Aminoacid functionalised magnetite nanoparticles Fe3O4@AA (AA = Ser, Cys, Pro, Trp) as biocompatible magnetite nanoparticles with potential therapeutic applications | Scientific Reports

Scientific Reports volume 14, Article number: 26228 (2024) Cite this article Metrics details Magnetic nanoparticles (MNPs) are of great interest for their wide applications in biomedical applications,

Scientific Reports volume 14, Article number: 26228 (2024) Cite this article

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Magnetic nanoparticles (MNPs) are of great interest for their wide applications in biomedical applications, such as bioimaging, antitumoral therapies, regenerative medicine, and drug delivery. The work aimed to obtain biocompatible magnetite nanoparticles coated with amino acids of the general formula Fe3O4@AA (AA = L-tryptophan, L-serine, L-proline and L-cysteine) for potential therapeutic application in anticancer drug delivery. The obtained materials were characterised using XRD, FTIR, DLS analysis, SEM, thermogravimetry (TG), differential scanning calorimetry (DSC), and UV–vis spectroscopy. The photocatalytic, cytotoxic and antimicrobial activity tests of the obtained materials were carried out. The choice of amino acid determines the properties of the material and its future use, for example, Fe3O4@Cys supports radical production, which may increase the efficiency of catalytic degradation, while tryptophan captures radicals, which may be an advantage in several biomedical applications. Fe3O4@Trp exhibited good antimicrobial activity (MBEC and MIC) against E. coli ATCC 25922, P. aeruginosa ATCC 27853 and C. albicans ATCC 10231 while Fe3O4@Pro exhibited the best results against S. aureus ATCC 25923.

Richard Feynman was the first scientist to introduce the notion of nanotechnology in his lecture entitled “There’s plenty of Room at the Bottom”. This concept has started a remarkable development in the area of nanotechnology. Nanotechnology is a science that deals with the preparation of nano-sized particles ranging from 1 to 100 nm employing diverse synthetic strategies, particle structure, and size modification1. Nanotechnology is one of the fastest-growing fields of scientific research, with significant progress being made in a range of applications. Nanoparticles are used in different areas such as molecular biology, physics, organic and inorganic chemistry, medicine, and material science because of their unique properties resulting from the effect of size and surface phenomena2,3,4. Nanoparticles and nanomaterials are increasingly being explored for their potential applications in medicine.

Currently, magnetic nanoparticles (MNPs) are of great interest for their wide applications in biomedical applications, such as bioimaging, antitumoral therapies, regenerative medicine and drug delivery, as they possess many unique properties such as metal-rich moieties, large surface area, and tunability of structural compositions4,5,6,7. The most promising MNPs are magnetite nanoparticles (Fe3O4), whose use is considered a breakthrough in biomedical applications such as magnetic drug and gene delivery, magnetic hyperthermia, magnetic resonance imaging (MRI), and theranostics8,9,10,11,12,13,14.

Magnetite (Fe3O4) has an inverse spinel structure in which the cations occupy tetrahedral and octahedral sites in the cubic lattice15,16. Tetrahedral sites (A) occupied by Fe3+ and the octahedral sites (B) occupied by both Fe3+ and Fe2+ form the basis for two antiparallel magnetic sublattices. The number of spins at sites A and B is unequal, resulting in ferrimagnetism in magnetite below the Curie temperature of 580 °C16. Fe3O4 magnetic nanoparticles exhibit superparamagnetism with high saturation magnetisation. It should be noticed that a characteristic feature of superparamagnetic nanoparticles is the presence of a single magnetic domain, thus enhancing their susceptibility to the magnetic fields, which allows the magnetic domains to form a variety of patterns with different orientations depending on the magnetic anisotropies and positions of nanoparticles17. The relationship between particle size and magnetic properties, such as coercivity (Hc), of Fe3O4 NPs has been widely reported. The critical size of magnetic NPs, which indicates the transition from a single- to multi-domain structure, was evaluated by the change in Hc with respect to the particle size. The particle size required to achieve superparamagnetism in Fe3O4 NP is widely estimated to be below 20 nm18,19,20,21, whereas the critical size for the formation of a multi-domain structure has been theoretically estimated to be 76 nm for cubic and 128 nm for spherical Fe3O4 NP22. Due to their magnetic properties, manipulating them using an external magnetic field is convenient in different applications such as: biosensor23,24,25, targeted therapy and diagnostics (theranostics)9,26,27, or combination of magnetic hyperthermia, anticancer drug delivery, magnetic resonance imaging (or other imaging technology)9,26,28,29,30, which could open up great potential in cancer treatment. Nanoparticles synthesised for drug delivery carrier should be stable and non-toxic. Because the body’s defences in biological systems function to trap any external species (equally living—e.g. microbial as non-living—e.g. inorganic particles), coating nanoparticles with biological molecules allows the body’s defence response to be avoided. The surface functionalisation of magnetite nanoparticles with a biological or chemical agent that binds to a specific target can significantly influence their biological properties30,31,32. Functionalisation magnetite nanoparticles with amino acids as coatings could design the next generation of intensified bioprocesses due to their chemical simplicity, surface activity, and biocompatibility33.

The mostly used functionalization agent for magnetite is PEG and, this which allow good stabilisation and, what is even more important, to mask the existence of these nanoparticles within the body and thus to avoid “the foreign body effect” and their premature degradation. Another important modification of the magnetic nanoparticles is related to the decoration with folate, especially because many cells (tumoral but also other cells specific for other diseases such as rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, Crohn’s disease, or atherosclerosis) have overexpressing receptors for folate (part of the vitamin B complex)14. Certainly, folate is needed by all the cells so, all the cells have this ability to recognise and to allow its internalization. This is why the decoration of the carriers with folate is currently used for intracellular transport of different drugs but, the specificity is in general done by the higher cellular rate of the tumoral cells as well as the overexpressing receptors for folate on the tumoral cells.

Along with the normal needs for vitamins, the cells also have other specific needs, including nutrients and amino acids, hidroxyacids, cetoacids, etc. could have similar effects even if there are only limited researches in the field and, unfortunately, most of them are just related to the the material point of view34,35,36,37. The functionalisation of the particles with amine groups would improve their interaction with large negatively charged cell membrane domains, significantly increasing the probability of surface interactions38,39,40.

This work aimed to obtain biocompatible magnetite nanoparticles coated with amino acids of the general formula Fe3O4@AA (AA = Trp (L-tryptophan), Ser (L-serine), Pro (L-proline), and Cys (L-cysteine)) for potential therapeutic applications as drug delivery carriers. For this purpose, the cytotoxicity of Fe3O4@AA nanoparticles, their antimicrobial activity, and photocatalytic activity (to investigate the ability of the material to produce reactive oxygen species, ROS) were evaluated. The obtained materials were characterised using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) analysis, scanning electron microscopy (SEM), differential scanning calorimetry (DSC-TG) and UV–vis spectroscopy. In addition, tests of the photocatalytic activity of the obtained materials were performed and, according to these results, new perspectives are opening in photocatalysis (including also for medical applications). These core@shell structure will be further tested in combined therapy of cancer (radical production with degradative activity + drug release), infections (native antimicrobial activity + degradative activity induced by the produced radicals) or other applications such as environmental applications involving simultaneous or successive adsorption and degradation of specific poluttants.

Magnetite nanoparticles were obtained starting from the following principal materials: iron (III) chloride—FeCl3 99% (Sigma Aldrich), iron chloride (II) terahydrate—FeCl2 × 4H2O 99% (Merk), L-tryptophan 99% (Roth), L-serine 99% (Roth), L-proline 98,5% (Roth) and (L-Cysteine, ICN Biochemicals), sodium hydroxide—NaOH (Sigma Aldrich), rhodamine B (RhB) 97% (Acros Organics), FBS (Biowest, France), DMEM/F-12 (Gibco, USA), penicillin–streptomycin (Gibco, USA), 0.25% trypsin- 0.03% EDTA, PBS (Gibco, USA), ethanol 96% (POCH, Poland), glacial acetic acid (Chempur, Poland) and neutral red assay (NR) (Sigma-Aldrich, United Kingdom). Yeast strains BY4741 were purchased from EUROSCARF (Frankfurt, Germany). All reagents were used without purification.

The synthesis of Fe3O4 NPs was carried out by the co-precipitation method in an alkaline solution using amino acids (L-tryptophan, L-serine, L-proline, and L-cysteine) as stabilizing agents. The synthesis of Fe3O4@AA started from Fe2+, Fe3+ inorganic precursors and AA, carefully controlling the reaction parameters (precursor concentration, pH, temperature, etc.).

In a Berzelius flask, 200 mL of distilled water was added, in which 5 g of AA and 25 g of NaOH were dissolved. On top of the basic solution obtained, 50 mL of aqueous solution, obtained by dissolving 8.70 g of FeCl2 × 4H2O and 14.2 g of FeCl3, was added dropwise under continuous stirring when a black precipitate appeared. After the mixture of precursors has been added, stirring is continued for another 3 h. Then, the black precipitate obtained was purified by successive washing and decantation to pH 7 and a chloride-free solution was achieved (Cl− ions were checked with AgNO3). After being washed, it was dried in an oven at a temperature 50 °C. The summary of the obtained Fe3O4@AA materials is presented in Table 1.

X-ray diffractions (XRD) were recorded on a PANalytical X’Pert Pro MPD analyser using Cu-Ka radiation of λ = 1.541874 Å, a hybrid monochromator 2 × Ge (220) for Cu, and a parallel-plate collimator on the PIXcel3D detector. Scanning was carried out within the 2 θ angle range between 10° and 80°, with an incidence angle of 0.5°, step size of 0.0256°, and a time for each step of 1 s. The Rietveld refinement was performed using the HighScore Plus software (version 3.0, PANalytical, Almelo, The Netherlands) to evaluate the nanoparticle crystallinity and crystallite size.

Infrared (IR) spectroscopy measurements were performed using a Thermo NicoletTM iSTM50 FT-IR spectroscope equipped with a diamond crystal-based ATR module capable of recording spectra over a range of 400–4000 cm−1. To obtain a better signal-to-noise ratio, the spectra were measured in 16 scan mode at a resolution of 8 cm−1. The acquisition rate was set to 3 scans/s using a DTGS type detector and an automatic beamsplitter for the ability to record the spectrum, especially in transmission mode, in the range 10–25,000 cm−1, that is, from far IR to near IR.

To measure the hydrodynamic diameter and zeta potential of Fe3O4@AA systems, samples were dispersed in deionized water (~ 6.9 pH) at a concentration of 0.3 mg/mL. For each sample, five acquisitions were measured using the DLS technique (DelsaMax Pro, Backman Coulter, Brea, CA, USA). The measurements were conducted simultaneously.

The morphology of the catalysts was observed with a field-emission scanning electron microscope QUANTA INSPECT F50 and FEI Helios NanoLab 650 microscope (FEI Company, Eindhoven, The Netherlands).

The simultaneous thermal analysis (STA) thermogravimetry (TG) & differential scanning calorimetry (DSC) for the samples was performed with a Netzsch STA 449C Jupiter apparatus. The samples were placed in an open crucible made of alumina and heated with 10 °C·min−1 from room temperature to 900 °C, under the flow of dried air of 50 mL min−1. An empty alumina crucible was used as a reference.

A Perkin Elmer LS55 (Perkin Elmer, Waltham, MA, USA) fluorimeter was used to record photoluminescence spectra (PL). An Xe lamp was used as an excitation source at ambient temperature. The excitation wavelength was 320 nm. The emission spectra were recorded in the 350–700 nm domain, with a scan speed of 200 nm min−1 and a 350 nm cut-off filter.

The PZC of Fe3O4@AA systems was measured according to Kocharova et al.41. For the determination of pHPZC, eleven vials were filled with 0.1 mol L−1 NaCl solution. The pH of each vial was adjusted with NaOH and HCl solutions to pH 2–12. Next, 10 mg of Fe3O4@AA were dispersed in each vial. The solution was constantly agitated at 240 rpm for 3 h at ambient temperature to reach equilibrium. The equilibrium pH value was measured with a multimeter (CPC 411, Elmetron, Poland) and the values were plotted against the initial pH values for both series. The PZC value was then obtained from the point at which the curve showing the final pH vs. the initial pH intersected the line y = x on the graph.

Photocatalysis was carried out in a laboratory reactor of LRS2 type (Heraeus, Hanau, Germany) equipped with a 150 W excimer lamp TQ150 as a light source (power density 4.696 mW cm−2 measured by digital lux meter Peak Tech 5025) surrounded by a water-cooling glass jacket. The 150 mg of Fe3O4@AA was added to a glass tube reactor containing 250 mL of 1 × 10–5 M rhodamine B (RhB) solution of pH = 9 ± 0.1 (adjusted with 0.1 M NaOH). The suspension was stirred for 30 min in the dark to equilibrate adsorption/desorption under oxygen or anaerobic conditions (Ar purging). After this time, the irradiation began and lasted 90 min. In some experiments, additional reagents (H2O2, CH3OH, HCOONa) were added to the reaction mixture as electron/hole scavengers. During the reaction, 2 mL samples were collected from the reactor at regular time intervals and centrifuged to separate the photocatalyst. The concentration of compounds was monitored using a UV–Vis spectrophotometer (VWR International, Radnor, Pennsylvania, USA) at 554 nm. External standards of six concentration levels ranging from 0.6 × 10–6 to 1 × 10–5 M were used to quantify RhB.

The removal efficiency of RB in photocatalytic degradation processes as a function of time is given by Eq. (1):

MIC assessment is performed using the method described in previous studies42,43. Decimal dilutions are made from each nanoparticle suspension, followed by inoculation with a standard microbial suspension (medium liquid volumetric ratio: microbial suspension = 10:1) obtained from fresh cultures. Also, following the same steps, blank and control samples are performed (sterility and microbial growth). The 96-well plates are incubated at 37 °C for 24 h. The determination of MIC values is performed both by macroscopic observation and by reading at 620 nm (the suspensions are transferred to a sterile plate after prior centrifugation at 5000X to remove the precipitate/ NPs deposited to avoid).

The semiquantitative assessment of the degree of adhesion to the inert substrate of the strains tested in this study is carried out using the crystal violet staining method. After determining the MIC values, 96-well plates are washed 3 times with phosphate buffer solution (PBS), fixed with methanol and stained with a 1% crystal violet solution. The dye included in the cells adhered to the walls of the plate is dissolved with acetic acid to determine the MBEC values (minimum biofilm eradication concentration). Spectrophotometric measurements were performed at 490 nm42,43.

The cytotoxicity of magnetite with selected amino acids (serine, cysteine, tryptophan, and proline) has been studied in two human cell lines—immortalised keratinocytes HaCaT and human malignant melanoma A-375. Cells were cultured in DMEM/F-12 cytotoxicity with 10% heat inactivated fetal bovine serum (FBS) 1% v/v penicillin-streptomycin and incubated at 37 °C under conditions 5% CO2, 95% humidity (Binder, Germany). The growth medium was changed twice a week, cells were passed at 80-90% confluence using 0.25% trypsin- 0.03% EDTA in calcium and magnesium-free phosphate-buffered saline (PBS).

Cells were seeded in a 96-well clear plate at a density of 7.5 × 103 cells/well (HaCaT) and 1 × 104 (A-375) in 100 µL culture medium and allowed to attach for 24 h at 37 °C. Subsequently the cell were treated with tested compound—Fe3O4@Ser, Fe3O4@Cys, Fe3O4@Pro, Fe3O4@Trp at concentration 0.1, 0.5, 1.0 mg/mL for 24 h. Nontreated cells were used as a control. The magnetite was sterilised using a UV lamp and sonication (90 s) (Omni Sonic Ruptor 250, USA). After exposure, the cytotoxicity test—neutral red assay (NR) was performed. Briefly, the medium with the compound tested was removed and replaced with a 2% neutral red solution in cell culture medium (100 µL/well), plate was incubated at 37 °C for 1 h. The cells were then rinsed with warm PBS and a permeabilized solution (50% destilied water, 49% ethanol 96% and 1% glacial acetic acid) (100 µL/well).

The plate was shaken (800 rpm) at room temperature for 25 minutes (Heidolph Inkubator 1000, Germany). Absorbance was measured at 540 nm against 620 nm using the TECAN Infinite 200 microplate reader (Grodig, Austria). The results were presented as a percentage of control counted from 9 replicates.

Yeast cells were grown in a standard liquid Yeast Extract–Peptone–Dextrose (YPD) medium (1% Difco yeast extract, 1% yeast bacto-peptone and 2% glucose) on a rotary shaker at 150 rpm or in solid YPD medium containing 2% agar. Experiments were carried out at 28 °C. Yeast strains BY4741 (MATa; his3; leu2; met15; ura3), sod1Δ (MATa; his3; leu2; met15; ura3; YJR104C::kanMX4), sod2Δ (MATa; his3; leu2; met15; ura3; YHR008C::kanMX4C) were grown to exponential phase (OD600nm between 0.8 and 1) and serially diluted to different cellular concentrations as indicated. Five microliters of each cell suspension was spotted onto agar plates containing various concentrations (0.1, 0.5, 0.75, 1.0 mg/mL) of tested compound—Fe3O4@Ser, Fe3O4@Cys, Fe3O4@Pro, Fe3O4@Trp. Growth was registered 48 h after incubation at 28 °C. All phenotypes described in this work were validated through three independent biological replicates.

The morphology of the catalysts is shown in Fig. 1a–e. The images show nanocrystal agglomerates formed by aggregation of small particles for all samples. An additional factor facilitating the formation of agglomerates is the presence of amino acids on the surface of nanoparticles, which act as a binder. Depending on the amino acid used, a change in the morphology of the MNPs can be observed (the smaller nanoparticles were obtained in the presence of Ser and Pro than other amino acids, established size < 100 nm, while in remain samples the particles sizes were approximately 100 nm).

SEM images of materials—Fe3O4 (a), Fe3O4@Ser (b), Fe3O4@Pro (c), Fe3O4@Cys (d), Fe3O4@Trp (e); XRD pattern for Fe3O4 and Fe3O4@AA (f); FTIR spectra for Fe3O4 and Fe3O4@AA nanoparticles (g); Fluorescence spectra of materials (h), in all graphs black—Fe3O4, red—Fe3O4@Trp, green—Fe3O4@Ser, dark blue—Fe3O4@Pro, light blue—Fe3O4@Cys.

XRD was used to establish the formation of magnetite as a pure phase. X-ray diffraction patterns (Fig. 1f) recorded on amino acid-coated Fe3O4 NPs indicate the presence of magnetite as a single phase with a crystallite size of 4–6 nm (Table S2). In the XRD diffractograms, the characteristic magnetite peaks can be observed at the 2θ angle values of 30.5°(220), 35.9°(311), 37°(222), 43.5°(400), 57.3°(511) and 63.1°(440), which fit the standard model of Fe3O4 well44. The diffractogram confirms the formation of a pure-phase cubic (space group: Fd-3 m) spinel structure. A slight shift to high angle diffraction of Fe3O4@AA (amino acids) NPs capped than the reference (JCPDS, File No. 19-0629) could be due to water adsorption on the surface of Fe3O4 NPs45. The lattice parameters were determined using Rietveld refinement and are presented in Table S2 (Supplementary Information).

The FTIR data reveal some differences between the spectra of the samples, especially compared to pure magnetite (Fig. 1g). The IR spectrum of Fe3O4 reveals two characteristic absorption bands for nanometric magnetite around 558 and 624 cm−146, which can be assigned to the Fe–O bond in the octahedral and tetrahedral geometry. The absorption bands around 1633 and 3448 cm−1 come from adsorbed water (proves the hydrophilicity of the surface, essential in medical applications), while in the 1000–1700 cm−1 range, the bands specific to the functional groups of amino acids appear.

The fluorescence spectra of Fe3O4 and Fe3O4@AA are shown in Fig. 1h. Magnetite fluorescence is generated by the hydroxyl groups on the surface of the nanoparticles, as well as by the surface crystalline defects. The coated with amino acids magnetite exhibits decreasing of fluorescent emission. Tryptophan had the least influence, while the greatest decrease in fluorescence intensity was observed for serine and proline. It is worth mentioning that according to thermogravimetric assessments, the amino acid content is 2.98% for Fe3O4@Ser and 0.69% for Fe3O4@Pro (so, the highest and lowest amino acid content) but quite the same fluorescence spectra. The main emission peak in the UV range is at 376 nm and is attributed to excitonic recombination. In the visible range, the peaks at 454 and 480 nm are due to network defects induced by vacancies or interstitial oxygen47.

The SEM images (Fig. 1a–e) recorded on the Fe3O4 as well as on the Fe3O4@AA show the nanostructured nature of these materials, but also the different degrees of agglomeration depending on the used amino acid similar as we proved previously on different Fe3O4@stabilizing agents (including amino acids)34. For all these magnetic nanoparticles, the shape is cvasispherical and their size is bellow 10 nm34,35, being is good agreements with the data we obtained for another series of Fe3O4@AA (the used amino acids were asparagine; aspartic acid and L-proline).

The zeta potential values are summarised in Fig. 2. One can conclude that the Fe3O4@Trp, Fe3O4@Ser, and Fe3O4@Cys nanoparticles were stable under measurements conditions48. Taking into account the value of the zeta potential of Fe3O4@Pro, it can be concluded that it is neutral, and hence the nanoparticles are susceptible to aggregation. This causes the hydrodynamic diameter of Fe3O4@Pro to be 3–4 times larger than the hydrodynamic diameter of the other coated samples. In the case of the other systems, the zeta potential values indicate that the MNPs can repel each other and provide adequate stabilisation. The hydrodynamic size of the Fe3O4@Trp, Fe3O4@Ser and Fe3O4@Cys samples is very close, between 137 and 170 nm. The value of the Pd index showed good stability of these systems, with the Pd index ranging from 0.23 to 0.33 (Table S6)49.

DLS data: hydrodynamic size (nm) of the samples and zetta potential (mV).

Corroborating DLS data with the SEM images, the particle size of these nanoparticles seems to be similar and this means that the agglomeration of the of Fe3O4@Pro is more important (also Pdi 0.33 for Fe3O4@Pro instead of 0.23–0.24 for the other samples while the zeta potential is of only − 0.66 mV).

The DSC-TG curves are presented in Fig. 3a and b. The diagrams illustrate the behaviour of the sample once the temperature changes. The TG curve shows three mass losses in the temperature ranges of room temperature—180, 180–500, and 500–900 °C. The primary mass loss corresponding to the endothermic peak in the temperature range 79–86 °C on the DSC curve, which is assigned to the removal of physically adsorbed water50. The second mass loss step, accompanied by exothermic effects with, with several maximums at 282–291 and 355–462 °C, is assigned to the degradative oxidation of the organic substance. In the second range, a weak peak can also be observed at 211 °C due to the oxidation of Fe(II) to Fe(III), magnetite being transformed into maghemite44. The last mass loss step is caused by the densification and total elimination of the molecules adsorbed on the surface of the nanoparticles, as well as the burning of the residual carbonaceous mass left after aminoacid45. The sharp exothermic effect in the range 549–588 °C is due to the physical transformation of maghemite into hematite, which is a specific effect of magnetite samples51. Detailed description of Fe3O4@AA TG-DSC analysis is available in the Supplementary Materials.

DSC-TG curves for Fe3O4 (a) and Fe3O4@AA nanoparticles (Fe3O4@Ser—black, Fe3O4@Cys—blue, Fe3O4@Trp—red, Fe3O4@Pro—purple) (b); 2D projection of the individual FTIR spectra for Fe3O4@Cys (top FTIR spectra at 120 °C; right the trace of CO2 for 2355 cm−1) (c), schematic representation of amino acid-functionalized magnetite nanoparticles (d).

The estimated aminoacids load on the magnetite nanoparticles as well as the mass loss and maghemite to hematite transformation temperature have been summarized in the Table 2.

For Fe3O4@Cys, the analysis of the gases resulting from the thermal analysis was also carried out, the 2D projection in the plane wavenumber/temperature of the individual FTIR spectra vs. temperature indicating the presence of water (first the water present as moisture is removed, then that adsorbed on the surface of the nanoparticles and finally that resulting from the condensation of hydroxyl groups on the surface). The presence of carbon dioxide generated by cysteine oxidation can also be observed in Fig. 3c. Figure 3d schematically shows magnetite nanoparticles with attached amino acids.

The photocatalytic efficiency is determined by the surface interactions between the organic compound and the catalyst. Adsorption of the organic compound on the surface of the catalyst significantly increases the catalysis efficiency. The surface charge of the dispersed in the water catalyst strongly determines the organic molecule-catalyst surface interaction. Point of Zero Charge (PZC) provides information about the pH value at which the catalyst has a net surface charge of zero. Usually, at pH values lower than PZC, the surface has a positive surface charge, while at pH values above PZC, the surface has a negative surface charge. The pHpzc values of amino acid-coated MNPs were above 7, whereas the bare was bellow pH 7 (Fig. S2a, Supplementary Information, Table 3). Rhodamine B was selected as a test substance in the photocatalysis process due to the fact of possessing a positive charge on amine nitrogen in solutions of pH > 7 (Fig. S2d).

The amount of adsorbed RhB on catalysts surface was calculated (Table 4) according to the Eq. (2):

where C0—initial RhB concentration (1 × 10–5 M), Ct—RhB concentration after equilibrium reached (30 min in dark), V—reaction mixture volume, m—catalyst mass.

RhB adsorption measurements on the surface of catalysts strongly correlate with the content of amino acids. The pHpzc of the catalysts changes according to changes in the PI value of the amino acids (Fig. S2b). The highest adsorption of RhB was observed on Fe3O4. Next was Fe3O4@Pro, which has a low amino acid content (0.69%). A negatively charged surface of the Fe3O4 catalyst facilitated the adsorption of RhB via positively charged amino groups. In the case of catalysts coated with amino acids, the situation is more complex. Since the degradation reactions were carried out at pH 9, to consider the catalyst surface-RhB interaction, it is necessary to analyse the ionic composition of the amino acids on the surface of the catalysts (Fig. S2c, Table S3). One can see that proline is mainly present in protonated form. In contrast, serine and cysteine are present in almost equal proportions as the protonated and deprotonated forms. Tryptophan occurs mainly in the protonated form. The diprotic species have been omitted because they do not exist under these conditions (pH 9). The RhB molecule is present in the reaction mixture as a zwitterion, the rhodamine B molecule can interact with the catalyst surface in two ways—via a positively charged nitrogen atom (Fig. S2d) or by an interaction between the surface of the RhB molecule and the catalyst by relatively weak London forces52. Amino acids covering the surfaces of the catalysts coordinate with iron ions through carboxyl, hydroxyl and/or thiol groups present in the side chains. For this reason, only amino groups are available for interactions with the rhodamine B molecule, which can interact with the carboxyl group present in the RhB molecule. Adsorption of RhB on catalysts was: Fe3O4@Cys < Fe3O4@Ser < Fe3O4@Trp < Fe3O4@Pro < Fe3O4. It indicates that both the content and the type of amino acid affect the adsorption of rhodamine B on the catalyst surface.

Reactive oxygen species (ROS), such as hydroxyl radicals (•OH), singlet oxygen (1O2), superoxide radicals (O2•−) and hydrogen peroxide (H2O2), are traditionally recognised for their ability to damage biological molecules. Photoexcitation of catalysts produces electrons in the conduction band (e−) and holes in the valence band (h+). Such a hole-electron pair is very unstable and can be recombined or taken part in oxidation and reduction reactions on the surface of the catalyst. The •OH radicals generated by the reaction of −OH anion or H2O molecule with photogenerated hole and O2•− anion radical created by the reaction of molecular oxygen with photogenerated electron can act as antimicrobial agents.

The photocatalytic experiments were carried out in six reaction systems summarised in Table 4.

One can see in Fig. 4a, the tested photocatalytic system (RS1) efficiency was low (degradation efficiency < 10% for all catalysts). The reaction of RhB with an electron should cause demethylation of amino groups and a hypsochromic shift of the dye’s absorption band (for Rh-110 λmax = 500 nm). The shift of the absorption band wasn’t observed. Thus, one can conclude that only oxygen dissolved in the reaction mixture reacts with an electron on the catalyst surface. However, the efficiency of this reaction is low due to the blocking of active sites on the catalyst by RhB molecules and amino acids. The O2•− and HOO• radicals are less reactive than hydroxyl radicals, so the efficiency of degradation is low. Possible processes taking place in RS1 on the catalyst surface are presented below:

RhB decay in RS1 (a); RhB decay in RS2 (b); Determination of kapp values in the RS2 system (c) for catalysts—Fe3O4@Pro (blue), Fe3O4@Ser (orange), Fe3O4@Cys (grey), Fe3O4@Trp (yellow), Fe3O4 (brown), RhB photolysis (green).

Introducing an additional reagent (H2O2) to the photocatalytic system (RS2 system) significantly improved the efficiency of photocatalytic degradation of RhB (Fig. 4b). The apparent degradation rate constant (kapp) was determined according to Eq. (9), assuming that the reactions occurring were of pseudo-first order.

where C0 and Ct are the initial concentration and the concentration at time t, respectively. The dependence of ln(Ct/C0) on time is represented by straight lines, as shown in Fig. 4c. Therefore, the degradation kinetics are consistent with the pseudo-first-order kinetic model (R2 > 0.95). The values of the degradation rate constants and the degradation efficiency are summarised in Table 5.

When the ratio of [OH−] to [H2O2] is around 0.02, no O2•− is obtained, but the reaction can produce53 oxygen (eq.10).

The ratio in studied reactions was 2.56 × 10−4 what indicate production of the superoxide ion radical. The ratio in the studied reactions was 2.56 × 10−4 which indicates production of the superoxide ion radical. Moreover, the pKa value of H2O2 is 11.62, which suggests an undissociated form of hydrogen peroxide present in the reaction mixture54.

Fe3O4@Cys shows the best degradation result in RS2. The functional groups in cysteine can interact with Fe(II) and/or Fe(III) on the magnetite surface. Both Fe2+ and Fe3+ bind with cysteine through bidentate chelation via sulfur and nitrogen atoms55. Cysteine (Cys) is found in iron-sulfur electron transfer proteins such as ferredoxins56. The reaction between cysteine and Fe(III) is a one-electron transfer process causing the formation of cysteine (RSSR) that may proceed as follows:

The best degradation result observed for the Fe3O4@L-cysteine catalyst may be due to the formation of additional radicals during the reaction:

The proposed equation is analogous to cysteine radical generation (Eqs. 15 and 16) described by Zhao et al.57 and Zhao et al.58.

Generated thiol radicals diffusing into the bulk of the solution can react:

The lowest degradation result obtained for the Fe3O4@L-tryptophane catalyst may be due to tryptophan (TrpH) radical scavenging properties (it scavenges reactive species such as e−, O2•− and HO• radicals) and the formation of stable radicals from tryptophan (Eqs. 19 and 20, Fig. S3, Supplementary Information). Trp• is a stable radical due to the delocalisation of the unpaired electron.

The reaction Fe3O4@Pro_Air (RS1), Fe3O4@Pro_Ar (RS4) and Fe3O4@Pro_Ar_CH3OH (RS5) indicates absence of reactive species, the observed degradation of RhB is negligible (Fig. 5a). The photolysis experiments (under aerated and deaerated conditions) indicate that H2O2 decomposition occurs mainly in reaction with O2•− and RhB*. Additionally, the O2•− is generated by the reaction of oxygen with excited RhB (RhB*) because the estimated reduction potential of catalysts is not enough negative to reduce oxygen. However, in the case of photolysis, the removal of oxygen from the system practically does not affect the rate of degradation of RhB.

RhB degradation (a) in RS1 (Fe3O4@Pro_Air—blue,), RS2 (Fe3O4@Pro_H2O2_Air—orange, Fe3O4_H2O2_Air—brown), RS3 (Fe3O4@Pro_Ar_H2O2—yellow), RS4 (Fe3O4@Pro_Ar—grey), RS5 (Fe3O4@Pro_Ar_CH3OH—light blue), RS6 (Fe3O4@Pro_Ar_HCOONa—green); determination of kapp values (b).

Significant degradation was observed in Fe3O4@Pro_Ar_H2O2, Fe3O4@Pro_Air_H2O2, Fe3O4_Air_H2O2. These results agree with adsorption results (the best adsorption on Fe3O4, and Fe3O4@Pro; Table 4). A comparison of the results for Fe3O4@Pro_Air_H2O2 and Fe3O4@Pro_Ar_H2O2 shows that the presence of oxygen in the reaction mixture improves the degradation efficiency (Fig. 5b, Table 6), and the kapp value of the aerated system is about 2,3-times higher than deaerated. It indicates the oxygen participation in the degradation reaction.

The hole potential (+ 1.9 V) should be sufficient to generate hydroxyl radicals from hydroxyl ions (+ 1.9 V) (not from water, EH2O/•OH = + 2.73 V)59. Alcohols are commonly used to quench hydroxyl radicals and/or holes (Eq. 21). The sodium formate works analogously (Eq. 22). However, the hole scavenger experiment does not indicate the formation of hydroxyl radicals in the hydroxyl ions reaction with the hole. It may be caused pHpzc of catalysts. The surface of the catalysts at pH 9 has a negative charge and repels hydroxyl ions.

Reactions with hole scavengers:

Hole scavengers can interact with the catalyst surface by hydrogen bonds. The potential of VB and scavenger redox potentials indicates the possibility of a hole-scavenger reaction.

Comparing the results for RhB degradation on the Fe3O4@Pro catalyst in aerobic and anaerobic conditions, it can be seen that the presence of oxygen (electron scavenger) does not affect the reaction rate. Furthermore, the use of hole scavengers (CH3OH, HCOONa, Ar, pH 9) did not change the reaction rate compared to the process carried out under aerobic conditions (Fe3O4@Pro, air, pH 9). This indicates that no reactive species, such as a superoxide radical or a hydroxyl radical, were formed on the catalyst.

Considering the obtained results, reactions occurring in the tested systems were proposed:

The reaction sequences presented above are visualised in Fig. S4 (Supplementary Information).

The analysis of the redox potentials collected in Table S3 shows that RhB* can donate an electron to the conduction band of the catalyst, while the hole can oxidise the RhB molecule to a radical cation. The conduction band potential is not sufficient to reduce oxygen to the superoxide anion radical, but it has sufficient potential to generate the HOO radical. Cysteine can react with the electron generated in the conduction band because the reduction potential of cysteine is lower than that of the conduction band.

The kinetic data available in the literature, summarised in Table S4, show that cysteine and tryptophan are effective when reacting with an electron, while proline and serine react at two orders of magnitude and an order of magnitude slower, respectively. Similarly, for the reaction with the hydroxyl radical, the reaction rate constant for tryptophan and cysteine is two orders of magnitude higher than for proline and serine. This supports our conclusion that tryptophan scavenges radicals generated during the photocatalytic process, resulting in the lowest degradation efficiency for the Fe3O4@Trp sample.

Following the MIC evaluation for the magnetite-based samples taken in the study, it can be seen that they determined the highest sensitivity on P. aeruginosa (Table 7). Most of the magnetic systems loaded with biologically active substances showed better antimicrobial activity compared to controls, and a synergistic effect was also observed. Among them, Fe3O4@Trp determined the highest sensitivity of E. coli, P. aeruginosa and C. albicans, with MIC values of 0.001 mg/mL. Fe3O4@Cys inhibited the adhesion of all strains studied.

The results of the MIC evaluation are confirmed by those of adhesion to the inert substrate. The lowest values of MBEC (Fig. 6) are in the case of P. aeruginosa, which was shown to be the most sensitive strain. Additionally, the lowest values of MBEC are between 0.01 and 0.001 mg/mL in the case of Fe3O4@Trp, Fe3O4@Cys, followed by Fe3O4@Ser. These samples showed the best antimicrobial activity against E. coli, P. aeruginosa, and C. albicans. Among all the studied samples, Fe3O4@Pro prevented the adhesion of S. aureus cells to the inert substrate, respectively the development of the mature and stable biofilm, presenting the most pronounced inhibitory effect (having MBEC values: 0.001 mg/mL).

MBEC for the Fe3O4@AA nanoparticles.

The data results are in good agreement with the literature, with the most effective modifications for inducing anti-adherent/antibiofilm activity being hydrophilic modifications. The antimicrobial activity of MNPs is due to their various interactions with bacterial cells (production of reactive oxygen species, cell wall damage, protein leakage, membrane depolarisation, DNA destruction, etc.)60,61,62. The pronounced inhibitory effect obtained is due to the small size of the NPs (< 20 nm)63,64 and their shape, resulting in a greater interaction with cells of the studied microbial species. Jin et al.65 determined a pronounced antibacterial activity of amino acid-functionalised MNPs on both Gram-positive and Gram-negative bacteria. Furthermore, Trujillo et al.66 demonstrated a high degree of inhibition of Fe3O4 nanoparticles functionalised with amino acids (arginine and lysine) in B. subtilis and E. coli. Additionally, amino acids are used as antibiofilm agents because of their ability to reduce antibiotic resistance. One such example is cysteine, known for its role in the regulation of metabolism and its synergistic effects with antibiotics67,68. Obtaining an inhibitory effect on the microbial strains in the study determines the potential of these MNPs to be used as antimicrobial and antibiofilm agents. On the basis of the results, it can be stated that by combining these supports, there are premises for the development of extended antimicrobial and antibiofilm activity, premises that will be verified in the next stages of the research.

Interestingly, our studies did not show any effect on unconventional and non-pathogenic budding yeast Saccharomyces cerevisiae. The applied wild-strain and isogenic haploid mutants lacking superoxide dismutase genes, SOD1 or SOD2, also did not show sensitivity to the tested nanoparticles. These studies suggest that the use of the tested materials may be safe for some eukaryotic cells, suggesting that the mechanism of action of these nanoparticles will require additional analysis. In natural environments, microorganisms form structures called biofilms, which adhere to various surfaces. Yeasts are no exception and can create biofilms in diverse environmental niches. Candida albicans is the most well-known yeast species capable of biofilm formation and is therefore extensively studied. Our research indicates that the applied nanoparticles may play a crucial role in biofilm destruction, while having no impact on yeasts that remain as individual cells, such as Saccharomyces cerevisiae. It is worth noting that yeast biofilms can affect food safety and other aspects, including medical infections69. Therefore, further investigations into the mechanisms of biofilm formation and methods of control are essential for a better understanding of this fascinating yeast trait.

We then directed the question whether the analysed magnetite nanoparticles are toxic to human cells in an in vitro system. In this study, we used two distinct human cell lines: HaCaT and A375. HaCaT cells, commonly used as a model to investigate skin biology and differentiation, are a spontaneously transformed aneuploid immortal keratinocyte cell line, originating from adult human skin70. In contrast, A375 is a cell line obtained from the skin of a 54-year-old patient diagnosed with malignant melanoma. This line, which exhibits epithelial morphology, is widely used in research fields such as toxicology and immunooncology, the study of signaling pathways, and for the testing or screening of potential anticancer agents71,72,73.

In this study, we have provided surprising data. The analysed magnetite nanoparticles did not show cytotoxic activity, in contrast to the control. As shown in Fig. 7, the most statistically significant changes occur in the case of using Fe3O4@Ser. In the case of using all concentrations, i.e., 0.1, 0.5, and 1.0 mg/mL, we demonstrated a significant increase in survival/proliferation (p < 0.001). A similar trend was observed for keratinocytes treated with Fe3O4@Cys. On the other hand, no significant changes in cell survival were observed after treating with Fe3O4@Pro. In the case of HaCaT cells treated with Fe3O4@Trp, a significant increase in survival was observed in the case of the lowest concentration (p < 0.05). Importantly, magnetite nanoparticles had no negative impact on keratinocytes in any of the nanoparticles or concentrations applied.

Effect of magnetic nanoparticles Fe3O4@Ser, Fe3O4@Cys, Fe3O4@Pro, Fe3O4@Trp MNPs on the viability of human keratinocytes HaCaT cells estimated by the Neutral Red assay. The cells were treated with MNPs in the concentration of 0.1, 0.5 and 1.0 mg/mL. Nontreated cell were used as a control. Data are expressed as median from at least three independent experiments. Error bars represent 25% and 75% percentiles. Statistical significance was assessed using ANOVA and Dunnett’s post hoc test (*p < 0.05, **p < 0.01, ***p < 0.001).

Similar observations were provided by the analysis of malignant melanoma cells treated with magnetite nanoparticles. As shown in Fig. 8, Fe3O4@Ser had a significant effect on survival/proliferation. In the case of all concentrations analysed, cells showed increased proliferation (p < 0.0001). In turn, treating A375 cells with Fe3O4@Cys, a significant increase in survival was demonstrated only for the lowest of the analyzed concentrations, i.e., 0.1 mg/mL (p < 0.001). Interesting, treating cells with Fe3O4@Trp, Fe3O4@Pro, an increase in survival was also observed, but these data were not statistically significant (Fig. 8).

Effect of magnetic nanoparticles Fe3O4@Ser, Fe3O4@Cys, Fe3O4@Pro, Fe3O4@Trp MNPs on the viability of malignant melanoma A375 cells estimated by the Neutral Red assay. The cells were treated with MNPs in the concentration of 0.1, 0.5 and 1.0 mg/mL. Nontreated cell were used as a control. Data are expressed as median from at least three independent experiments. Error bars represent 25% and 75% percentiles. Statistical significance was assessed using ANOVA and Dunnett’s post hoc test (**p < 0.01, ***p < 0.001).

Although cell division acceleration is an uncommon phenotype, certain bioactive compounds effectively enhance proliferation rates. Recent in vitro studies demonstrated that silver- and gold-doped materials had no cytotoxic effects on osteosarcoma MG-63 cells. Remarkably, even at low concentrations, these materials promoted significant cell proliferation74. Recent research findings indicate that ultrasmall 9 nm Fe3O4 nanoparticles (NPs) were efficiently internalised by breast cancer cells and localised within the cell nucleus. This internalization led to the inhibition of DNA synthesis by inducing S-phase arrest. Furthermore, Fe3O4 NPs triggered the production of reactive oxygen species and caused oxidative damage by disrupting the expression of antioxidant-related genes. Consequently, this disruption resulted in increased cell apoptosis and suppressed cell proliferation, highlighting the potential of Fe3O4 NPs as a therapeutic agent75. Thus, magnetite nanoparticles coated with amino acids neutralise cytotoxic effects, leading to a protective effect. Amino acids play a crucial role in various metabolic processes within human cells. They are involved in protein synthesis, serve as precursors for several important molecules, and contribute to cellular proliferation76. Amino acids play a crucial role as central regulators in tumours, participating in multifaceted bidirectional interactions that encompass signal pathways, the tumour microenvironment, and epigenetic modifications77,78. Therefore, we believe that amino acid-functionalised magnetite nanoparticles can deliver amino acids to cells and significantly alter metabolism in both normal and cancer cells. Therefore, our study strongly highlights the potential of amino acid-functionalised magnetite nanoparticles as targeted drug carriers for cancer therapy.

The use of an amino acid as a component of the precipitation bath during the synthesis of Fe3O4 nanoparticles results in their presence on the surface of the material. The amount and type of amino acid strongly influence the surface properties of the magnetic nanoparticles. The choice of amino acid determines the properties of the obtained material and its future use, e.g., Fe3O4@Cys supports the production of radicals, which may increase the efficiency of catalytic degradation, or can be used together with other components in combined therapy of cancer, infections, etc. Fe3O4@TRP captures radicals, which may be an advantage in several biomedical applications to reduce the hazardous radicals or to use it in association with some components for wellbeing such as probiotics. For the first time, the differences in photocatalytic properties depending on the amino acids attached to the Fe3O4 surface were discussed in detail. Fe3O4@Trp exhibits good antimicrobial activity (MBEC and MIC) against E. coli ATCC 25922, P. aeruginosa ATCC 27853 and C. albicans ATCC 10231 while Fe3O4@Pro exhibits the best results against S. aureus ATCC 25923.

Fe3O4@AA nanoparticles exhibit promising potential for biomedical applications, as they did not show any cytotoxic activity. On the contrary, they significantly enhanced cell proliferation compared to the control group. This observation suggests new possibilities for the utilisation of magnetite nanoparticles in various biomedical fields, especially in scenarios where an increase in cell proliferation is advantageous. All these systems can be further use as drug delivery systems in anti-infectious therapies. However, comprehensive studies are required to elucidate the mechanisms that underpin these effects. This understanding will be instrumental in optimising the application of magnetite nanoparticles for specific purposes.

The authors confirm that the data supporting the findings of this study are available within the article and/or its supplementary materials. The raw data that support the findings of this study are available on request from the corresponding author, [DF].

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The authors acknowledge the financial support of the “Magnetic smart drug delivery systems for theranostic using a personalized approach (SmartACT)” project PN-III-P1-1.1-TE_2021-1342, contract TE 96 din 17/05/2022.

Department of Science and Engineering of Oxide Materials and Nanomaterials, Faculty of Chemical Engineering and Biotechnologies, National University of Science and Technology Politehnica Bucharest, 1-7 Gh Polizu Street, 011061, Bucharest, Romania

Spoială Angela, Motelica Ludmila, Ilie Cornelia-Ioana, Chircov Cristina, Surdu Adrian-Vasile, Trușcă Doina Roxana & Ficai Anton

National Centre for Micro and Nanomaterials and National Centre for Food Safety, Faculty of Chemical Engineering and Biotechnologies, National University of Science and Technology Politehnica Bucharest, Spl. Independentei 313, 060042, Bucharest, Romania

Spoială Angela, Motelica Ludmila, Ilie Cornelia-Ioana, Ficai Denisa, Chircov Cristina, Surdu Adrian-Vasile, Trușcă Doina Roxana, Oprea Ovidiu Cristian & Ficai Anton

Department of Inorganic Chemistry, Physical Chemistry and Electrochemistry, Faculty of Chemical Engineering and Biotechnologies, National University of Science and Technology Politehnica Bucharest, 1-7 Gh Polizu Street, 050054, Bucharest, Romania

Ficai Denisa & Oprea Ovidiu Cristian

Institute of Medical Sciences, University of Rzeszów, Warzywna 1a, 35-310, Rzeszów, Poland

Pieńkowska Natalia & Galiniak Sabina

Institute of Biology, University of Rzeszow, Zelwerowicza 4, 35-601, Rzeszów, Poland

Mołoń Mateusz & Kisala Joanna

Academy of Romanian Scientists, 3 Ilfov Street, 050045, Bucharest, Romania

Motelica Ludmila, Ficai Denisa, Oprea Ovidiu Cristian & Ficai Anton

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Conceptualization: Ficai Denisa, Joanna Kisała, Anton Ficai Methodology: Ludmila Motelica (FTIR analysis), Cornelia Ioana Ilie (Quantitative evaluation of antimicrobial activity), Cristina Chircov (DLS analyses), Adrian Vasile Surdu (XRD analysis, Roxana Doina Trusca (SEM analysis), Natalia Pieńkowska (Cell culture and Fe3O4@AA cytotoxicity assay, Yeast growth condition and spot test assay), Mateusz Mołoń (Cell culture and Fe3O4@AA cytotoxicity assay, Yeast growth condition and spot test assay), Joanna Kisała (photocatalytic tests), Ovidiu Cristian Oprea (thermal analysis TG-DSC) Statistical analysis: Mateusz Mołoń Investigation: Denisa Ficai and Ludmila Motelica (sinthesys of aminoacids based Fe3O4 magnetic nanoparticles)Natalia Pieńkowska (Cell culture and Fe3O4@AA cytotoxicity assay, Yeast growth condition and spot test assay), Sabina Galiniak (Cell culture and Fe3O4@AA cytotoxicity assay, Yeast growth condition and spot test assay), Joanna Kisała (photocatalytic tests) Writing—Original Draft Preparation: Angela Spoiala, Cornelia Ioana Ilie, Ludmila Motelica, Denisa Ficai, Mateusz Mołoń, Sabina Galiniak, Joanna Kisała, Ovidiu Cristian Oprea, Anton Ficai Writing—Review & Editing: Denisa Ficai, Ovidiu Cristian Oprea, Anton Ficai, Joanna Kisała Supervision: Denisa Ficai, Anton Ficai Funding acquisition: Denisa Ficai.

Correspondence to Ficai Denisa.

The authors declare no competing interests.

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Angela, S., Ludmila, M., Cornelia-Ioana, I. et al. Aminoacid functionalised magnetite nanoparticles Fe3O4@AA (AA = Ser, Cys, Pro, Trp) as biocompatible magnetite nanoparticles with potential therapeutic applications. Sci Rep 14, 26228 (2024). https://doi.org/10.1038/s41598-024-76552-1

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Received: 23 April 2024

Accepted: 15 October 2024

Published: 31 October 2024

DOI: https://doi.org/10.1038/s41598-024-76552-1

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