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28 January 2026

18 Pages

Biocompatible Dipeptide-Based Nanogels Incorporating Cinnamic Acid for Applications in Skin Disorder Therapy

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,
and
1
Gülhane Faculty of Pharmacy, Department of Pharmaceutical Toxicology, University of Health Sciences Turkey, 06018 Ankara, Türkiye
2
Gülhane Faculty of Pharmacy, Department of Pharmaceutical Chemistry, University of Health Sciences Turkey, 06018 Ankara, Türkiye
3
Gülhane Faculty of Pharmacy, Department of Analytical Chemistry, University of Health Sciences Turkey, 06018 Ankara, Türkiye
*
Author to whom correspondence should be addressed.
Pharmaceutics2026, 18(2), 173;https://doi.org/10.3390/pharmaceutics18020173
This article belongs to the Section Nanomedicine and Nanotechnology
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Abstract

Background/Objectives: Skin-related disorders such as melanoma, premature aging, and chronic wounds significantly impact individuals’ quality of life and psychological well-being. Melanoma, due to its high metastatic potential and poor response to conventional chemotherapeutic agents, remains a major clinical challenge. Additionally, skin aging and impaired wound healing continue to drive the demand for novel therapeutic strategies and bioactive formulations. Methods: In this study, cinnamic acid (CA), a naturally occurring compound with known anti-inflammatory and antioxidant properties, was incorporated into biocompatible Fmoc-FF dipeptide-based nanogels to improve its stability and therapeutic efficacy. The antitumor effects of CA and CA-loaded nanogels were evaluated using human melanoma (SK-MEL-30) cells, while wound healing activity was assessed on human keratinocyte (HaCaT) cells. Results: The results demonstrated that CA exhibited significant activity against melanoma cells and promoted wound healing, with enhanced effects observed when delivered via Fmoc-FF nanogels. Conclusions: These findings suggest that CA-loaded peptide nanogels represent a promising platform for multifunctional treatment approaches targeting various skin disorders.

1. Introduction

The skin, as the largest organ of the human body, serves as a critical barrier against environmental insults. However, with advancing age and exposure to ultraviolet (UV) radiation and various chemical agents, its structural and functional integrity deteriorates. This degradation not only leads to physiological consequences but also has a significant impact on appearance, contributing to increased psychological distress, including anxiety and depression—particularly in a society that increasingly values aesthetics [1,2].
Among chronic skin conditions, melanoma remains the most aggressive and lethal form of skin cancer, accounting for nearly 80% of skin cancer-related deaths due to its high metastatic potential [3]. Although current treatment options such as chemotherapy and radiotherapy are clinically utilized, their limited success rates and severe adverse effects have intensified the search for alternative therapeutic strategies [4]. In this context, “green therapeutics” derived from natural compounds are gaining considerable attention due to their multifunctional bioactivities and lower toxicity profiles [5].
Wound healing is a complex physiological process involving inflammation, cellular proliferation, collagen deposition, and tissue remodeling [6]. The use of anti-inflammatory and pro-collagen agents, particularly of herbal origin, has been explored as a safer and effective approach to enhance wound repair, reducing the limitations posed by synthetic drugs [7].
Cinnamic acid (CA), a natural phenolic compound found in cinnamon bark (Cinnamomum cassia), ginseng (Panax ginseng), and various fruits and grains, exhibits a broad spectrum of pharmacological activities, including antioxidant, anti-inflammatory, anticancer, antimicrobial, and neuroprotective effects [8,9,10]. Its non-tolerance-inducing nature and ability to regulate cancer pathways have prompted its investigation in anticancer drug development [11]. CA has limited aqueous solubility and is prone to photodegradation and metabolic instability, which limit its therapeutic application [12].
Nanotechnology-based drug delivery systems have demonstrated the ability to enhance the solubility, stability, and therapeutic performance of hydrophobic compounds like CA [13]. Among these, the use of biocompatible, biodegradable, and high-loading-capacity nanostructures is particularly important for safe and effective drug delivery [14].
Recent advances have emphasized the potential of bioinspired materials, such as amino acids and peptides, in designing self-assembled nanostructures for drug delivery, given their inherent compatibility with biological systems and functional versatility [15]. In particular, dipeptide-based supramolecular hydrogels, formed via noncovalent interactions, offer promising features including low cost, ease of synthesis, biodegradability, and self-healing properties [16,17,18]. Among these, Fmoc-diphenylalanine (Fmoc-FF) has emerged as one of the most widely studied and efficient low molecular weight gelators with excellent structural stability and biocompatibility [19,20]. Fmoc-FF nanogels, being amphiphilic and self-assembling in aqueous media, encapsulate CA within their hydrophobic domains, shielding it from aqueous degradation pathways and enhancing apparent solubility.
Topical application of peptide-based nanogels, such as Fmoc-FF, presents a unique advantage in treating skin-related disorders, offering localized action, sustained drug release, and minimal systemic toxicity [16]. Based on these considerations, this study aimed to develop and characterize CA-loaded Fmoc-FF nanogels, and to evaluate their dual therapeutic potential: anti-melanoma efficacy in human SK-MEL-30 melanoma cells, and wound-healing effects in HaCaT keratinocyte cells. The investigation included assessments of cytotoxicity, genotoxicity, oxidative stress, tyrosinase activity, inflammation, and wound closure to provide a comprehensive understanding of the therapeutic potential of the designed nanoplatform.

2. Material and Methods

2.1. Apparatus

Morphological characterization of the nanoparticles was performed using Transmission Electron Microscopy (TEM) at an accelerating voltage of 200 kV (Jeol 2100F RTEM, Tokio, Japan). The structural properties of the formulations were investigated using FTIR spectroscopy (PerkinElmer IR, Sacramento, CA, USA). Particle size and size distribution of the dipeptide-based nanogels were measured using photon correlation spectroscopy (PCS) (Nicomp Z3000, Entegris, Billerica, MA, USA).
To evaluate cytotoxicity, reactive oxygen species (ROS) production, and immunotoxic responses, absorbance measurements were performed using a microplate reader (BioTek Instruments, Winooski, VT, USA). For genotoxicity assessment, slides stained with ethidium bromide (EtBr) were visualized under a fluorescence microscope (Leica®, Leica Microsystems GmbH, Wetzlar, Germany) equipped with a 40× objective lens and connected to a CCD camera. Image capture and analysis were carried out using Comet Analysis Software (Version 4.0, Kinetic Imaging Ltd., Liverpool, UK). Apoptotic responses were evaluated using flow cytometry (Beckman Coulter, Brea, CA, USA). Biorender was used to create the graphical summary and OriginPro 8.0 was used to draw the graphs.

2.2. Chemicals

All reagents are analytical grade reagents unless stated otherwise. Aqueous solutions were prepared in ultrapure water (18.3 μΩ cm). Fmoc-FF was obtained from Bachem (Bachem, Torrance, CA, USA). The following reagents were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA): cinnamic acid (CA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (DCF-DA), xanthine, disodium ethylenediaminetetraacetic acid (Na2EDTA), hexafluoroisopropanol (HFIP), ammonium sulfate ((NH4)2SO4), ethanol, methanol, low-melting point agarose (LMA), normal melting point agarose (NMA), dimethyl sulfoxide (DMSO), ethidium bromide (EtBr), phosphate-buffered saline (PBS) tablets, N-lauroylsarcosine sodium salt, and Triton X-100.
All cell culture reagents, including high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with L-glutamine and sodium pyruvate, 0.25% trypsin-EDTA, fetal bovine serum (FBS), penicillin-streptomycin, and sterile phosphate-buffered saline (PBS), were obtained from Serena (Pessin, Germany). Enzyme-linked immunosorbent assay (ELISA) kits targeting interleukin-8 (IL-8), nuclear factor kappa B (NF-κB), and tumor necrosis factor-alpha (TNF-α) were sourced from FineTest (Wuhan, China), while the tyrosinase-specific ELISA kit was procured from Elabscience Biotechnology Inc. (Houston, TX, USA). IsoLab Chemicals (Langweid am Lech, Germany) supplied 2-propanol. The β-galactosidase senescence detection kit was purchased from Merck (Darmstadt, Germany), and the Annexin V-FITC Apoptosis Detection Kit was obtained from Serva (Heidelberg, Germany).

2.3. Synthesis of Fmoc-FF Nanogels Encapsulating Cinnamic Acid

To prepare the surfactant mixture, we weighed equal amounts (w/w) of TWEEN® 60 and SPAN® 60 and added them to 10 mL of mineral oil. The mixture was then heated to 50 ± 2 °C in a water bath under gentle magnetic stirring to allow complete melting and homogeneous dissolution of SPAN® 60 into the oil phase. Once a clear and uniform surfactant-oil solution was obtained, it was immediately used in the nanoemulsion preparation step.
Fmoc-FF nanogels were synthesized using a nanoprecipitation-based emulsion method with slight modifications to encapsulate cinnamic acid (CA). Initially, 10 mg of CA was dissolved in 1 mL of ethanol under sonication. Separately, 10 mg of Fmoc-FF peptide was dissolved in 1 mL of DMSO to ensure complete solubilization. These two organic phases were then mixed and added dropwise into 10 mL of mineral oil containing a pre-heated (50 °C) surfactant mixture of SPAN® 60 and TWEEN® 60 (1:1, w/w) under continuous magnetic stirring at 1000 rpm to form a coarse emulsion. The mixture was further sonicated for 5 min (amplitude 40%, 5 s on/off pulse) to obtain a uniform nanoemulsion. The resulting system was stirred for 2 h to allow nanogel formation via self-assembly of Fmoc-FF and encapsulation of CA. For blank Fmoc-FF nanogels (without CA), the same procedure was followed excluding the addition of cinnamic acid. The final nanogel dispersion was washed with cold ethanol and centrifuged at 10,000 rpm for 10 min to remove excess oil and surfactants. The pellet was resuspended in distilled water and subjected to lyophilization for storage. The prepared nanogel suspension was washed 3 times in hexane medium to remove excess oil and then any remaining hexane was removed under vacuum. The UV-Vis spectrophotometer was used to determine the loading efficiency (LE) of the CA-loaded nanogels. The solution’s spectrum over the range 400–200 nm was recorded, and an absorption maximum at 271 nm was identified [21].
The LE (%) was calculated using the formula
LE (%) = (Cs/Cb) × 100
where Cb represents the initial CA amount, and Cs denotes the remaining CA after encapsulation.
In vitro drug release evaluation was performed using 15 mL phosphate-buffered saline (PBS, pH 7.4) as the dissolution medium. A total of 10 mL PBS was used per sample in sealed dialysis tubes (MWCO: 3.5 kDa). The release was monitored over a 24 h period, with aliquots withdrawn at the following time points: 1, 2, 3, 6, 12, 18, and 24 h. At each time point, 1 mL of release medium was withdrawn and replaced with an equal volume of fresh PBS to maintain sink conditions. The withdrawn samples were analyzed immediately. The amount of released cinnamic acid (CA) was quantified using a UV–Vis spectrophotometric method at λ = 270 nm, based on a calibration curve constructed with known concentrations. The method was validated for linearity, precision, and accuracy within the working range of 1–25 µg/mL. Blank nanogel samples and PBS were used as controls to correct for background absorbance [22].

2.4. Cell Culture

SK-MEL-30 (passage 17–21) and HaCaT (passage 9–10) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) enriched with 10% fetal bovine serum (FBS), 1–2% L-glutamine, sodium pyruvate, and 1% penicillin-streptomycin. The cultures were maintained in a humidified atmosphere at 37 °C with 5% CO2 and 95% relative humidity to ensure optimal growth conditions. Upon reaching approximately 80% confluency, cells were passaged using 0.25% trypsin-EDTA and transferred into appropriate culture vessels for subsequent experiments.
After enzymatic detachment, cell counts were determined using a cell counting chamber, and the suspensions were subsequently diluted with fresh culture medium to achieve the desired seeding densities. Detailed information regarding the number of cells seeded, the types of culture plates utilized, and the concentrations of CA and CA-loaded Fmoc-FF nanogels employed in each experimental setup are presented in Table 1.
Table 1. Summary of cell culture conditions, cell numbers, plate types, and concentrations of treatments used in each experiment.

2.4.1. Evaluation of Cytotoxic Effects

The cytotoxic potential of Fmoc-FF, CA, and CA-loaded Fmoc-FF nanogels on melanoma cells was assessed using the MTT assay. For this purpose, cells were treated with varying concentrations of each compound and incubated for 24 h. Following incubation, the media were discarded, and 0.5 mg/mL MTT solution was added to each well. Cells were then incubated for an additional 4 h to allow for formazan crystal formation. Afterward, the MTT solution was removed, and dimethyl sulfoxide (DMSO) was added to dissolve the formazan. The resulting color intensity was quantified spectrophotometrically at 570 nm.
A serum-free DMEM medium served as the negative control. Cell viability was calculated based on the absorbance values, and the results were expressed as a percentage relative to the control group using the formula below [23]:
Viability = (Absorbance of test well/Absorbance of negative control well) × 100

2.4.2. Assessment of ROS Formation in Treated Cells

Intracellular ROS production was evaluated using the H2DCFDA assay. SK-MEL-30 cells were seeded in appropriate culture plates and incubated for 24 h. Following incubation, cells were treated with Fmoc-FF, CA, and CA-loaded Fmoc-FF nanogels for 1 h. After treatment, the plates were transferred to ice, the media were carefully removed, and the wells were rinsed with 100 µL of phosphate-buffered saline (PBS).
To prevent photobleaching, all subsequent steps were performed in the dark. A volume of 100 µL of H2DCFDA solution was added to each well, and the plates were incubated at 37 °C for an additional 1 h. After staining, the wells were again placed on ice, the dye solution was removed, and cells were washed with 100 µL PBS. Finally, 100 µL of PBS was added to each well prior to measurement.
PBS served as the blank control, culture medium as the negative control, and 100 µM hydrogen peroxide (H2O2) was used as the positive control. Fluorescence intensity was measured using a microplate reader at an excitation wavelength of 485 nm and emission at 520 nm [24].

2.4.3. Preparation of Cell Lysates

To investigate alterations in tyrosinase activity and immunotoxicity-related biomarkers, SK-MEL-30 cells were seeded and incubated for 24 h to ensure proper adhesion. Following this period, the cells were exposed to different concentrations of Fmoc-FF, CA, and CA-loaded Fmoc-FF nanogels for an additional 24 h. After treatment, cells were detached using 0.25% trypsin-EDTA and harvested by centrifugation at 1000 g for 10 min. The resulting cell pellets were then resuspended in 1 mL of PBS and homogenized for further analyses.
For cell lysis, 400 μL of lysis buffer containing 1% protease inhibitor cocktail (aprotinin, bestatin, leupeptin, and pepstatin A) was added to each sample, and the mixture was pipetted thoroughly on ice to ensure complete lysis. The lysates were then centrifuged at 4000× g for 15 min, and the resulting supernatants were aliquoted and stored at −80 °C for subsequent analysis of oxidative stress markers and immunotoxicity-related cytokines [25].

2.4.4. Determination of Tyrosinase

To assess changes in tyrosinase levels, SK-MEL-30 cells were seeded and incubated for 24 h to allow for adhesion. After attachment, cells were treated with various concentrations of Fmoc-FF, cinnamic acid (CA), and CA-loaded Fmoc-FF nanogels for 24 h. Following treatment, the cells were detached using trypsin and collected by centrifugation at 1000× g for 10 min. The resulting cell pellets were resuspended in 1 mL of phosphate-buffered saline (PBS) and lysed as described in Section 2.4.3. The homogenates obtained were subsequently used for the quantification of tyrosinase levels. For the analysis of tyrosinase levels, commercial ELISA kits specifically designed for each target were utilized, following the manufacturers’ protocols. The concentrations of the biomarkers were quantified by measuring the absorbance at 450 nm, which reflects the colorimetric intensity generated in each well.

2.4.5. Evaluation of Immunotoxicity Biomarker Alterations

To investigate treatment-induced alterations in immunotoxicity biomarkers, SK-MEL-30 cells were seeded into culture plates and incubated for 24 h to facilitate cellular attachment. Following adhesion, the cells were exposed to varying concentrations of Fmoc-FF, CA, and CA-loaded Fmoc-FF nanogels for an additional 24 h incubation period. Following treatment, cells were detached using trypsin and harvested by centrifugation at 1000× g for 10 min. The resulting cell pellets were resuspended in 1 mL of phosphate-buffered saline (PBS) and lysed as described in Section 2.4.3. The homogenates obtained were then used to assess immunotoxicity biomarkers.
The levels of nuclear factor kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), and interleukin-8 (IL-8) were measured as immunotoxicity indicators using specific ELISA kits. All procedures were conducted in accordance with the manufacturers’ instructions. Absorbance values were recorded at 450 nm to quantify biomarker levels based on the intensity of color development in the assay wells.

2.4.6. Genotoxicity Studies

To assess the genotoxic potential of the test compounds, SK-MEL-30 cells were seeded and incubated for 24 h to enable proper adhesion. Following attachment, cells were exposed to Fmoc-FF, cinnamic acid (CA), and CA-loaded Fmoc-FF nanogels for a duration of 4 h. A serum-free culture medium served as the negative control throughout the experiment. Following the incubation period, the treatment medium was removed, and the wells were washed with phosphate-buffered saline (PBS). Cells were then detached using trypsin-EDTA and collected into suspension. The cell suspension was mixed with trypan blue, and viable cells were counted using a Neubauer counting slide. The cell concentration was adjusted with medium to obtain 10,000–20,000 cells per slide.
A total of 50 µL of the adjusted cell suspension was mixed with 100 µL of 1% low-melting point agarose (LMA) maintained at 37 °C ± 0.5 °C. This mixture was layered onto slides previously coated with 1% normal melting point agarose (NMA) and covered with a coverslip. After solidification on ice for 5–10 min, the coverslips were gently removed, and the slides were placed vertically in racks immersed in cold lysis solution (pre-chilled at 4 °C) for 1 h.
Electrophoresis was carried out using a pre-chilled electrophoresis buffer. After the lysis step, the slides were carefully positioned in the electrophoresis chamber with the agarose-coated side facing upward. The slides were then allowed to equilibrate in the buffer for 20 min without applying any electric current, ensuring optimal conditions for subsequent DNA migration. Electrophoresis was then conducted at 25 V and 300 mA for 20 min. Upon completion, the slides were carefully removed and immersed in PBS for 15 min, followed by sequential dehydration in 50%, 75%, and 99% ethanol for 5 min each.
To visualize DNA damage, slides were stained with ethidium bromide (EtBr). For each sample, 100 cells per slide were analyzed using a fluorescence microscope equipped with the Perceptive Instruments COMET Assay IV Analysis System (Perceptive Instruments Ltd., Staffordshire, UK). DNA damage was quantified based on the tail intensity parameter, which reflects the percentage of DNA migrated into the comet tail during electrophoresis [26].

2.4.7. Apoptosis Determination

SK-MEL-30 cells were seeded in 6-well plates and incubated for 24 h to ensure adequate cell attachment. After this period, the cells were exposed to Fmoc-FF, CA, and CA-loaded Fmoc-FF nanogels for an additional 24 h incubation. A serum-free medium was used as the negative control.
Following the completion of the treatment period, cells were harvested, and apoptosis analysis was performed using flow cytometry (Beckman Coulter, Brea, CA, USA) in combination with an Annexin V-FITC/propidium iodide (PI) double staining apoptosis detection kit (Serva, Heidelberg, Germany). The procedure was carried out according to the manufacturer’s instructions. The proportion of early and late apoptotic, necrotic, and viable cells was determined based on Annexin V and PI fluorescence signals.

2.4.8. Senescence-Associated β-Galactosidase Staining Assay

To assess cellular senescence, the senescence-associated β-galactosidase (SA-β-gal) staining method was employed to determine the proportion of SA-β-gal-positive cells. SK-MEL-30 cells were seeded into culture plates and incubated for 24 h to enable proper adhesion. Afterward, cells were treated with CA and CA-loaded Fmoc-FF nanogels for an additional 24 h. Culture medium alone served as negative control. Upon completion of the treatment, cells were fixed with 4% paraformaldehyde at 4 °C for 30 min. Following fixation, cells were rinsed four times with cold PBS, then incubated overnight at 37 °C with X-Gal staining solution adjusted to pH 6.0. After the final PBS wash, stained cells were examined under a light microscope for qualitative and quantitative evaluation of senescence.

2.4.9. In Vitro Scratch Assay for Wound Healing Evaluation

HaCaT cells were seeded into 6-well plates and incubated for 24 h to ensure proper attachment. Once a confluent monolayer was achieved, a linear scratch was created across the center of each well using a sterile pipette tip to simulate a wound. CA-loaded Fmoc-FF nanogels were then applied at previously determined non-cytotoxic concentrations. Wells treated with serum-free medium served as the negative control. The plates were incubated at 37 °C under standard culture conditions. Images of the scratch area were captured at 0, 6, 24, and 48 h using a light microscope, and wound closure was quantitatively analyzed using ImageJ 1.51 software [27,28].

2.5. Statistical Analysis

Statistical analyses were performed using the SPSS for Windows software, version 23.0 (SPSS Inc., Chicago, IL, USA). The distribution of continuous and discrete variables was assessed using the Kolmogorov–Smirnov test, while the homogeneity of variances was evaluated using Levene’s test. For comparisons between two groups with normally distributed data, Student’s t-test was applied. For multiple group comparisons, one-way ANOVA followed by the least significant difference (LSD) post hoc test was used. The significance level of p  <  0.05 was considered statistically significant. Data are presented as mean ±  standard deviation (SD). All results were obtained from three independent experiments; each performed in duplicate.

3. Results and Discussion

The morphological characteristics of both unloaded and CA-incorporated Fmoc-FF dipeptide gels were examined in this study. TEM was utilized to analyze particle size and structural morphology. The observations indicated that unloaded Fmoc-FF particles primarily exhibited a spherical shape, whereas CA-loaded counterparts displayed an elongated, rod-like morphology with sizes exceeding 500 nm (Figure 1B). Dynamic light scattering (DLS) analysis indicated that the size distributions of the empty Fmoc-FF structures ranged from 100 to 300 nm, whereas the CA-loaded Fmoc-FF structures had particle sizes between 250 and 500 nm. The zeta potential measurements showed values of −38.30 ± 1.25 mV for empty Fmoc-FF and −41.86 ± 1.15 mV for CA-loaded Fmoc-FF. The observed negative zeta potentials are indicative of successful CA incorporation, likely attributed to the negatively charged C-terminal groups of the Fmoc-FF dipeptide structure. The presence of CA in the formulation medium may further amplify this negative charge, potentially due to its inherent surface ionization at pH values above neutrality or the presence of residual surfactants. This pronounced negative surface charge plays a crucial role in maintaining colloidal stability by preventing nanoparticle aggregation, thereby supporting the potential utility of these formulations for in vivo drug delivery applications. Following the morphological and surface characterization, structural and thermal properties were examined using Fourier-transform infrared (FTIR) spectroscopy. The amide I region showed peaks at 1691 cm−1 and 1690 cm−1 for Fmoc-FF and CA-loaded Fmoc-FF nanogels, respectively, indicative of antiparallel β-sheet or β-turn peptide structures due to the carbamate group. The C=O absorption is clearly show from the alkene C=C stretching absorption band which peaks at 1624 cm−1. Variations in peak intensities in this region provide further evidence for this structural organization. C-N stretching bands in the 1230–1300 cm−1 range correlate with the amide III region. Sharp peaks in the 1000–1500 cm−1 range, attributed to C-C aliphatic carbons, were observed in both spectra. The presence of peaks at 2929 cm−1 and 2850 cm−1 confirms the vibration of the -CH2 group. Peaks at 3307 cm−1 and 3303 cm−1 in the FTIR spectra correspond to primary N-H stretching vibrations for Fmoc-FF and CA-loaded Fmoc-FF nanogels, respectively. Additionally, the peaks observed between 3100 and 3500 cm−1 in the CA-loaded Fmoc-FF nanogel spectra are likely associated with water content. Shifts in peak positions further support interactions between CA and the dipeptide structure (Figure 1E).
Figure 1. Characterization of nanogel formulations: (A) TEM image of Fmoc-FF nanogels; (B) TEM image of CA-loaded Fmoc-FF nanogels; (C) Size distribution profile of Fmoc-FF nanogels; (D) Size distribution profile of CA-loaded Fmoc-FF nanogels; (E) FTIR spectra of free cinnamic acid (CA), unloaded Fmoc-FF, and CA-loaded Fmoc-FF nanogels; (F) Cumulative drug release of the CA loaded Fmoc-FF nanogel and kinetic model parameters.
For the drug release study, the amount of CA released at various time points over 24 h was measured using UV-Vis spectrophotometry, focusing on the absorption maximum at 271 nm. The simulated release study demonstrated that approximately 40% of the loaded drug was released within 24 h (Figure 1F).
Fmoc-FF nanogels provide sustained and localized release, which maintains therapeutic levels of CA over time, as also confirmed by our release studies (~40% release over 24 h). This helps avoid rapid degradation and clearance, potentially leading to higher bioactivity at the target site. Moreover, nanostructured peptide hydrogels have been reported to enhance cell internalization and facilitate endosomal escape, which can amplify intracellular effects of the loaded compound. When the data were calculated for the R2 value according to the Zero Order, First Order, Higuchi, and Korsmeyer–Peppas models for the purpose of kinetic model examination. Here, it can be said that the release behavior occurred according to Higuchi, and Krosmeyer models. The results shown here are more consistent with the Higuchi and Korsmeyer-Peppas models for the release mechanism and also demonstrate the release mechanism from the nanogel system. It is believed that the model was created to demonstrate the release properties from a biological gel matrix. The release behavior is believed to be both diffusion-controlled and matrix-erosion-driven.
The structural transition is likely driven by π–π stacking interactions and hydrogen bonding between cinnamic acid (CA) and the aromatic moieties of Fmoc-FF. The aromatic ring of CA can participate in π–π stacking with the Fmoc moiety, altering the self-assembly behavior and shifting the balance from spherical micelle-like aggregates toward elongated fibrillar/rod-like structures. CA also contains a carboxylic acid group, which may form hydrogen bonds with the amide or carbamate groups in the Fmoc-FF backbone, further modulating the assembly pathway. These cooperative interactions may promote the formation of more anisotropic supramolecular architectures, leading to increased aspect ratio and the rod-like morphology observed under TEM. These behaviors are consistent with prior reports indicating that guest molecule insertion into self-assembling peptide systems can profoundly influence nanostructure geometry.
After characterization and determination of release properties of the obtained nanoparticle structures, cytotoxic effects on SK-MEL-30 cells were evaluated in order to evaluate the effects of the structures on melanoma. As seen in Figure 2A, no important effect of Fmoc gels alone on melanoma was observed, while CA loading on the gels increased the effects of CA on melanoma. Analysis of the cell images presented in Figure 2B–D reveals notable alterations in both cell morphology and density, particularly in the group treated with CA-loaded Fmoc-FF nanogels at a concentration of 10 µg/mL. Furthermore, the IC50 values were determined as 599.643 µg/mL for Fmoc-FF, 107.9394 µg/mL for CA, and 35.8392 µg/mL for CA-loaded Fmoc-FF nanogels, indicating enhanced cytotoxic efficacy upon CA encapsulation. There are studies showing the effects of CA and its synthesized derivatives on melanoma cells [29,30]. However, considering that melanoma affects the skin, our study is the first to our knowledge to demonstrate the effects of CA-loaded into topical, biocompatible and nature-inspired nanogels on melanoma.
Figure 2. (A) Cytotoxicity of Fmoc-FF, cinnamic acid (CA), and CA-loaded Fmoc-FF nanogels on SK-MEL-30 melanoma cells, as assessed by the MTT assay, (B) Light microscopy image of untreated SK-MEL-30 cells (control), (C) SK-MEL-30 cells treated with 25 µg/mL free CA, (D) SK-MEL-30 cells treated with 25 µg/mL CA-loaded Fmoc-FF nanogels; Results were expressed as mean ± standard deviation.
After determining the concentrations of CA and CA-loaded Fmoc-FF that can be effective on melanoma cells through cytotoxicity studies, the effects of CA and CA-loaded Fmoc-FF nanogels on the events that are important in melanoma pathogenesis were evaluated. While it is known that ROS are released in cancer types such as melanoma, treatment strategies are being developed to kill cancer cells by increasing ROS release in order to eliminate these health effects [31]. Therefore, the effects of the structures obtained within the scope of our study on ROS release were evaluated. When Figure 3 was examined, it was found that CA-loaded Fmoc-FF nanogels increased ROS formation compared to CA with the increase in concentration.
Figure 3. Relative intracellular ROS levels in SK-MEL-30 cells following exposure to hydrogen peroxide (H2O2) and CA-loaded Fmoc-FF nanogels; DMEM without serum was used as the negative control (N.C.); Results are presented as mean ± standard deviation.
The tyrosinase enzyme plays a role in melanin synthesis, which is produced by melanocytes, which are special skin cells. With the increase in tyrosinase enzyme activity, the possibility of melanin pigment production and ultimately melanoma formation increases. CA, which has been used for many years due to its positive effects on skin health, affects the tyrosinase enzyme and causes the inhibition of melanogenesis [32,33]. As in previous studies, the effects of CA on tyrosinase enzyme [34] were found in our study in a concentration dependent manner, and this effect was found to increase when loaded into Fmoc-FF nanogels (Figure 4).
Figure 4. Tyrosinase levels of SK-MEL-30 cells exposed to CA and CA-loaded Fmoc-FF nanogels; N.C.: Negative control (DMEM without serum); Results were expressed as mean ± standard deviation; * significant difference between N.C.; ** significant difference between groups (p < 0.05).
It is claimed that anti-inflammatory agents may also be useful in the treatment of melanoma. [35,36]. Therefore, how this feature of CA, which has been shown to have anti-inflammatory effects in studies [37], will affect melanoma cells was also evaluated within the scope of our study. It is noteworthy that NF-ΚB, TNF-α, and IL-8 levels in melanoma cells were reduced by both CA and CA-loaded Fmoc-FF nanogels, but this reduction was more pronounced in cells to which CA-loaded Fmoc-FF nanogels were applied (Figure 5A–C).
Figure 5. The effects of CA and CA-loaded Fmoc-FF nanogels on immunotoxicity biomarker levels in SK-MEL-30 cells (A) NF-κB; (B) TNF-α; (C) IL-8; N.C.: Negative control (DMEM without serum); Results were expressed as mean ± standard deviation; * significant difference between N.C.; ** significant difference between groups (p < 0.05).
DNA damage is repaired by DNA repair mechanisms in cells, and when damaged lesions are successfully repaired, cells continue to live. In order to kill cancer cells with DNA damage, DNA repair mechanisms found in cancer cells but not in normal cells are used. At this point, it can be said that genotoxicity is useful in anticancer treatment in terms of causing cancer cells to die [38]. In this study, the potential genotoxic effects of CA, previously reported to possess antigenotoxic properties [39], were evaluated in melanoma cells. Using the Comet assay, DNA damage levels were assessed following treatment with both free CA and CA-loaded Fmoc-FF nanogels. The results demonstrated a concentration-dependent increase in DNA damage, suggesting that, under certain conditions, CA and its nanoformulations may exhibit genotoxic effects in melanoma cells. At this point, it can be thought that the ability of the especially CA-loaded Fmoc-FF structures to kill melanoma cells is related to the increase in genotoxic effect (Figure 6A–D).
Figure 6. Comet assay images and genotoxicity results for SK-MEL-30 cells: (A) N.C.; (B) 10 µg/mL CA; (C) 10 µg/mL CA-loaded Fmoc-FF nanogel; (D) Genotoxicity results of in SK-MEL-30 cells; N.C.: Negative control (DMEM without serum); * significant difference between N.C.; ** significant difference between groups (p < 0.05).
Inducing apoptosis remains an effective strategy for treating cancer. Melanoma cells can also self-destruct through apoptosis [40]. Inducing apoptosis in melanoma cells is considered a promising therapeutic strategy for melanoma treatment [41]. In our study, we assessed the pro-apoptotic effects of CA and CA-loaded Fmoc-FF nanogels in melanoma cells. The results indicated that both free CA and its nanoformulation significantly enhanced apoptotic activity (Figure 7). These findings suggest that the developed nanogel formulation may contribute to melanoma therapy by promoting apoptosis.
Figure 7. Flow cytometry analysis of annexin V-FITC/PI staining of (A) negative control cells (serum-free DMEM), (B) CA, (C) Fmoc-FF, (D) CA-loaded Fmoc-FF, (E) Percentages of live, early apoptotic, late apoptotic, and necrotic cells.
The aromatic π–π stacking between CA and Fmoc-FF moieties likely contributes to non-covalent stabilization of the nanostructure and may enhance the local concentration of CA in proximity to cell membranes or intracellular targets. This supramolecular organization can alter the pharmacodynamics of CA, increasing cellular ROS production and apoptosis as observed in our study.
It is claimed that one of the steps before cell death is cellular senescence [42]. Our findings demonstrated that treatment with cinnamic acid (CA) and CA-loaded Fmoc-FF nanogels led to an increase in the number of β-galactosidase-positive (senescent) SK-MEL-30 cells compared to the negative control. Notably, this effect was more prominent in cells treated with the CA-loaded Fmoc-FF nanogel formulation, suggesting an enhanced senescence-inducing potential of the nanoformulated system. Based on this result, we can think that CA-loaded Fmoc-FF structures cause melanoma cells to age and cause their cells to die (Figure 8).
Figure 8. (A) β-senescence (+) cell numbers of SK-MEL-30 cells treated with 10 µg/mL CA and 10 µg/mL CA-loaded Fmoc-FF nanogel; N.C.: Negative control (DMEM without serum); * significant difference between N.C. (p < 0.05). Microscopy images of SA-β-gal-positive cells in: (B) N.C.; (C) 10 µg/mL CA; (D) 10 µg/mL CA-loaded Fmoc-FF nanogel; scale bar: 200 µm; The bright spots in the photographs indicate β-senescence (+) cells.
Wound healing is a highly complex and regulated biological process, and several studies have reported that phytochemical compounds can positively influence this process [43]. In our study, the impact of CA-loaded Fmoc-FF nanogels on an in vitro wound model using HaCaT cells was investigated. The results revealed that treatment with the nanogel at a concentration of 10 µg/mL significantly enhanced wound closure and reduced the wound area more effectively than the other tested concentrations (Figure 9), suggesting its potential to accelerate the healing process.
Figure 9. Evaluation of wound healing activity of CA-loaded Fmoc-FF nanogels in HaCaT cells using an in vitro scratch assay. DMEM without serum was used as the negative control (N.C.). Representative images and quantitative analysis demonstrate enhanced wound closure at a concentration of 10 µg/mL. Scale bar: 200 µm.

4. Conclusions

There is an increase in the incidence of skin cancer caused by external factors. Among these cancer cases, the agents used in the treatment of melanoma, which can metastasize and is the most dangerous, have low treatment efficacy and high undesirable effects, and alternative agents have been sought treatment. At this point, compounds obtained from plants stand out because they have fewer toxic effects compared to synthetic components, but due to their poor bioavailability and solubility properties, they have been loaded into drug carrier systems and used.
Due to the importance that people give to their appearance today, there has been a significant increase in the number of products used against skin disorders. The use of herbal compounds, which are claimed to have a low probability of causing undesirable effects in the content of these products, is increasing.
In this study, the therapeutic potential of cinnamic acid (CA)-loaded Fmoc-FF dipeptide nanogels—designed as nature-inspired, biocompatible, and topically applicable systems—was evaluated in the contexts of melanoma treatment and wound healing. Comprehensive analysis of the experimental findings revealed that the CA-loaded Fmoc-FF nanostructures exerted more pronounced effects on melanoma cells than free CA alone. These effects included enhanced cytotoxicity, increased ROS generation, greater genotoxicity, stronger anti-tyrosinase activity, as well as more effective anti-senescence, pro-apoptotic, and anti-inflammatory responses. It was also determined that they had pronounced wound healing effects on human keratinocyte cells. Therefore, we can talk about the beneficial effects of using CA in the peptide-based drug delivery system. However, for general use, further in vitro and in vivo studies are needed to elucidate and confirm the basic mechanisms.
A major limitation of this study is the absence of in vivo data, which is critical to fully assess the therapeutic efficacy, pharmacokinetics, and biocompatibility of the developed nanogel system. Future studies will focus on animal models to validate the in vitro findings presented here.
Although the formulation showed enhanced cytotoxic and genotoxic effects specifically in melanoma cells, the potential off-target consequences of increased ROS generation—particularly in surrounding healthy keratinocytes or fibroblasts—should be carefully evaluated in future studies. In this regard, selective targeting strategies (e.g., tumor microenvironment-responsive carriers) or dual delivery systems may be required to enhance therapeutic specificity.

Author Contributions

Conceptualization, H.E. and M.G.B.; Methodology, H.E. and M.G.B.; Software, H.E. and M.G.B.; Validation, J.S., B.K., H.E. and M.G.B.; Formal analysis, J.S., B.K., H.E. and M.G.B.; Investigation, H.E. and M.G.B.; Resources, M.G.B.; Data curation, H.E. and M.G.B.; Writing—original draft, H.E. and M.G.B.; Writing—review & editing, H.E. and M.G.B.; Visualization, H.E. and M.G.B.; Supervision, M.G.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author(s).

Acknowledgments

The authors gratefully acknowledge Aylin Üstündağ (Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology) for her kind support in supplying HaCaT cells. The authors also thank Esra Emerce (Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology) for providing the SK-MEL-30 cell line used in this study.

Conflicts of Interest

No potential conflict of interest was reported by the authors.

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