SPMD) is essential for numerous cellular functions and crucial for sustaining diverse biological activities. However, its antioxidant capabilities are relatively weak. In this study, we overcame this limitation by examining the antioxidant and cellular effects of ascorbic acid (AA)-conjugated spermidine (AA-SPMD) in human skin keratinocyte and fibroblast cells. AA-SPMD was successfully fabricated using an optimized design and synthetic approach, and its stability, antioxidant activity, cellular responses, and collagen I production were evaluated. In addition, we assessed the protective effects of AA-SPMD from hydrogen peroxide and UVA-induced oxidative damage in human skin cells. The AA-SPMD showed high stability under rigorous conditions and exhibited strong antioxidant activity. AA-SPMD showed no cytotoxic effect even at a concentration of 1 mM. In addition, it can increase the rate of cell proliferation and migration in skin cells without reducing the inhibition of human keratinocytes (HaCaT) and human dermal fibroblasts (HDF) at concentrations of 10 μM. Moreover, AA-SPMD can increase the amount of collagen I synthesized in HDF cells, thereby influencing cell proliferation and migration. Based on our in vitro study, AA-SPMD is expected to be more effective than AA or SPMD alone, indicating its potential utility in biomedical and cosmetic applications.

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Full text

1. Introduction

Polyamines (PAs), such as putrescine, spermidine (SPMD), and spermine, are naturally occurring cations present in all living cells and are indispensable for several cellular functions. The intracellular contents of each PA are tightly controlled by coordinated regulation of their biosynthesis, catabolism, and membrane transport pathways [1,2,3,4,5,6]. SPMD, a naturally occurring PA, is particularly important for preserving cellular homeostasis across diverse organisms [7]. The chemical structure of SPMD influences various biological processes, such as nucleic acid stabilization, cell proliferation resulting in tissue regeneration, enzymatic modulation, and regulation of translation [1,5,7,8]. SPMD has been reported to promote mitochondrial activity and protein homeostasis while exerting anti-inflammatory and mild antioxidant effects [9]. Furthermore, recent reports have shown that the external supplementation of SPMD at relatively high doses relative to the intracellular contents exhibits several beneficial effects on age-related diseases in various biological model organisms [10,11]. For example, the dietary supplementation of SPMD extends lifespan across species [10,12,13], promotes cardioprotection [10] and neuroprotection [14,15,16], stimulates antineoplastic immunity [17], and potentially averts immune senescence via the induction of memory T-cell formation [18,19]. Several of these geroprotective activities have been causally linked to the ability of SPMD to maintain proteostasis by stimulating cytoprotective macroautophagy [20,21,22]. Despite these advantages, SPMD exhibits relatively weak antioxidant capabilities.

Ascorbic acid (AA) is a vital water-soluble nutrient that is crucial for numerous physiological processes, including skin collagen synthesis [23] and anti-inflammatory [24,25] and depigmentation actions [26,27]. In addition, it represents the primary water-soluble non-enzymic antioxidant in human tissues [27,28,29,30,31]. However, unlike some plants and animals, humans cannot synthesize AA endogenously owing to a specific enzymatic deficiency in l-glucono-gamma lactone oxidase, which is an enzyme responsible for catalyzing the passage terminal in AA synthesis [28,29,30,31,32]. Therefore, humans rely on their dietary intake or vitamin supplements to obtain AA for preventing diseases and maintaining overall well-being [31,32]. AA is highly unstable in aqueous solutions under conditions of high temperature, high pH, and light exposure [23], and it is readily degraded into inactive products by specific enzymes and metal ions, resulting in no biological activity [33,34,35].

As mentioned above, SPMD and AA have different biological activities. As such, functionalized SPMD-based biomaterials, such as hydrogels [36,37,38] and nanomaterials [39], and AA derivatives, such as ascorbyl glucoside and ascorbyl palmitate [40,41], have been developed separately to date. Considering these points, this study aimed to develop a functionalized SPMD that simultaneously possesses the biological activity of AA.

In this study, we proposed an optimized fabrication method for ascorbyl (AA)-conjugated SPMD (ascorbyl SPMD; AA-SPMD) to obtain the biological functionalities of AA in SPMD and evaluated it at the cellular level in vitro. The stability and antioxidant activity of AA-SPMD were confirmed using a heat stability test, a 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant capacity assay, and a total antioxidant capacity (TAC) assay. The biocompatibility and cell proliferation and migration abilities of AA-SPMD were also evaluated in human skin cells, dermal keratinocytes, and fibroblasts. Furthermore, we assessed the protection ability of AA-SPMD against oxidative damage induced by hydrogen peroxide (H_2_O_2_) and UVA. In addition, we confirmed the ability of collagen synthesis in skin fibroblasts to evaluate its potential applications in the biomedical field.

2. Results and Discussion

2.1. Heat Stability of AA-SPMD

The stability of AA-SPMD was investigated by conducting heat stability tests for 1 week and comparing the results with those of pure AA, as shown in Figure 1. The stability test results revealed that AA was completely oxidized, disappearing at 50 °C on the first day. By contrast, the stability of the AA in AA-SPMD was dramatically enhanced and maintained at over 90% for 7 days, as the most reactive sites of the AA molecules were shielded by the covalent bonding between the molecules and SPMD matrix at these sites. This indicates that AA alone is thermally unstable and oxidizes quickly; however, the stability of AA in AA-SPMD can be maintained extensively (more than 7 days) without thermal instability. The heat stability of AA-SPMD can be useful in biomedical and cosmetic therapy.

2.2. Antioxidant Activity of AA-SPMD

We investigated the antioxidative effects of AA-SPMD using various AA-SPMD concentrations (0, 12.5, 25, 50, 100, and 200 μM) and compared the results with those of the AA and SPMD samples. The radical scavenging activity of AA-SPMD was observed in dose-dependent manners as shown in Figure 2. Although the antioxidant capacity of AA-SPMD was lower than that of AA, it remained significantly higher at all concentrations than that of SPMD alone. The IC_50_ values of AA and AA-SPMD were calculated to be 17.8 and 24.25 μM, respectively. However, SPMD alone did not exhibit DPPH radical scavenging activity. These results show that SPMD alone has no antioxidant effect; however, chemically conjugating AA with SPMD provides an antioxidant capacity via free radical scavenging.

Meanwhile, the antioxidative activity of AA-SPMD using the TAC assay also showed similar results to the DPPH assay, as outlined in Table 1. The IC_50_ value of AA-SPMD was calculated to be 232.42 μM. This value was lower than that of the positive control, Trolox, indicating that AA-SPMD possesses stronger antioxidant activity than the positive control.

Based on these antioxidant data, the results demonstrated that chemically bound AA-SPMD retained the antioxidant capacity of AA, highlighting its potential in applications requiring antioxidant function.

Therefore, AA-SPMD can be used as an additive or adjunct in biomedical, cosmetic, and dietary applications; however, a more in-depth study of the antioxidant activity of AA-SPMD using different methods to assess its biological antioxidant ability is required.

2.3. Cytotoxicity of AA-SPMD in Skin Keratinocyte and Fibroblast Cells

The biocompatibility of AA-SPMD was confirmed by performing cytotoxicity tests on two types of skin cells: keratinocytes (HaCaT) and primary cultured fibroblasts (HDF). The cell viabilities of AA, SPMD, and AA-SPMD did not decrease at all concentrations in the HaCaT cell, as shown in Figure 3a,c; however, the HDF showed cytotoxicity to 100 and 1000 μM SPMD (30% and 21% cell viability, respectively) and to 1000 μM AA (92% cell viability), as shown in Figure 3b,d. However, AA-SPMD showed no cytotoxicity at any concentration for both skin cells. This indicates that the chemical conjugation of AA and SPMD reduced the cytotoxicity of SPMD to HDF at high concentrations.

At a suitable dosage, AA is typically considered safe and can be used in skin fibroblast experiments; however, AA is known to exhibit cytotoxicity in fibroblast cells at high concentrations (over 100–200 μM) [42], as shown in Figure 3b,d. Meanwhile, SPMD exhibits cytotoxicity in fibroblast cells at a high dose (1 mM). However, at 1 mM concentration, AA-SPMD showed no cytotoxicity to HDF. Theoretically, the synthesis ratio of AA and SPMD is 1:1; that is, 1 mM AA-SPMD contains 1 mM AA and SPMD, respectively. Therefore, cytotoxicity should typically be observed in fibroblasts; however, no toxicity was observed in our experimental results. Although the exact mechanism for this observation is currently unknown, we believe that cytotoxicity to HDF is eliminated by the chemical bonds between AA and SPMD.

These results demonstrate that AA-SPMD has excellent biocompatibility without any cytotoxicity, even at high concentrations, and can be used in the biomedical and cosmetic fields; however, further investigations are required to elucidate its molecular mechanisms.

2.4. Proliferation of Skin Cells by AA-SPMD

Proliferations of skin cells, keratinocytes, and fibroblasts, are essential to maintaining the integrity of skin tissues, repairing wounds, and renewing the skin barrier [42,43,44,45,46,47,48,49,50]. Therefore, we evaluated the cell proliferation effects of AA-SPMD using HaCaT and HDF cells over an extended period (5 days), as shown in Figure 4.

The HaCaT and HDF cells were incubated at various concentrations of AA, SPMD, and AA-SPMD for 5 days. The cell proliferation results were similar across both cell types; however, slight differences were observed. In particular, the proliferation rates of the HaCaT (109.5%) and HDF (110.3%) cells increased at a concentration of 10 μM AA-SPMD; however, AA-SPMD inhibited cell proliferation in both cell types at concentrations of 100–1000 μM. SPMD-treated groups (0.1 and 1 μM) in HDF also showed cell proliferation effects of 110.5 and 112%, respectively, and AA exhibited cell proliferation effects at concentrations of 100 μM for HaCaT and 10 μM for HDF. However, cell inhibitions in both samples by AA and SPMD were also confirmed at high concentrations (100–1000 μM). Thus, the conjugation of AA and SPMD appears to promote proliferation in both skin cell types at specific concentrations (10 μM for both cells), while avoiding the growth inhibition observed with high-dose AA or SPMD alone.

2.5. In Vitro Wound-Closure Ability of AA-SPMD

As shown in Figure 5, the migration ability of HaCaT and HDF cells was investigated using an in vitro wound-closure assay for 24 h to evaluate the wound healing effect of AA-SPMD on human skin cells. In AA-SPMD-treated cells, the wound widths in both cell types decreased gradually relative to the control; however, they were not significantly different from those observed with AA and SPMD alone. These results indicated that AA-SPMD maintained the cell migration abilities of AA and SPMD, which are known to increase fibroblast migration [42,43,44].

2.6. Production of Collagen I by AA-SPMD in Skin Fibroblast Cells

Collagen is indispensable for maintaining skin structure and plays a critical role in wound healing because it contributes to the biomechanical properties of skin tissue, such as structural stability [45,46]. Moreover, collagen contributes to the recruitment of skin cells, such as epithelial keratinocytes and dermal fibroblasts, during skin tissue remodeling [47]. In particular, collagen I is the most prevalent protein in the human body and is a major structural component of fibrous connective tissues [48]. Among the cells in fibrous connective tissues, HDFs in the dermis play vital roles in skin repair and remodeling by proliferating and migrating to the wound area, followed by the synthesis of collagen I and elastin and the formation of robust actin bundles in myofibroblasts [49,50].

Therefore, we conducted an experiment to confirm the synthesis of collagen I from HDF using an anti-collagen I antibody, which stains the collagen alpha-1 chain in HDF, by immunostaining to explain the effects of cell proliferation and wound healing in HDF.

As shown in Figure 6, AA-SPMD increased collagen I synthesis at all concentrations compared with AA and SPMD alone and significantly increased collagen I synthesis at concentrations of 1 and 10 μM. The amounts of collagen I synthesized by AA-SPMD at 1 and 10 μM were calculated to be 114.3 and 112.8%, respectively. Meanwhile, AA, which is known to aid collagen synthesis, showed a collagen synthesis rate of 97–102%, whereas SPMD showed a collagen synthesis rate of 100–104%. Both AA and SPMD showed similar or slightly higher levels than the control group at all concentrations, as illustrated by the fluorescence images in Figure 6b. These results demonstrate that AA-SPMD can increase the amount of collagen I synthesized in HDF cells, thereby influencing cell proliferation and cell migration or wound healing. AA-SPMD is expected to be more effective than AA or SPMD alone.

2.7. Protective Effects of AA-SPMD from H2O2-Induced Oxidative Damage and UVA Irradiation in Skin Cells

As shown in Figure 7, AA-SPMD exhibited protective effects against H_2_O_2_-induced oxidative damage in HaCaT and HDF cells. The cell viabilities in both cells were relatively higher for AA-SPMD (77% for HaCaT and 65.1% for HDF cells) than those of the H_2_O_2_-treated group (68.1% for HaCaT and 35.1% for HDF cells) and AA-treated group (46.1% for HaCaT and 63.5% for HDF cells); however, the SPMD-treated group showed higher cell viability for the HaCaT cells (90.1% for HaCaT and 52.5% for HDF cells) compared with that of AA-SPMD.

These data suggest that both SPMD and AA-SPMD confer protection against H_2_O_2_-induced oxidative stress in skin cells. AA-SPMD alleviated cytotoxicity by reducing H_2_O_2_-induced oxidative damage in both skin cell types.

In this study, we focused on comparing phototoxicity by UVA irradiation and evaluated the ability of protection of AA-SPMD against UVA-induced phototoxicity in skin cells using the WST-8 method.

As shown in Figure 8, UVA irradiation reduced cell viability in both cells owing to UVA-induced phototoxicity. The cell viabilities (73% for HaCaT cells and 62% for HDF cells) of AA-SPMD were slightly higher than those of the control (UVA treated; 67% for HaCaT cells and 51% for HDF cells); however, the cell viabilities (78% for HaCaT cells and 70% for HDF cells) of AA in both cells were significantly higher than those of the control and AA-SPMD. Conversely, SPMD showed no protective effect against UVA-induced phototoxicity in either cell type. These results demonstrate that, compared with the control, AA significantly increases the cell viability in both cells by protecting against UVA-induced phototoxicity. In addition, AA-SPMD has a certain protective effect against UVA irradiation. This suggests that AA-SPMD has anti-photoaging potential in human skin cells in vitro.

These results on the protective effects against H_2_O_2_-induced oxidative stress and UVA-induced phototoxicity indicate that AA-SPMD has antioxidative and anti-photoaging potential in vitro. However, these findings suggest that additional investigations are warranted to further elucidate its molecular mechanisms.

Overall, we successfully fabricated a functionalized SPMD conjugating with AA using an optimized design and synthetic approach to leverage the numerous biological functions of AA, such as antioxidant activity [27,28,29,30,31], collagen I synthesis [23,51,52,53], anti-inflammatory [24,25], depigmentation actions [26,27], and skin cell proliferation [52,53].

We confirmed the high stability and strong antioxidant activity of AA-SPMD, thereby overcoming the instability of AA and successfully imparting its antioxidant function to SPMD.

Furthermore, we confirmed that AA-SPMD can potentially increase cell proliferation and migration in skin cells via increased cell viability in both cells and collagen I production in HDF cells. Although they have different mechanisms for increasing cell viability, AA and SPMD are known to increase skin cell proliferation [1,5,7,8,52,53]. Therefore, we conjugated SPMD with AA to obtain a synergistic effect on cellular activity. We confirmed the synergistic effects of AA-SPMD on cell proliferation and collagen I production at specific concentrations. However, we could not observe the synergistic effects of AA-SPMD on cell migration. These results indicate that AA-SPMD still possesses the individual cell proliferation abilities of AA and SPMD in both skin cells, as well as the collagen I production capability of AA [23,51,52,53] in HDF cells.

In addition, we assessed the protective effects of AA-SPMD against H_2_O_2_- and UVA-induced oxidative damage in human skin cells because AA is known to be a cytoprotective agent against H_2_O_2_- and UVA-induced oxidative damage [28,54,55,56,57] in skin cells, and SPMD is also known to exhibit cytoprotective autophagy in female germline stem cells (FGSCs) in vitro and ameliorates cellular senescence of FGSCs induced by H_2_O_2_ [58].

In the cytoprotective study against H_2_O_2_-induced oxidative damage, AA-SPMD exhibited a certain protective effect in the HaCaT and HDF cells; however, SPMD exhibited stronger cytoprotective effects than AA alone and AA-SPMD. Meanwhile, for UVA-induced oxidative damage, AA exhibited photoprotective effects with significantly increased cell viability in both cells, and AA-SPMD showed certain photoprotective effects against UVA irradiation. However, SPMD showed no protective effect against UVA-induced phototoxicity in either cell type. Based on these cytoprotective studies against oxidative damage, we confirmed that AA-SPMD simultaneously possesses certain cytoprotective and photoprotective effects against H_2_O_2_- and UVA-induced oxidative stress. Conversely, SPMD exhibited cytoprotective effects against H_2_O_2_-induced oxidative damage, with no such findings for UVA-induced oxidative damage, and AA exhibited stronger photoprotective effects against UVA irradiation compared with H_2_O_2_-induced oxidative damage.

3. Materials and Methods

3.1. Materials

All chemicals used for the synthesis of AA-SPMD were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

3.2. Synthesis of AA-SPMD

3.2.1. Synthesis of N1, N5-Bis-Boc-Spermidine

To obtain the AA-SPMD, the N^5^-Bis-Boc-spermidine was considered a key intermediate and synthesized using a multistep strategy (four steps), as shown in Scheme 1.

Synthesis of Compound 1 (N-Boc-1,4-diaminobutane)

First, N-Boc-1,4-diaminobutane was synthesized and prepared by adding a solution of di-tert-butyldicarbonate (Boc_2_O) (2.17 mL, 9.4 mmol) in CHCl_3_ (50 mL) dropwise over 2 h to a solution of 1,4-diaminobutane (9.45 mL, 94.4 mmol) in CHCl_3_ (100 mL), and the mixture was stirred overnight. The organic layer was then washed with distilled water (DW, 3 × 30 mL) and dried over MgSO_4_. And then, the solvent residue was removed under reduced pressure to afford the pure product as a colorless oil (yield = 90%). The compound was characterized by ^1^H NMR, which showed the expected signals, δ: 7.11 (brs, 1H), 3.63 (t, 2H), 2.84 (t, 2H), 2.21 (brs, 2H), 2.02 (brs, 2H), 1.55 (t, 2H), 1.25 (s, 9H), as shown in ^1^H NMR (400 MHz, CDCl_3_).

2.Synthesis of Compounds 2 and 3 (Tert-butyl (4-((tert-butoxycarbonyl)amino)butyl)(cyanomethyl)carbamate)

Next, a solution of carbamate 1 (2.08 g, 11.05 mmol) in methanol (20 mL) was added to acrylonitrile (0.87 mL, 12.15 mmol) and stirred at room temperature (RT) for 7 h. Thin-layer chromatography revealed that the starting material was consumed (DCM:MeOH = 10:1). A Boc_2_O (2.4 mL, 11.05 mmol) was then added, and the mixture was stirred for an additional 6 h at RT. The residual solvent was removed under vacuum, and the residue was treated with dilute hydrochloric acid (20 mL) and extracted with EtOAc (20 mL × 3). The combined organic layers were washed with brine, dried over Na_2_SO_4_, and concentrated. Then, the mixture was purified using silica gel chromatography (hexane:EtOAc = 1:1) as a colorless oil (yield = 93%). The compound was characterized by ^1^H NMR. The NMR spectra showed the expected signals, as shown in ^1^H NMR (400 MHz, CDCl_3_) δ: 4.79 (brs, 1H), 3.35 (t, 2H), 3.16 (t, 2H), 3.05–2.97 (m, 2H), 2.55–2.43 (m, 2H), 1.50–1.40 (m, 4H), 1.35 (s, 9H), 1.32 (s, 9H).

3.Synthesis of N^1^, N^5^-Bis-Boc-spermidine

Finally, a suspension of Raney Ni (2 g/10 mL in ethanol) was added to Compound 3 (1 equiv., 1.4 mmol) and slowly to NH_4_OH (4 mL). The mixture was stirred under a hydrogen atmosphere (20 bar) at RT for 24 h. Subsequently, the Raney Ni was removed by filtration through a Celite pad, and the solution of filtration was evaporated.

The obtained solid was dissolved in dichloromethane and washed twice with 2.5M NaOH and DW. Subsequently, the solution was dried over anhydrous Na_2_SO_4_ and concentrated under reduced pressure to obtain N^1^, N^5^-Bis-Boc-spermidine as a colorless oil (yield = 88%). The compound was characterized by ^1^H NMR. The NMR spectra showed the expected signals, as shown in ^1^H NMR (400 MHz, CDCl_3_) δ 4.58 (brs, 1H), 3.23–3.09 (m, 6H), 2.68–2.62 (m, 2H), 2.08–1.95 (m, 2H), 1.70–1.61 (m, 2H), 1.60–1.48 (m, 2H), 1.41 (s, 18H).

3.2.2. Synthesis and Purification of AA-SPMD

AA-SPMD was synthesized using a multistep strategy, as shown in Scheme 2.

Synthesis of Compound 5

Compound 5 was obtained by adding a mixture of AA acetonide 4 (5 g, 23.13 mmol) and DIPEA (4.8 mL, 27.8 mL) in DMSO (20 mL) to ethyl bromoacetate (2.6 mL, 23.13 mmol). The mixture was stirred at RT for 6 h, quenched with aq. sat. NaHSO_4_ (20 mL), and extracted with EtOAc (20 mL × 3). The organic layers were combined, washed with brine, and dried over Na_2_SO_4_. Then, the mixture was concentrated and purified using silica gel chromatography (hexane:EtOAc = 1:1) as a colorless oil (yield = 82%). The NMR spectra showed the expected signals, as shown in ^1^H NMR (400 MHz, CDCl_3_) δ 5.91 (brs, 1H), 5.04–4.91 (ABq, 2H), 4.71 (d, 1H), 4.34–4.27 (m, 3H), 4.21 (t, 1H), 4.09–4.06 (m, 1H), 1.42 (s, 3H), 1.39 (s, 3H), 1.32 (t, 3H).

2.Synthesis of Compound 6

Compound 6 was synthesized by adding LiOH (1.7 g, 40.5 mmol) to a solution of Compound 5 (3.53 g, 11.7 mmol) in THF/H_2_O (3:1, 40 mL) at 0 °C. The mixture was stirred at 0 °C for 30 min, acidified with 3N-HCl, and extracted with EtOAc (20 mL × 5). The organic layers were combined, washed with brine, and dried over Na_2_SO_4_. Then, the mixture was concentrated and used in the next step without further purification in its white form (yield = 95%). The NMR spectra showed the expected signals, as shown in ^1^H NMR (400 MHz, MeOD-d_4_) δ 5.09–4.82 (ABq, 2H), 4.92–4.76 (d, 1H), 4.34–4.30 (m, 1H), 4.21 (t, 1H), 4.05–4.02 (m, 1H), 1.37 (s, 3H), 1.35 (s, 3H).

3.Synthesis of Compound 7 and AA-SPMD

Compound 7 was obtained by adding HOBt (0.59 g, 4.38 mmol) and EDC·HCl (1.26 g, 6.57 mmol) to a solution of Compound 6 (1.2 g, 4.38 mmol) in CH_2_Cl_2_ (20 mL) and stirring for 10 min at 0 °C. Subsequently, the N^1^, N^5^-Bis-Boc-spermidine (4) (1.81 g, 5.25 mmol) was added to the mixture and stirred for 2 h at RT The reaction mixture was quenched with saturated aqueous NaHSO_4_ (20 mL) and extracted. The organic layer was washed with brine, dried over MgSO_4_, filtered, and concentrated under vacuum. The mixture was then used in the next step without further purification in its white form. Finally, Compound 7 was dissolved in 4M HCl in Dioxane/H_2_O (9/1, 10 mL) and stirred for 2 h at RT. The solvent was concentrated under reduced pressure to obtain AA-SPMD (8) as a white solid (yield = 95%). The NMR spectra in Figure 9 show the expected signals, as shown in ^1^H NMR (400 MHz, MeOD-d_4_) δ 5.15–4.87 (ABq, 2H), 4.95 (d, 1H), 4.01 (t, 1H), 3.70 (d, 2H), 3.42 (t, 2H), 3.05–2.97 (m, 6H), 1.95–1.77 (6H). MS (ESI^+^) m/z for C_15_H_27_N_3_O_7_ [M + H]^+^ was 361.4 (calculated) and experimentally determined to be 362.2.

3.3. Stability Test

The heat stability of pure AA in AA-SPMD was evaluated by monitoring the value of AA retention at different periods. Briefly, 100 μM AA-SPMD powder (containing 100 μM AA) and 100 μM pure AA were separately added into vials containing 10 mL of decarbonated water and sealed carefully with the caps. The vials were maintained in an oven at a strictly controlled temperature of 50 °C to ensure rigorous conditions. Finally, the AA content in AA-SPMD was analyzed via high-performance liquid chromatography on days 1, 3, and 7. The chromatographic analysis was performed using an HPLC system (Nanospace Si-2, Osaka Soda Co. Ltd., Osaka, Japan) with 220 and 245 nm wavelengths. Chromatographic separations were conducted using a C18 (CAPCELL PAK, 4.6 μm × 250 mm, particle size 5 μm, Osaka Soda Co. Ltd.). The mobile phase comprises water with 0.05% trifluoroacetic acid (TFA) and acetonitrile with 0.05% TFA. The flow rate was kept constant at 1 mL/min.

3.4. Evaluation of the Antioxidant Activity of AA-SPMD

We evaluated the antioxidant activity of AA-SPMD using two methods: DPPH radical scavenging assay and TAC assay.

The DPPH radical scavenging activity of AA-SPMD was conducted using a commercial assay kit (BO-DPH-200, Biomax, Ltd., Guri-si, Republic of Korea), following the manufacturer’s protocol. AA, SPMD, and AA-SPMD were prepared at various concentrations (0–200 μM) and added into a 96-well plate. Adding the working solution of DPPH and assay buffer solution, the plate was incubated at RT for 30 min in the dark. Finally, the optical absorbance was measured at 517 nm using a microplate reader (Cytation-3; Agilent Technologies, Inc., Santa Clara, CA, USA). The radical scavenging activity was then calculated and expressed relative to a standard curve of Trolox.

The TAC assay was performed using a total antioxidant capacity assay kit (BO-TAC-200, Biomax, Ltd.) according to the manufacturer’s instructions. Various concentrations (0, 42, 83, 167, 340, and 670 μM) of AA, SPMD, and AA-SPMD were dissolved in Dulbecco’s phosphate-buffered saline (DPBS), and 100 μL of each solution of the sample was added to 100 μL of copper reagent and reaction buffer. Subsequently, the mixture was incubated at RT for 30 min, and the absorbance was measured at 450 nm with a multimode microplate reader. Final absorbance values were obtained by subtracting the blank, and the antioxidant activity was calculated from the standard curve of Trolox.

3.5. Cytotoxicity Assessment of AA-SPMD

In this study, two types of human skin cells, HaCaT and HDF, were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany) and CEFO Co. (Seoul, Republic of Korea). The cells were maintained in Dulbecco’s modified Eagle’s medium containing a 10% fetal bovine serum (FBS) and a 1% antibiotic antimycotic solution for HaCaT and in a human mesenchymal stem cell (MSC) growth medium (CEFOgro-MSC) containing 10% FBS and a 0.5% antibiotic antimycotic solution for HDF. Both cells were cultured at 37 °C in an incubator containing 5% CO_2_.

The in vitro biocompatibilities of AA, SPMD, and AA-SPMD were assessed by performing cytotoxicity tests on HaCaT and HDF, as previously described [59,60,61]. Pre-cultured cells at 2.0 × 10^5^ cells/well for HaCaT and at 1.2 × 10^5^ cells/well for HDF in 24-well plates were incubated with various concentrations (0, 0.1, 1, 10, 100, and 1000 μM) of AA, SPMD, and AA-SPMD for 24 h. The cell viabilities were detected using a solution of the colorimetric assay kit (WST-8 Cell Counting Kit, Biomax, Ltd.), calculated by detecting the optical absorbance at 450 nm using a microplate reader (Cytation-3), and quantified as the percentage of viable cells relative to the untreated control. Furthermore, live-cell populations of HaCaT and HDF were observed using EZ-view live-cell staining kits (Biomax, Seoul, Republic of Korea) according to the manufacturer’s protocol. Fluorescence images of each cell were captured after incubating with a calcein-AM solution (final concentration, 6.7 μM) for 20 min at 37 °C using a live-cell imaging system (Lionheart FX; Agilent Technologies, Inc., CA, USA) with a 4× high-contrast objective lens.

3.6. Cell Proliferation by AA-SPMD in Human Skin Cells

The in vitro proliferation rates of human skin cells treated with AA, SPMD, and AA-SPMD were also evaluated using the WST-8 Cell Counting Kit, as described above. The two types of skin cells, at 2.0 × 10^4^ cells/well for HaCaT and 4.0 × 10^3^ cells/well for HDF, were incubated with various concentrations (0, 0.1, 1, 10, 100, and 1000 μM) of AA, SPMD, and AA-SPMD at 37 °C for 5 days. The viabilities of cells were detected using a WST-8 solution, as described above, to confirm the cell proliferation rate.

3.7. In Vitro Wound-Closure Assay by AA-SPMD

The ability of skin keratinocytes and fibroblasts to migrate to culture plates under different experimental conditions was evaluated using an in vitro wound-closure assay. The assay was employed in a wound-closure test for HaCaT and HDF cells. The cells were plated in a 24-well plate at 2.5 × 10^5^ cells/well for HaCaT and at 1.5 × 10^5^ cells/well for HDF and incubated for 24 h. Subsequently, a vertical wound ~800–1000 µm wide in each well was generated using a 2.5 mL pipette tip, and the cell debris and media were carefully washed and exchanged with fresh media. The cells were then incubated with 10 μM AA, SPMD, and AA-SPMD at 37 °C. An initial image was taken at that time (time = 0 s), and the wound closure was recorded for 24 h using a live-cell imaging system (Lionheart FX) as described above. The distance between the sides of the wound was measured at three points in each well (in triplicate, n = 3) to evaluate the wound closure from the acquired images. The cell migration data were analyzed using Gen5 software (Ver. 3.17, Agilent Technologies, Inc., CA, USA) by calculating the percentage of wound closure at each point relative to the control.

3.8. Analysis of Collagen I Levels by Immunostaining

The levels of collagen I synthesized from HDF by AA-SPMD were analyzed by fluorescence microscopy and immunostaining. HDF (8 × 10^4^ cells/well) was cultured on a black 24-well plate (ibidi, μ-plate 24 well) for 24 h. The medium was then replaced with a fresh one containing various concentrations (0, 1, 10, and 100 μM) of AA, SPMD, and AA-SPMD, and the plate was further incubated for 24 h. Subsequently, the cells in the plate were washed with DPBS, fixed with a methanol solution (100%) for 5 min, permeabilized with 0.1% Triton X-100 solution for 5 min, and then blocked with 1% bovine serum albumin solution containing 10% normal goat serum and 0.3M glycine in 0.1% PBS-Tween solution for 1 h. After blocking the cells, the blocking solution was changed with a rabbit monoclonal antibody (ab138492, Abcam), detecting collagen I at 1:350 dilution in a blocking solution, and incubated overnight at 4 °C. The plate was washed, and the cells were stained with 2 μg/mL of goat anti-rabbit IgG secondary antibody (Alexa Fluor 488, ab150081 Abcam) for 1 h in the dark. Finally, the cells were stained with Hoechst 33342 to detect nuclear DNA. The fluorescence intensity in each well was measured using the well area scan (5 × 5) mode in a multimode microplate reader (Cytation-3) for collagen I at 485/528 nm and nuclear DNA at 350/461 nm. The total amount of synthesized collagen I was normalized by the amount of collagen I synthesized per cell. Furthermore, fluorescence images of green fluorescence (collagen I) were captured using a live-cell imager (Lionheart FX) with a 4× objective lens and fluorescence optics (excitation/emission at 469/525 nm for collagen I and 377/447 nm for nuclear DNA).

3.9. Protective Effects of AA-SPMD from H2O2- and UVA-Induced Oxidative Damage

The protective effect of AA-SPMD from oxidative damage in the HaCaT and HDF cells was confirmed by treatment with H_2_O_2_ and UVA as follows: First, pre-cultured HaCaT (1.5 × 10^5^ cells/well) and HDF (1.2 × 10^5^ cells/well) cells were incubated with 100 μM AA, SPMD, and AA-SPMD for 8 h. Subsequently, the cells were treated with 400 μM H_2_O_2_ and further incubated for 20 h. Finally, the cells were incubated with a WST-8 solution, as described above, to confirm cell viability.

For UVA irradiation, pre-cultured HaCaT (1.6 × 10^4^ cells/well) and HDF (1.0 × 10^4^ cells/well) cells in 96-well plates were washed with DPBS. Each cell was treated with 100 μM AA, SPMD, and AA-SPMD and incubated for 4 h. Subsequently, the cells were washed with DPBS, changed with fresh media, and irradiated with a blue light-emitting diode (UVA LED, 365 nm) at 15 mW/cm^2^ for 20 min. The UVA-irradiated cells were washed with DPBS and changed with fresh media again and incubated for 24 h. After incubation, cell viability was confirmed using the WST-8 assay described previously.

3.10. Statistical Analysis

All data are presented as the mean ± standard deviation (SD), and statistical analyses were conducted using a paired t-test to compare the negative control (NC) with each sample concentration using SPSS statistics (IBM SPSS Statistics, ver. 29.0, NY, USA). Statistical significance was defined as p < 0.05 (* p < 0.05; ** p < 0.005, *** p < 0.0005).

4. Conclusions

In this study, we verified the effects of chemically synthesized AA-SPMD on the in vitro capabilities in terms of stability, antioxidant capacity, cell proliferation and migration without cytotoxicity, protective capacity against H_2_O_2_-induced oxidative stress and phototoxicity by UVA, and production of collagen I as an additive or adjunct in biomedical, cosmetic, or dietary applications.

The results revealed that AA-SPMD has no cytotoxicity even at 1 mM, and it can increase the rate of cell proliferation and migration in skin cells without reducing the inhibition of HaCaT and HDF cells at concentrations of 10 μM. In addition, compared with the control, AA significantly increased the cell viability in both cells by protecting against UVA-induced phototoxicity. AA-SPMD also demonstrated a certain protective effect against UVA irradiation, suggesting that AA-SPMD has anti-photoaging potential in in vitro human skin cells.

Chemically conjugated AA-SPMD is expected to be more effective than AA and/or SPMD alone in terms of its antioxidant and cellular activity. The study findings suggest its potential applications in the biomedical and cosmetic fields. However, additional mechanistic studies, along with in vivo animal studies, are necessary to verify the biological and physiological activities of AA-SPMD before its use in biomedical and cosmetic applications.