Upcycled Carbon Dots as Multifunctional Boosters for Broad-Spectrum Photostable Sunscreens

Abstract

Sustainable ultraviolet (UV) filters that couple photoprotection with antioxidant activity are needed. Carbon dots (CDots) derived from agro-industrial waste have emerged as promising candidates. CDots were prepared from Coffea canephora (coffee leaf) residues by a one-pot microwave route and characterized by UV–Vis, FTIR, and TEM. Antioxidant capacity was determined by CUPRAC and DPPH-EPR. The photoprotective efficacy was assessed in vitro by diffuse reflectance spectrophotometry before and after solar-simulator exposure. Nearly spherical CDots (3.3 ± 0.7 nm) displayed a 4.16 eV optical bandgap and broad absorption from 200 to 400 nm. At 10 μg mL⁻¹, CDots exhibited 24.62 ± 0.19% antioxidant activity relative to Trolox by CUPRAC, while by DPPH-EPR, they showed 99.9 ± 12.5% of radical quenching at 240 µg mL⁻¹. Addition of 4.5% w/w (dry basis) CDots to the sunscreen system increased the in vitro SPF from 26 ± 13 to 161 ± 8 (p < 0.05) while maintaining the critical wavelength at 380 ± 0.64 nm. After 30 min of irradiation, the SPF dropped only 10%, versus 44% for the CDots-free sample (control), indicating superior photostability. Coffee leaf CDots acted as an efficient broadband UV absorber and antioxidant that markedly enhanced and stabilized a conventional sunscreen formulation. The work positions waste-derived CDots as an eco-friendly, next-generation multifunctional ingredient, aligning with circular economy principles.

1. Introduction

Prolonged exposure of human skin to sunlight has significant potential to cause both short- and long-term molecular and structural alterations. In the short term, effects include erythema (skin redness), which activates melanocytes and enhances melanin production, leading to skin darkening [1]. Prolonged exposure, on the other hand, can trigger photoaging, characterized by the irreversible loss of skin elasticity, and contribute to the development of skin cancer [2,3]. UVA rays penetrate deeply into the dermis, causing functional and structural alterations, while UVB rays are more energetic and are associated with sunburns and damage to the epidermis and superficial dermis [4]. Prolonged exposure of the skin to ultraviolet (UV-A and UV-B) rays induces immunosuppression, generating oxidative stress and inflammation through the production of reactive oxygen species (ROS), such as hydroperoxyl (HO₂•), superoxide (O₂•⁻), and hydroxyl (•OH) radicals. These radicals can alter the structure and/or function of proteins, damage DNA, and promote genetic mutations, which, combined with immune system suppression, increase the likelihood of cancer cell development [5]. Although the body’s innate antioxidant defense can neutralize ROS, this system may become overwhelmed, leading to a state of oxidative stress or immunosuppression, which can even trigger carcinogenesis [6].

Over the years, sunscreens have been widely used to reduce damage caused by sun exposure, preventing burns, edema, and skin cancer. Sunscreen efficacy is defined by its ability to protect the skin against UV-induced burns, with its performance level indicated by the sun protection factor (SPF) [7]. They are divided into inorganic and organic filters. Conventional sunscreens typically contain organic filters, such as derivatives of salicylates, cinnamates, benzophenones, and dibenzoylmethanes, while inorganic filters are mostly metal oxides, primarily titanium dioxide (TiO₂) and zinc oxide (ZnO) [8,9].

Although most sunscreens are considered safe for topical use, some individuals experience sensitivity, skin irritation, and allergic reactions [10]. In addition, environmental concerns have been raised, as significant concentrations of chemical filters such as oxybenzone and octinoxate have been detected in coastal and ocean waters, linked to the use of sunscreens. Studies indicated that these compounds can have toxic effects on corals, contributing to bleaching and reef degradation [11].

A material that has not been extensively explored in this field is Carbon Dots (CDots). CDots are a versatile class of nanomaterials composed of small carbon particles, typically smaller than 10 nanometers, characterized by an internal structure comprising sp²/sp³-hybridized carbon and surface groups, such as hydroxyl (-OH), carboxyl (-COOH), and amine (-NH₂), that influence their dispersibility, stability, and reactivity. These particles have been the subject of intense multidisciplinary research due to their promising luminescent properties, good chemical stability, high biocompatibility, biodegradability, high dispersibility in different solvents, ease of synthesis, and low cost [12], making them versatile for a wide range of applications [13].

There are various methodologies for synthesizing carbon nanoparticles depending on the desired properties. These include top-down approaches such as arc discharge, laser ablation, and electrochemical oxidation, and bottom-up strategies, such as combustion, hydrothermal treatment, and microwave-assisted solvothermal processes [14]. CDots can be obtained from a wide range of carbon sources, including agro-industrial residues, like rice husk, white pepper, sugarcane bagasse, coconut husk, corn oil, among others [15]. Their chemical structure and surface functionalities depend directly on the synthesis method and the precursor material employed [16].

Plant extracts can serve as a complex carbon source, naturally enriched with a variety of organic functional groups that facilitate the surface functionalization of the nanoparticles. For instance, studies using HPLC-DAD have identified numerous organic compounds in alcoholic extracts of Coffea canephora leaves, including chlorogenic acids, caffeine, trigonelline, and various other secondary phenolic compounds [17]. Beyond their rich chemical composition, the use of coffee leaf extracts represents a sustainable strategy to valorize agricultural residues. Given that coffee is one of the most widely consumed beverages worldwide, large quantities of leaves are generated as waste during cultivation and processing. By employing these leaves as a precursor for carbon dots, it is possible to convert this abundant biomass into high-value nanomaterials while reducing environmental impact and promoting circular economy practices.

Plant-derived CDots stand out for their antioxidant and anti-inflammatory activities, as well as their ability to protect against UV radiation—properties that originate from the bioactive compounds present in the plant precursors. These characteristics allow tailoring their properties for specific applications, including cosmetic formulations for application in the skin aging control, treatment of skin inflammations, and photoprotection [18]. Moreover, the possibility of synthesizing CDots from agro-industrial waste aligns with the growing demand for sustainable and environmentally friendly products, offering an innovative alternative to conventional UV filters. In the agro-industrial context, CDots can be employed not only as a strategy for waste valorization but also as nanofertilizers, contributing to light conversion and nutrient delivery [19]. In addition, they can provide solar photoprotection by mitigating UV-B stress in plants [20], as excessive sunlight exposure may impair photosynthetic activity and overall plant development through both direct and indirect mechanisms [21].

Recent research has explored the potential of CDots synthesized from carrot juice via a continuous microflow method as green and effective sunscreen agents. Their efficacy as a UV filter agent was assessed by incorporating them into an emulsion and measuring the sample’s UV transmittance. A cream containing 5% by mass of these CDots showed excellent photoprotective performance, transmitting only 2.7% of UVB and 4.9% of UVA radiation, which was reported to be superior to some commercial agents [22].

In a study by Campalani et al. 2022 it was observed that CDots obtained from fish scales, when incorporated at 5% by weight into a gelatin film, with negligible impact on the film’s transparency, blocked almost 70% of UV radiation, as determined by UV transmittance spectroscopy [23].

Chen et al. [24] synthesized L-cysteine-derived CDots that filtered 99% UVC, 97% UVB, and 86% UVA at 0.5 mg mL⁻¹. These nanodots mitigated oxidative damage and delayed UVB-induced aging in zebrafish, suggesting dual physical and biological photoprotection.

Chatzimitakos et al. (2020) produced CDots from the micro-alga Dunaliella salina; dry powders displayed in vitro SPF values of 13–18, and, when dispersed in model emulsions, the nanodots significantly boosted overall SPF without cytotoxic effects, demonstrating their potential as natural primary filters [25].

Given the above, the development of photoprotective alternatives that not only reduce UV-induced damage but also provide antioxidant action and minimize environmental impacts becomes essential. In this context, the incorporation of nanomaterials such as CDots into photoprotective formulations emerges as a promising approach. Due to their chemical properties and potential upcycling from agro-industrial waste, plant-derived CDots are being investigated as sustainable adjuncts to complement conventional UV filters. Here, we evaluate coffee leaf CDots as multifunctional boosters and assess their in vitro antioxidant and broadband UV-shielding performance in a model sunscreen.

2. Experimental Section

2.1. Reagents

Glycerin and ethanol (70%) were provided by Dinâmica (Indaiatuba, Brazil). Butyl methoxydibenzoylmethane (BMDBM) and ethylhexyl p-methoxycinnamate (EHMC) were donated by Symrise (São Paulo, Brazil). Crodafos^® CES [cetearyl alcohol (and) dicetyl phosphate (and) ceteth-10 phosphate] and Optiphen^® [phenoxyethanol (and) caprylyl glycol] were sourced from Croda (Barcelona, Spain) and Ashland (São Paulo, Brazil), respectively. Caprylic/capric glyceride was acquired from AQIA (São Paulo, Brazil). Dulbecco’s modified Eagle’s medium with high glucose (DMEM) was purchased from Biowest (Nuaillé, France). Trypsin, penicillin–streptomycin solution, fetal bovine serum, dimethyl sulfoxide (DMSO), and thiazolyl blue tetrazolium bromide (MTT) were acquired from Sigma–Aldrich (Saint Louis, MO, USA). Ultrapure water was used, and all ingredients and reagents were utilized as received, without further purification.

2.2. Methods

2.2.1. Synthesis of Carbon Dots

The preparation of CDots was carried out using an adaptation of methodologies reported in the literature [26,27]. Agricultural waste, specifically coffee plantation leaves (Coffea canephora Pierre ex A Froehne), commonly known as Conilon/Robusta coffee, was used as the carbon source. The leaves were donated by the experimental farm of UFES, São Mateus campus (18°40′23″ S, 39°51′22″ W, 36 m a.s.l.), Espírito Santo state, Brazil.

Initially, 60 g of coffee leaves were macerated and immersed in 600 mL of 70% w/w ethanol, followed by magnetic stirring for 4 h at room temperature. The mixture was then filtered, and the solvent was evaporated on a heating plate at a controlled temperature (70–80 °C). The resulting solid was suspended in 150 mL of distilled water and heated in a household microwave oven (PMO23BB, 900W, Philco, São Paulo, Brazil) until complete evaporation. The residue was then resuspended in 200 mL of distilled water, centrifuged at 15,000 rpm, and filtered through 0.22 μm cellulose acetate membranes. The dry residue of the CDots aqueous suspension was determined by a gravimetric method adapted from the standard “Loss on Drying” procedure [28]. known volume of the suspension was placed in a pre-weighed glass container and dried in an oven at 105 °C until a constant weight was achieved. This was confirmed by successive weighings after cooling in a desiccator. The dry residue was calculated by subtracting the initial weight of the empty container from the final weight, and the concentration was expressed in mg/mL.

2.2.2. Nanoparticle Characterization

The nanoparticles were characterized using UV/Vis Absorption Spectroscopy, Transmission Electron Microscopy (TEM), and Fourier-transform infrared (FTIR) spectroscopy. The characterization via UV/Vis absorption spectroscopy was performed using a GENESYS 10S UV/Vis Spectrophotometer (Thermo Scientific, Waltham, MA USA) in the wavelength range of 200 to 800 nm. The sample suspensions containing CDots were diluted in water at a 1:10 ratio and transferred to a quartz cuvette, suitable for ultraviolet range analysis. TEM images were acquired using a Jeol 2100 TEM-MSC (Tokyo, Japan) microscope operating at 200 kV. The samples were sonicated in isopropyl alcohol (1:10, v/v) and deposited onto copper grids with an ultrathin carbon film (Ted pella, Inc., Redding, CA, USA) FTIR analyses were performed using a Shimadzu IRPrestige-21 spectrometer (Kyoto, Japan), employing the KBr pellet method. Photoluminescence study was performed using a Jasco FP8600 spectrofluorophotometer (Tokyo, Japan).

2.2.3. Cytotoxicity Assessment

Cell Viability Assay

Human epidermal immortalized keratinocyte cells (HaCat) were purchased from CLS Cell Lines Service (Eppelheim, Germany) and routinely cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM highglucose), supplemented with 10% fetal bovine serum and 1.0% penicillin-streptomycin solution, under air with 5% CO₂ at 37 °C. Approximately 6 × 10³ cells were cultured per well in 96-well plates. After seeding, cells were treated for 48 h with different concentrations of the CDots (10, 100, 250, 500, and 1000 μg mL⁻¹). CDots were dispersed in phosphate-buffered saline (PBS), with PBS used as a negative control and dimethyl sulfoxide (DMSO) as a positive control. Afterwards, 200 μL of MTT solution (0.5 mg mL⁻¹) in culture medium was added to each well and incubated for 4 h at 37 °C. After this incubation, the wells were washed with PBS, and 200 μL of DMSO was added to solubilize the formed crystals, which were then detected by colorimetric assay at 570 nm using a microplate reader (Synergy™ HTX Multimode Reader from Agilent BioTek Instruments (Santa Clara, CA, USA). The colorimetric assay was based on the ability of viable cells to metabolize MTT into dark blue formazan crystals. Three independent experiments were performed, each with four replicates [29].

Membrane Stabilization Assay—Hemolysis Test

The hemolytic activity of CDots was assessed using a spectrophotometric method with adaptations [30,31]. The samples were dispersed in phosphate-buffered saline (PBS) with glucose (100 μg mL⁻¹) at several concentrations (5; 10; 25; 75; 100; 150; 300; 500; 800; 1000 μg mL⁻¹). Fresh sheep blood was obtained from Ebefarma^® (Itaborai, RJ, Brazil) and the red blood cells (RBC) were subsequently separated. A volume of 50 μL of the erythrocyte suspension was added to each tube containing 950 μL of the sample solution; followed by incubation for 30 min. Distilled water was used as a positive control and PBS with glucose served as a negative control. After the incubation period, the tubes were centrifuged at 3000 rpm for 5 min (FANEM^®, Guarulhos, Brazil). A volume of 200 μL of the supernatants was transferred to 96-well plates and the liberated hemoglobin in the supernatant was measured in the microplate reader Kasuaki (Wuxi Hiwell-Diatek Instruments Co., Wuxi, China) at a wavelength of 540 nm. The results were compared with the negative control and thus established the percentage of erythrocyte membrane stabilization.

2.2.4. Evaluation of Antioxidant Activity

Free Radical Scavenging Capacity by the Copper Reduction Method

The antiradical assay was performed based on the methodology proposed by [32], with several modifications. The CUPRAC (Cupric Reducing Antioxidant Capacity) method relies on the electron transfer between the antioxidant substance and the cupric ion (Cu²⁺), resulting in its reduction to the cuprous ion (Cu⁺) by reducing agents, such as antioxidants. The cuprous ion, in turn, forms a stable complex with neocuproine, thereby increasing the absorbance at 450 nm [32,33,34].

In microcentrifuge tubes, 250 μL of the following solutions were added: copper (II) chloride (10⁻² M, aqueous solution), neocuproine (7.5 × 10⁻³ M, ethanolic solution), NH₄Ac buffer (pH 7.0), and either the sample or a freshly prepared Trolox solution. The CDots samples were tested at concentrations of 75, 150, 300, and 600 µg mL⁻¹. After incubation for 1 h at 28 °C, the absorbance was measured at 450 nm using a UV-Vis spectrophotometer (model 5100, Shanghai Metash Instruments, Shanghai, China) with 1.4 mL quartz cuvettes and a 1 cm optical path.

The cupric ion reducing capacity was expressed in Trolox equivalent antioxidant capacities (TEAC) and calculated from a calibration curve obtained by linear regression, constructed using different concentrations of Trolox (0.7821, 1.5643, 3.1285, 6.2570, 12.5150, 18.7720, and 25.029 µg mL⁻¹) in an aqueous solution containing 1% Tween 20 (r² = 0.9952). TEAC was determined by the equation:

TEAC = ABS ÷ 0.012 − 0.833

(1)

where ABS represents the absorbance of the sample at a wavelength of 450 nm.

Antioxidant Activity by Electron Paramagnetic Resonance

Antioxidant activity was also determined by electron paramagnetic resonance (EPR) using a Bruker spectrometer (EMXplus, Ettlingen, Germany) equipped with a cylindrical cavity operating in the X-band (9.8 GHz) high sensitivity cavity (Bruker ER 4119HS, Ettlingen, Germany). The experimental parameters were set to a modulation amplitude/frequency of 1 G/100 kHz and a microwave power of 20.00 mW. The EPR spectra were recorded at room temperature over a magnetic field range of 336 to 366 mT under ambient conditions. Free radical content was determined by spectra double integration [35].

For the analysis of the samples, the DPPH radical was dissolved in ethanol (250 µM) and used as a reference [36]. Aliquots of the CDots samples were added to the DPPH solution, yielding final concentrations of 6, 12, 24, 30, 60, 72, and 240 µg mL⁻¹. The samples were kept at room temperature for 60 min, protected from light, and subsequently frozen in liquid nitrogen until measurement. For analysis, 50 μL of each sample was deposited into Blauband microcapillaries (Intramark, Wertheim, Germany). The mean inhibitory concentration (IC₅₀), corresponding to the concentration required to scavenge 50% of the DPPH free radicals, was determined from a plot of the relative DPPH concentration (C_rel) as a function of the CDots concentrations (μg mL⁻¹). The C_rel values were calculated according to Equation (2):

C_rel (%) = (DI_EPR/DI_ref) × 100

(2)

where DI_EPR and DI_ref represent the double integral of the CDots/DPPH solution and the reference sample, respectively. DI_EPR represents the reference concentration of DPPH, that is, at the onset of the reaction (time zero) [37].

2.2.5. Sunscreen Samples

Sunscreens were prepared as oil-in-water emulsions according to Neto et al., 2023, with some modifications [38]. The controls (CF) were the formulations prepared without adding CDots. CDots concentration is expressed on a dry-extract basis. Formulations were prepared by incorporating 10% (w/w) of the CDot-containing extract. The extract’s dry residue, determined gravimetrically, was 45% (w/w). The qualitative and quantitative (%, w/w) compositions of the sunscreens are described in

Table 1. Qualitative and quantitative (% w/w) compositions of the sunscreens.

Ingredients (INCI)	Function	Control (%)	CDots (%)
Phenoxyethanol (and) caprylyl glycol (Optiphen®)	Preservative	1.0	1.0
Cetearyl alcohol (and) dicetyl phosphate (and) ceteth—10 phosphate (Crodafos® CES)	Self-emulsifying wax	6.0	6.0
Butyl Methoxydibenzoymethane	UVA filter	5.0	5.0
Ethylhexyl Methoxycinnamate	UVB filter	10.0	10.0
CDots (dry-mass)	Active	-	4.5
Purified water	Vehicle	*	*

* Sufficient to complete 100.0%.

.

2.2.6. In Vitro Photoprotective Efficacy and Photostability Assay

The samples were evaluated by diffuse reflectance spectrophotometry using an integrated sphere (Labsphere UV-2000S^® Ultraviolet Transmittance Analyzer, North Sutton, NH, USA), following the method of Marcelino et al. [39]. Aliquots of 1.3 mg cm⁻² were weighed and applied uniformly to the surface of 25 cm² polymethyl methacrylate plates (PMMA). After, the plates were allowed to dry for 30 min in the dark before measurements. Five different points were measured on each plate in triplicate, employing a freshly prepared glycerin-coated plate as the blank. The data were processed using the UV-2000^® software over a wavelength range of 290–400 nm with a scan rate of 1.0 nm. The results were converted into in vitro sun protection factor (SPF) and critical wavelength (λc) values.

The formulations’ functional photostability was assessed by comparing in vitro SPF values obtained before and after exposure to simulated solar radiation, in accordance with reports [40,41].

Samples were exposed to a UV irradiance of 55 W m⁻² for 90 min (total dose of 297 kJ m⁻²) in an Atlas Suntest^® CPS+ (Atlas Material Testing Technology, Mt Prospect, IL, USA) solar simulator equipped with a xenon lamp and a set of coated and special UV glass filters, reproducing outdoor global solar radiation in the 300–400 nm range. After irradiation, efficacy parameters were recalculated and compared with pre-stress values [42,43].

2.2.7. Statistical Treatment

The statistical analysis was performed using Minitab^® software (Version 21.1.0), adopting a significance level of 5% (α = 0.05). All assays were conducted at least in triplicate (n ≥ 3). Results were expressed as mean ± standard deviation. Normality was assessed using graphical methods (histograms) and the Kolmogorov–Smirnov test. Homoscedasticity was confirmed with Levene’s test. Differences in effects among multiple samples will be evaluated by one-way analysis of variance (ANOVA) followed by the Tukey post-test.

3. Results and Discussion

3.1. Synthesis and Characterization

In this work, we employed a green, cost-effective, and rapid approach to synthesize carbon dots using agro-industrial waste and a household microwave. This strategy enhances process scalability while promoting waste recycling and adding value to an otherwise discarded byproduct. Thus, an alcoholic extract of Coffea canephora leaves was employed as the carbon precursor for the synthesis of CDots. A previous study using HPLC-DAD reported a detailed chromatographic profile of Coffea canephora leaf extracts, which identified a variety of organic compounds, including chlorogenic acids, caffeine, trigonelline, and secondary phenolic compounds [17]. Such compounds are rich in carbon, nitrogen, and oxygen, providing an excellent feedstock for carbon dot formation. During the microwave-assisted synthesis, these molecules undergo carbonization, leading to the nucleation and growth of carbonaceous nanodomains. Simultaneously, the presence of oxygenated and nitrogen-containing species in the extract promotes the in situ functionalization of the CDots’ surface with organic groups.

FTIR spectroscopy was employed to identify the components, and functional groups present on the surface of the samples. Figure 1 displays the FTIR spectra of the CDots, showing bands centered at 2896 and 2976 cm⁻¹, which are attributed to the C–H stretching vibrations [44]. A broad and intense peak at 3427 cm⁻¹ is observed, corresponding to the symmetric and asymmetric stretching vibrations of –OH and N–H groups, with the broadening attributed to hydrogen bonding. Additionally, a peak at 1643 cm⁻¹ is assigned to the C=O stretching vibration, likely from carboxylic acid groups. The band at 1383 cm⁻¹ may correspond to the asymmetric stretching of C–O–C or to C–H bending, possibly associated with methyl groups. Peaks at 1047 and 1086 cm⁻¹ are assigned to symmetric and asymmetric stretching vibrations of C–O–C bonds [45,46]. Meanwhile, the peak at 668 cm⁻¹ indicates an out-of-plane bending vibration of aromatic C–H bonds [47]. These results suggest that the surface of the CDots derived from coffee leaves can contain carboxyl, hydroxyl, and amine functional groups.

The presence of hydroxyl, carboxyl, and amine groups, as revealed by FTIR analysis, can significantly enhance the hydrophilicity of the carbon nanoparticles, which improves their dispersion and uniform distribution within cosmetic formulations, such as sunscreens. In practical terms, this enhanced solubility can enable lighter formulations and more pleasant skin feel by reducing the need for large amounts of oily components typically used in conventional sunscreens [48]. Furthermore, recent studies have demonstrated that the presence of such functional groups imparts polymer-like characteristics to CDots, enabling them to effectively retain water molecules through absorption and swelling processes [18,49]. These surface moieties are also capable of participating in hydrogen bonding or electrostatic interactions with organic UV filters, leading to enhanced photostability and reduced photodegradation upon UV exposure. Additionally, surface functional groups such as carboxyl and hydroxyl moieties can act as hydrogen donors, reacting with unstable free radicals. Overall, the radical scavenging capacity of CDots is generally attributed to both the hydrogen-donating ability of their surface edge groups and the electron transfer processes occurring within their sp²-conjugated core [50].

The aqueous suspension of CDots was also analyzed using a UV–Vis spectrophotometer. As shown in Figure 2A, the CDots exhibited multimodal absorption spanning the 200–800 nm range. A high-intensity band centered at 228 nm was observed, which can be attributed to the π–π transition of sp² domains in the carbon core [51]. The band centered at 276 nm may be associated with the n → π transition in the C=O bond [52], which can arise from surface functional groups such as carboxyl and carboxylate groups [53]. The absorption bands at longer wavelengths are typically related to surface states and nanoparticle functionalization with nitrogen- or oxygen-containing groups [54], which originate from the carbon source used during the synthesis.

The optical bandgap of a material represents the minimum energy required to promote an electron from the ground state to an excited state. Therefore, materials with a larger optical bandgap require higher-energy photons to induce electronic transitions. The bandgap energy can be estimated from the absorption spectrum of the material using the Tauc equation [55].

$$\alpha \hslash \nu =A{\left(\hslash \nu -E_g\right)}^n$$

(3)

where α is the absorption coefficient, ℏ is Planck’s constant, ν is the frequency of the incident light, A is a material-dependent constant, E_g is the optical bandgap energy, and n is an exponent that depends on the nature of the electronic transition—n = 1/2 for direct bandgap and n = 2 for indirect bandgap. In this study, the bandgap value was estimated by constructing a Tauc plot (see Figure 2B). In this plot, the linear portion of the curve is extrapolated to intersect the x-axis, and the corresponding energy value gives the bandgap, which in this case was found to be 4.16 eV. A material with such a wide optical bandgap requires high-energy photons to trigger electronic transitions, meaning it absorbs less low-energy radiation, such as visible light and most UVA and UVB radiation, making it a promising candidate for photoprotection applications [56,57].

To further characterize the optical properties of the synthesized CDots, their photoluminescence (PL) emission spectrum was measured under excitation at 350 nm. As seen in Figure 2A, a broad PL band ranging ~400–600 nm with a maximum at 445 nm was observed. This characteristic and widely reported blue emission is commonly attributed to radiative recombination through surface defect states and functional moieties containing oxygen and nitrogen [58,59]. Such groups introduce mid-gap energy levels, allowing radiative recombination. The UV–vis absorption profile and the surface-state-dominated PL are in good agreement with the FTIR results, which suggested the presence of abundant oxygenated and nitrogen-containing groups on the nanoparticle surface.

The morphology and size of the synthesized carbon dots (CDots) were investigated using high-resolution transmission electron microscopy (HRTEM). Figure 3A–C show images of the nanoparticles (in high-contrast), which exhibit an approximately spherical shape and a monodisperse distribution. The size distribution histogram presented in Figure 3D reveals that the particle diameters range from 1.8 to 5.3 nm, with an average diameter of 3.3 ± 0.7 nm, based on statistical analysis of a representative number of particles. These characterization results confirm the successful formation of nearly spherical nanoparticles via a bottom-up microwave-assisted synthesis route. This approach stands out as a more environmentally friendly, practical, and cost-effective alternative when compared to conventional synthetic methods, which often require high temperatures, toxic solvents, and prolonged reaction times [26].

3.2. Cytotoxicity Detection and Hemocompatibility

Cell viability was quantified by the MTT colorimetric assay on HaCaT cells following 48 h exposure to a dry-weight CDots stock (874.6 µg mL⁻¹) serially diluted to 10–1000 µg mL⁻¹. A clear, concentration-dependent decline in metabolic activity was observed, with viability decreasing progressively as the CDots concentration increased (Figure 4A). These findings demonstrate that the CDots exerted a concentration-dependent cytotoxicity effect on keratinocytes.

To further investigate the observed cytotoxicity, membrane-stabilization studies were conducted using erythrocytes (RBCs). The hemolysis assay aimed to evaluate the mechanical stability of the RBC membrane in the presence of the test compounds. In our study, the CDots sample (dry residue 103.55 mg mL⁻¹) was tested at concentrations ranging from 5 to 1000 μg mL⁻¹ (Figure 4B).

The cell viability by MTT revealed that varying the concentrations of CDots induced progressively significant reductions in cell viability, yielding viability values of 70.37 ± 8.0, 56.8 ± 2.5, 48.4 ± 3.6, 29.5 ± 1.6 and 6.2 ± 0.8% for 10, 100, 250, 500, and 1000 µg·mL⁻¹, respectively (mean ± SD; n = 3). Based on linear regression of percent viability versus CDots concentration within the 100–500 µg·mL⁻¹ range, the IC₅₀ was 206.9 ± 36.5 µg·mL⁻¹ (mean ± SD; n = 3).

Chatzimitakos et al. [25] evaluated the cytotoxicity of CDots obtained by Dunaliella salina microalgae derivatives in HaCaT cells at concentrations ranging from 100 to 800 μg mL⁻¹ using the crystal violet assay. After 24 h of incubation, cell viability remained near 100% for concentrations up to 600 μg mL⁻¹, and only at 800 μg mL⁻¹ was a significant reduction in viability observed. Ozdemir et al. [60] reported a selective cytotoxic effect of CDots derived from Echinophora tenuifolia on different cell lines. Exposures from 0 to 100 μg mL⁻¹ over 24, 48, and 72 h showed that, in L929 mouse fibroblasts, viability remained high (≈ 80.31%) even at the highest concentration (100 μg mL⁻¹) after 72 h, with no statistically significant differences versus control at concentrations ≤ 25 μg mL⁻¹. In HepG2 human hepatocellular carcinoma cells, however, CDots induced a pronounced, dose-dependent cytotoxicity, i.e., after 72 h, viability dropped to 27.87% at 100 μg mL⁻¹.

A review by Nagoc et al. (2023) likewise noted that plant-derived CDots exhibit low cytotoxicity at concentrations up to 200 μg mL⁻¹ [18].

CDots were evaluated in human dermal fibroblasts, where cell viability remained above 85% at concentrations up to 1000 µg·mL⁻¹ [54]. Similarly, in normal rat myoblast L6 cells, cell viability (MTT assay) was not affected even at high concentrations of CDots (200 µg·mL⁻¹) after 48 h of exposure [61].

Although several reports indicated relatively low cytotoxicity of CDots across diverse cell types, the studies demonstrated that higher concentrations may be substantially harmful, particularly to epithelial cells, such as HaCaT. This variability likely reflects differences in experimental conditions and CDots composition, which can affect their cellular uptake and intracellular interactions [18].

HaCaT studies with near-infrared CDs from plant sources showed no significant difference until 200 µg·mL⁻¹ [62] and broader tolerability (viability ~60% at 1 mg·mL⁻¹; projected LC₅₀ ≈ 1660 µg·mL⁻¹) [63]. Together with our IC₅₀ in the 10²–10³ µg·mL⁻¹ range, these reports suggest that cytotoxicity is strongly influenced by precursor and surface functionalities. Because HaCaT-specific literature is still scarce, we restrict cross-cell-line comparisons to qualitative context and indicate when synthesis routes or surface chemistries differ from those used in our study.

To further assess the CDots’ compatibility, their hemolytic activity against RBCs was investigated. This assay is a key indicator of membrane-damaging potential, as RBC lysis is analogous to lysosomal leakage and can induce tissue inflammation [64]. The assay revealed that even at the highest concentration tested (1000 µg/mL), the CDots induced less than 7.8% hemolysis, indicating minimal membrane-damaging potential (Figure 4B). Although a statistically significant difference (p < 0.05) compared to the negative control was observed at concentrations from 300 µg/mL upwards, the low overall percentage of lysis confirmed that the CDots exhibited excellent hemocompatibility. This finding agrees with previous reports, such as that of Wang et al. [51], who observed significant hemolysis (>5%) only at much higher concentrations (above 2500 µg/mL).

4. Antioxidant Activity

The antioxidant potential of the CDots was assessed by its cupric ion (Cu²⁺) reducing capacity and free-radical scavenging activity, measured by electron paramagnetic resonance (EPR), with Trolox used as the reference. Trolox, a water-soluble form of vitamin E, is an antioxidant widely employed as a reference standard due to its well-established activity [65]. In the linear regression analysis using the least-squares method, the linear correlation coefficient obtained from the Trolox curve was r = 0.9999 (Figure 5A), which is considered highly acceptable (r > 0.99) and indicative of adequate linearity profile. Additionally, the Pearson coefficient of determination (r²) was 0.9952 for the evaluated concentrations, confirming a strong linear correlation between absorbance and the concentration of the antioxidant sample.

Antioxidant activity was also evaluated by electron paramagnetic resonance (EPR), employing the DPPH radical as a marker. The CDots sample were systematically dispersed, and different concentrations were tested until reaching a range in which the samples exhibited a significant distinction in antioxidant activity. As illustrated in Figure 5B, the double integral of the EPR spectra represents the intensity of the DPPH signal, which is proportional to the remaining free radical population in the sample comparable with standard Trolox.

The CDots reducing capacity (6% w/v, dry mass) was evaluated at concentrations of 75, 150, 300, and 600 μg mL⁻¹, corresponding to 7.64, 12.28, 24.14, and 48.31 μg mL⁻¹ (r² = 0.999) of TEAC, respectively (Figure 5A,B), as calculated by the TEAC equation. At an isolated concentration of 10 μg mL⁻¹, the samples exhibited 24.62% antioxidant activity relative to Trolox, in addition to displaying dose-dependent antioxidant activity.

The results in Figure 5C,D indicated that increasing the concentration of Trolox and CDots progressively reduced the double integral of the DPPH-EPR signal, evidencing their antioxidant action through the consumption of free radicals, a dose-dependent effect was observed, and CDots showed greater capacity than Trolox to scavenge DPPH radicals. With a 99.89% reduction in the initial DPPH concentration (250 µM) in the presence of 240 μg mL⁻¹ of CDots solution (equivalent to 14.4 μg mL⁻¹ of CDots dry mass). This finding reinforces the material’s high antioxidant capacity, corroborating the results obtained from the CUPRAC assay and highlighting the potential of CDots as effective free radical scavengers [18,22].

The combined results from antioxidant activity methods confirmed that CDots exhibited significant antioxidant activity, both via the reduction in cupric ions (CUPRAC) and the inactivation of free radicals (EPR-DPPH). This performance suggested that CDots may serve as a potent antioxidant, with potential biomedical and technological applications. Further studies are required to elucidate the underlying mechanisms of this activity and to assess their efficacy in more complex biological matrices.

Ozdemir et al. [60] produced red-emissive carbon quantum dots from the medicinal herb Echinophora tenuifolia by a rapid microwave route. Antioxidant performance assessed with the CUPRAC assay revealed a reducing power comparable to, and in some dilutions exceeding, that of the crude plant extract, while the dots retained high photostability and biocompatibility, underscoring their dual imaging/antioxidant promise.

Kasif et al. [66] reported a one-step hydrothermal synthesis of N,S-codoped carbon dots. Electron paramagnetic resonance(EPR) spectroscopy showed these dots scavenged >80% of model free radicals at sub-milligram-per-milliliter levels, combined with negligible cytotoxicity toward mammalian cells, the data highlight their suitability as potent antioxidant nanocarriers for biomedical and cosmetic applications.

Li et al. [22] investigated carbon dots synthesized via a green microfluidic process using carrot juice as a feedstock. The authors applied the DPPH• radical-scavenging assay to quantify the antioxidant capacity of the CDs. Their results demonstrated a high level of free-radical removal, with even low concentrations of CDs causing a significant decrease in DPPH• absorbance, indicative of efficient radical neutralization. Moreover, the study reported low cytotoxicity of these nanomaterials, highlighting their potential as multifunctional agents in sunscreen formulations: beyond UV-blocking, they serve as powerful free-radical scavengers capable of mitigating oxidative processes associated with cutaneous photoaging.

5. In Vitro SPF

The in vitro functional evaluation of SPF is used as a preliminary assessment in sunscreen development. The in vitro SPF of the samples containing CDots, together with well-established organic UV filters (BMDBM and EHMC) at their maximum concentrations the European cosmetic regulation (Regulation (EC) No. 1223/2009) [67], was determined using the standardized method proposed by (COLIPA, 2009) [40]. The critical wavelength (cλ) is another key parameter in efficacy evaluation, as it indicates whether the sunscreen achieves broad-spectrum protection. It corresponds to the longest UV wavelength that a sunscreen can effectively absorb/filter and is calculated as the point in the absorption spectrum at which the area under the curve reaches 90% of the total sample absorption within the 290–400 nm range [68]. The U.S. Food and Drug Administration (FDA) defines 370 nm as the minimum cλ for broad-spectrum sunscreens, which must also exhibit an SPF of at least 15 [69].

The formulation containing 4.5% CDots (dry residue) achieved a 619% increase in SPF value (from 26 ± 13.0 to 161 ± 7.8). With respect to critical wavelength (λc), both the control and CDots containing formulations exceeded 380 nm (

Table 2. In vitro sun protection factor (SPF) and critical wavelength (cλ) values of the control cream and the same formulation enriched with CDots before and after solar simulation exposure.

Before Irradiation			After Irradiation (Min)
			30		60		90
	SPF	cλ (nm)	SPF	cλ (nm)	SPF	cλ (nm)	SPF	cλ (nm)
Control	26 ± 13.0	381 ± 0.7	14.58 ± 2.2	379.2 ± 0.8	_	_	_	_
CDots	161 ± 7.8 A	380 ± 0.6	145.22 ± 10.1 A	378 ± 0.5	26.28 ± 5.2 B	377 ± 1.0	14.26 ± 2.2 B	375

Legend: SPF—sun protection factor; cλ—critical wavelength. Values are mean ± SD (n = 3 plates). Different superscript letters within a same line denote significant differences (one-way ANOVA, Tukey, p < 0.05, n = 3 plates). Formulations: Control—BMDBM 5% and EHMC 10%; CDots—control formulation + CDots 4.5% (w/w, dry basis).

).

With respect to photostability, the CDots-enriched sample retained 90.2% of its initial SPF after 30 min of solar-simulator exposure, whereas the control preserved only 56% under the same conditions. After 90 min, the CDots sample provided measurable protection (14.3 ± 2.2; 9% of the initial SPF), a value comparable to that of the control after merely 30 min. A decrease in SPF after irradiation is expected, especially in the control sample containing BMDBM/EHMC, as BMDBM, a widely used UVA filter, is known to undergo photodegradation due to multiple isomerization pathways upon UV exposure [70]. However, following 30 min of exposure, the control formulation fell below the SPF threshold, while the CDots-enriched sunscreen continued to satisfy the requirement.

As shown in Table 2, both the CDots sample and the control were classified as effective for broad-spectrum photoprotection (cλ above 370 nm and SPF higher than 15). The UVA/UVB absorbance ratio was 0.766 for the CDots formulation versus 0.717 for the control (pre-irradiation), indicating a modest shift toward UVA attenuation that may contribute to more balanced broad-spectrum coverage.

Recent literature indicated that CDots are emerging as multifunctional cosmetic ingredients capable of broad-spectrum UV absorption, ROS suppression, and formulation transparency. A 2023 review on plant extract-derived CDots emphasized their inherent antioxidant, anti-inflammatory, and UV-filtering properties, positioning them as sustainable alternatives for next-generation sunscreens [18].

Although some studies have suggested photoprotective activity, to the best of our knowledge, none of them have evaluated the functional in vitro SPF using diffuse reflectance spectrophotometry with an integrating sphere, as performed in our investigation. The photoprotective performance of modified CDots, synthesized from citric acid and nitrogen compounds, was confirmed through both in vivo human trials and theoretical analysis. The material prevented skin erythema at radiation doses 5x the minimal required (5 MED) and showed a theoretically calculated SPF of approximately, 22 [71].

The synergistic effect of CDots with traditional inorganic UV filters has also been investigated. Researchers developed transparent composite films by combining CDots with nanocellulose and ZnO nanostructures. The results indicated that the addition of CDots significantly enhanced the UV-filtering capability of the films compared to those containing only nanocellulose and ZnO. This demonstrated that CDots can act as effective enhancers for inorganic filters, improving the overall photoprotective performance of the final material [16].

6. Limitations and Challenges

Despite the promising results, this study has limitations that pave the way for future research. While CDots are widely reported as biocompatible, their environmental safety depends on dose, surface chemistry, environmental concentration, and transformation. The evaluation of photoprotective efficacy was conducted exclusively through in vitro methods. Therefore, the UVA Protection Factor (UVA-PF) was not quantified, as determination is ordinarily paired with in vivo SPF testing, which we deferred pending completion of our safety assessment.

Although the antioxidant activity and SPF boost are clear, the exact photophysical mechanisms by which CDots interact with organic UV filters to enhance photostability remain to be elucidated. As for future perspectives, conducting cytotoxicity assays is suggested to ensure safety for cosmetic application, and environmental risks to large scale is a point raised for future exploration

Optimizing the CDots concentration in the formulation is another important avenue, aiming to balance maximum efficacy, cost, and sensory attributes. Finally, the long-term physicochemical stability of the final formulation should be investigated to ensure its commercial viability.

Accordingly, we report in vitro SPF as a comparative screening outcome on PMMA plates that mimic skin microrelief. Therefore, the absolute values reported should not equated with in vivo SPF. Performance should be validated using methods compliant with current regulatory requirements.

7. Conclusions

Coffee leaf-derived CDots were prepared via a rapid, low-cost microwave process combining nanometric size, broad UV absorption, and potent antioxidant activity. When incorporated at 4.5% (w/w, dry basis) into a sunscreen system, they increased the in vitro SPF by more than six-fold while preserving a critical wavelength ≥ 380 nm and limiting SPF loss to 10% after 30 min of intense artificial UV exposure. These findings highlighted CDots as sustainable, multifunctional boosters that enhance efficacy and photostability of conventional formulations, providing a credible pathway toward greener, high-performance sun-care products. Further in vivo efficacy, safety, and stability studies are warranted to translate this laboratory’s evidence into commercial and regulatory success.