1. Introduction
Carbon dots (C-dots), a new “zero-dimensional” nanomaterial, have become one of the most prominent members of the carbon materials family due to their excellent luminescence, great photostability, small size, low toxicity, and biocompatibility [
1]. In particular, green carbon dots derived from natural resources have received extensive attention due to their unique benefits, including abundant sources, cost-effectiveness, and environmental friendliness [
2].
Bottom-up green approaches based on the use of renewable biomass for producing C-dots are particularly attractive since high-valued nanomaterials can be obtained from low-value precursors, contributing to a circular economy [
3,
4]. Regarding their unique properties, green C-dots have shown tremendous potential applications in several areas such as biomedicine, (bio)sensors, photocatalysis, optoelectronics, and bioimaging [
1,
5].
Among the various green sources explored, microalgae (
µAlgae) have prominent advantages with respect to their ease in growth and ability to survive in extreme conditions of pH and temperature that make them a promising green low-cost feedstock for the large-scale synthesis of nanoparticles [
6]. Additionally, their richness in compounds that benefit human health, including lutein, astaxanthin,
β-carotene, and unsaturated fatty acids, provide this biomass source with enormous potential to produce carbon materials with varied physicochemical and morphological properties [
7,
8].
In this work, we highlight the synthesis of green C-dots (µAlgae-CDs) via a one-pot hydrothermal treatment of Porphyridium cruentum biomass, a red marine microalgae. Results concerning the biocompatibility and antioxidant capabilities of the new nanomaterials will also be presented.
2. Materials and Methods
2.1. Materials
The biomass of Porphyridium cruentum microalgae used in the experiments was produced in flat-panel photobioreactors at Allmicroalgae’s industrial facilities and was kept refrigerated at 5 °C until use. All reagents were used as received and the solvents were of analytical grade and/or were purified by standard methods. In all experiments, ultrapure water was used (Milli-Q, Millipore, Burlington, MA, USA).
2.2. Methods
A high-pressure reactor (Parr model 4560) equipped with pressure, temperature, and stirring sensors/controllers (Parr, model 4843), was used for hydrothermal carbonization of the biomass.
The structural characterisation of the as-prepared µAlgae-CDs was accomplished by FTIR on a Bruker Vertex 70 as KBr pellets, while for UV-Vis analysis the aqueous dispersions were directly employed after appropriate dilution on a Jasco UV V-750 spectrophotometer using 1 cm quartz cells.
Fluorescence spectra were obtained on a Perkin Elmer LS45 fluorimeter using a 1 cm quartz cuvette at 25 °C in right angle (RA) geometry. Fluorescence quantum yields (
ΦF) were measured in aqueous solutions using quinine sulphate in 0.01 M H
2SO
4 (
ΦF = 0.54) as a reference standard [
9,
10]. The optical density of the samples was kept below 0.05 at the excitation wavelength to prevent homo-inner filter effects. The microscopy fluorescence images were obtained on a fluorescence microscope Olympus BX51.
2.2.1. Synthesis of Carbon Dots (CDs)
The biomass was fed into an inox vessel with water and ethylenediamine (ED) as an additive in several ratios. The hydrothermal carbonization of the biomass was achieved at the desired temperature (200–250 °C) for a certain period (4–6 h), under air-equilibrated conditions, using wet biomass (after removing the nutrient via centrifugation) or dry biomass achieved from the wet biomass.
The as-synthesized µAlgae-CDs were cooled to room temperature and the reaction mixture was filtered through a 0.2 μm cellulose membrane, yielding aqueous dispersions. Low-polarity molecular species were removed by liquid–liquid extraction with CH2Cl2/AcOEt.
2.2.2. In Vitro Cytotoxicity Assays and Cell Imaging
The viability of Vero (ATCC™ CCL-81) and HeLa (ATCC™ CCL-2) cell lines was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [
11]. Cells were seeded in a 96-well plate in Dulbecco’s Modified Eagle Medium (GIBCO
®, Thermo Fisher Scientific). After spreading, cells were treated with the
µAlgae-CDs concentrations of 0.02–4000 µg mL
−1 and incubated at 37 °C under a 5% CO
2-enriched atmosphere. After incubation for 18 h, an MTT solution was added into each well and microtiter plates were incubated. The concentration of blue formazan crystals produced by live cells by reduction of MTT in mitochondria were determined by UV-Vis spectroscopy. The absorption spectrum was acquired, and the absorbance was read at 570 nm using 650 nm as reference.
Cellular uptake of µAlgae-CDs was observed by fluorescence microscopy.
2.2.3. Evaluation of Antioxidant Capacity
The antioxidant capacities of the
µAlgae-CDs were evaluated using the ABTS radical cation method [
12]. The reaction was conducted at room temperature in the dark for 6 min and the absorbance was measured at 655 nm. The radical scavenging effect was calculated using the following equation:
where A
c is the absorbance of the negative control and A
s is the absorbance of the sample solution. Trolox was used as positive control.
3. Results and Discussion
3.1. Synthesis and Surface Characterization
The µAlgae-CDs were synthesized using a one-step hydrothermal method in a high-pressure reactor using an aqueous suspension from Porphyridium cruentum biomass (4.0 g) and ED as the nitrogen source (ED/biomass ratio = 0.16) under stirring. After a dwell time of 6 h at 250 °C, the resultant material was filtrated through a cellulose membrane, resulting in a yellowish-brown liquor.
The FTIR spectrum of
µAlgae-CDs (
Figure 1) showed an intense broad band at 3430 cm
−1 from O-H, with a shoulder near 3270 cm
−1 and N-H stretching, and weak bands at ~2930–2858 cm
−1, assigned to C-H bonds. The presence of an intense band at 1656 cm
−1 is attributed to conjugated C=O and carboxylate functionalities, and C=C stretching. C-H bending bands at 1440 cm
−1 (CH
2) and 1370 cm
−1 (CH
3) are also present.
3.2. Optical Characterization
Aqueous dispersions of
µAlgae-CDs were prepared in appropriate concentrations for the optical assays. The UV-Vis absorption spectrum of
µAlgae-CDs showed a long absorption edge in both the UV and visible regions (300–600 nm) (
Figure 2a).
The aqueous dispersions of
µAlgae-CDs displayed a maximum excitation and emission at 346 and 438 nm, respectively (
Figure 2a), and exhibited a blue color when irradiated with 366 nm ultraviolet light (
Figure 2a, inset).
The
μAlgae-CDs synthesized with ED as a nitrogen source using wet and dry biomass of
P. cruentum showed relevant fluorescence quantum yields (0.07 <
ΦF < 0.16), revealing a strong dependence between
λexc and
λem (
Figure 2b) and excellent photostability (not shown).
3.3. Antioxidant Performance of µAlgae-CDs
An important property of C-dots is their antioxidant activity, i.e., the ability to act as a free radical scavenger. The antioxidant capacity of
µAlgae-CDs at 5 mg mL
−1 was evaluated with the ABTS radical inhibition assay (
Table 1).
The results showed that the
µAlgae-CDs obtained from wet biomass (Entry 1,
Table 1) had better biological properties, even though they revealed a lower quantum yield as compared to the carbon dots achieved from dry biomass (Entry 2,
Table 1).
3.4. Toxicity Evaluation and Cell Imaging
Using MTT assays, the biocompatibility of
μAlgae-CDs against two epithelial cell lines, Vero (kidney epithelial cells extracted from an African green monkey) and HeLa (human cervical tumor cells), was evaluated over a concentration range of 0.02 to 4000 µg mL
−1 (
Figure 3 and
Figure 4, respectively).
Even for high levels of µAlgae-CDs, no significant cytotoxicity was observed for both cell lines, showing cell viability higher than 80%.
After incubation with
µAlgae-CDs for 18 h at 37 °C, both cell lines were observed using fluorescence microscopy and the images in
Figure 5 were collected.
4. Conclusions
The µAlgae-CDs were prepared from biomass of the red microalgae Porphyridium cruentum using an ecofriendly method and showed good photostability and relevant quantum yield.
The ABTS assay showed high efficacy in radical scavenging, suggesting good antioxidant properties of the µAlgae-CDs.
The µAlgae-CNDs did not exhibit cytotoxicity against Vero and HeLa cells, and by using fluorescence microscopy we observed that µAlgae-CNDs were internalized by both cell lines, opening up new applications for these nanomaterials.
Author Contributions
Conceptualization, A.I.C., J.V.P. and P.D.B.; methodology, J.V.P., F.M.H.C. and M.L.L.; investigation, I.C., F.M.H.C. and M.L.L.; resources, A.I.C.; data curation, A.I.C., I.C., M.C.S., P.D.B. and S.M.; writing—original draft preparation, A.I.C., M.C.S., P.D.B. and S.M.; writing—review and editing, A.I.C., J.V.P., M.C.S., P.D.B. and S.M. All authors have read and agreed to the published version of the manuscript.
Funding
We are grateful to Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecno-logia e Ensino Superior (FCT/MCTES) for financial support (UIDB/00616/2023, UIDP/00616/2023 and UIDP/04035/2020), and Instituto Politécnico de Lisboa (IPL/IDI&CA_2022/µAlgae2Dot_ISEL).
Data Availability Statement
Data sharing is not applicable to this article.
Acknowledgments
We thank Allmicroalgae-Natural Products, S.A. for µAlgae biomass supply.
Conflicts of Interest
The authors declare no conflict of interest.
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