1. Introduction

2. Materials and Methods

3. Results

3.1. Characterization of Nanoparticles

3.1.1. ELS and DLS Results

The zeta potential is a measure of the electrostatic repulsion between particles in a solution. A higher absolute value of zeta potential indicates greater stability. Both the CA-modified and FA-modified iron oxide nanoparticles and the electrohydraulic discharge-processed CA-SPIONs had negative zeta potentials from -19 mV to -31 mV, at neutral pH (6.5). This suggests that these nanoparticles are well-dispersed in solution and less likely to aggregate. The Bare SPIONs had a positive zeta potential of 22 mV, indicating a change in surface charge due to the CA coating or FA conjugation.

Figure 1. Particle size and ζ potential of Bare SPIONs; CA-SPIONs, FA-SPIONs, CA-SPIONs-EHD and FA-SPIONs-EHD (a) Particle size (b) ζ potential.

Figure 1. Particle size and ζ potential of Bare SPIONs; CA-SPIONs, FA-SPIONs, CA-SPIONs-EHD and FA-SPIONs-EHD (a) Particle size (b) ζ potential.

The hydrodynamic diameter refers to the size of a particle in solution, including any surrounding hydration layer. The CA-modified iron oxide nanoparticles and the electrohydraulic discharge-processed CA-SPIONs had smaller hydrodynamic diameters of 119 nm and 92 nm, respectively, compared to FA-modified iron oxide nanoparticles and the electrohydraulic discharge-processed FA-SPIONs had a larger hydrodynamic diameter of 148 nm and 138 nm, respectively. However, all the samples were relatively smaller than Bare-SPIONs, with 243 nm particle diameter, likely due to the stabilization by the CA Molecules on the nanoparticle surface.

3.1.2. XRD Results

The X-ray diffraction (XRD) is a technique used to identify the crystal structure of a material. The XRD patterns of all synthesized samples (CA-SPIONs, FA-SPIONs, CA-SPIONs-EHD and FA-SPIONs-EHD) showed the same characteristic peaks corresponding to the cubic inverse spinel structure of magnetite (Fe₃O₄). This indicates that the modification process with citric acid and the conjugation with folic acid did not alter the fundamental crystal structure of the iron oxide nanoparticles.

Figure 2. Diffraction patterns of the synthesized samples: CA-SPIONs, FA-SPIONs, CA-SPIONs-EHD and FA-SPIONs-EHD.

Figure 2. Diffraction patterns of the synthesized samples: CA-SPIONs, FA-SPIONs, CA-SPIONs-EHD and FA-SPIONs-EHD.

3.1.3. TEM Results

Transmission electron microscopy (TEM) is a technique used to visualize the morphology and size of nanoparticles. The TEM images revealed that the synthesized nanoparticles had a spherical morphology with a narrow size distribution. The CA-modified iron oxide nanoparticles (CA-SPIONs) appeared slightly sparse compared to the FA-modified nanoparticles (FA-SPIONs) which is consistent with the DLS results.

Figure 3. TEM images of (a) CA-modified and (b) FA-modified iron oxide nanoparticles.

Figure 3. TEM images of (a) CA-modified and (b) FA-modified iron oxide nanoparticles.

3.1.4. VSM Results

Vibrating sample magnetometry (VSM) is a technique used to measure the magnetic properties of materials. The magnetization curves of the synthesized magnetic nanofluids showed that both bare and citric-acid-modified iron oxide nanoparticles exhibited superparamagnetic behavior. This means that they are magnetic only when an external magnetic field is applied and lose their magnetism when the field is removed. This property is important for potential applications in targeted drug delivery, as the nanoparticles can be guided to the tumor site using an external magnetic field.

Figure 4. Magnetization curves of magnetic fluids with bare SPIONs and Citric acid coated magnetite nanoparticles were measured on a Vibrating Sample Magnetometer at room temperature under an applied field up to 3 Tesla.

Figure 4. Magnetization curves of magnetic fluids with bare SPIONs and Citric acid coated magnetite nanoparticles were measured on a Vibrating Sample Magnetometer at room temperature under an applied field up to 3 Tesla.

3.1.5. UV-Vis Optical Studies

UV-Vis spectroscopy is a technique used to identify and quantify the presence of specific molecules based on their absorption of light at different wavelengths. The UV-Vis absorption spectra of the DOX-loaded samples showed a characteristic absorption peak at around 480 nm, which is the absorption peak of doxorubicin. The intensity of this peak increased with increasing doxorubicin loading concentration, confirming the successful loading of doxorubicin onto the CA-SPIONs. This is important for the development of a drug delivery system, as it demonstrates that the nanoparticles can effectively encapsulate and carry the anticancer drug.

Figure 5. UV-VIS spectra of magnetic nanoparticles containing Bare SPIONs, citric acid coated, Folic acid conjugated, electro-hydraulically treated and DOX loaded NPs (with different ratio of DOX to magnetite in nanofluid (parity - 1: 1; 1: 5; 1:10).

Figure 5. UV-VIS spectra of magnetic nanoparticles containing Bare SPIONs, citric acid coated, Folic acid conjugated, electro-hydraulically treated and DOX loaded NPs (with different ratio of DOX to magnetite in nanofluid (parity - 1: 1; 1: 5; 1:10).

3.1.6. FTIR Analysis

The FTIR analysis is crucial for confirming the successful synthesis and characterization of the nanoparticles. It provides evidence for the presence of the desired coating, such as citric acid (CA) or conjugation, as folic acid (FA), on the iron oxide nanoparticles. The distinct peaks observed in the spectra of CA-SPIONS and FA-SPIONS, compared to the bare SPIONs, confirm the successful attachment of these coatings. Additionally, the presence of doxorubicin peaks in the DOX-CA-SPIONS spectrum indicates successful drug loading onto the nanoparticles. The FTIR results provide valuable information for optimizing the synthesis process and ensuring the desired properties of the nanoparticles for their intended therapeutic applications.

Figure 6. FTIR spectra of pure citric acid (CA), pure folic acid (FA), bare SPIONs (${Fe}_3{O}_4$); CA-SPIONs, pure DOX, DOX-FA-SPION and DOX-CA-SPION.

Figure 6. FTIR spectra of pure citric acid (CA), pure folic acid (FA), bare SPIONs (${Fe}_3{O}_4$); CA-SPIONs, pure DOX, DOX-FA-SPION and DOX-CA-SPION.

3.2. Results of in vitro experiments

following steps were performed:

• Cultivation of cell lines;
• Obtaining the required concentration for cell research (10,000 cells/mL);
• Making different combinations of nanoparticle-drugs systems (table 1);
• Incubation of cells and nanofluid in an environment as close as possible to the organism (37°C, 5% CO₂);
• Cytotoxicity assessment of samples at 0, 24 and 48 hours using MTT-method.

Incubation of cells is carried out at 0 hours, 24 hours and 48 hours. Then the test substances were added: - S0-uncoated nanoparticles at 1.00 mg/mL, 0.50 mg/mL, and 0.20 mg/mL;

- S1-(SPIONs-CA) – citric acid coated nanoparticles at 1.00 mg/mL, 0.50 mg/mL, and 0.20 mg/mL;

- S2-(SPIONs–HYD-CA) – electrohydraulically treated + citric acid coated nanoparticles at 1.00 mg/mL, 0.50 mg/mL, and 0.20 mg/mL;

- DOX-doxorubicin at 1.84 mM, 0.92 mM, and 0.37 mM;

- S3-(SPIONs-CA-Dox) - nanoparticles coated with citric acid and functionalized with Doxorubicin at 0.50 mg/mL, 0.25 mg/mL, and 0.10 mg/mL (1:1 ratio), 0.83 mg/mL, 0.42 mg/mL, and 0.17 mg/mL (1:5 ratio), and 0.91 mg/mL, 0.45 mg/mL, and 0.18 mg/mL (1:10 ratio);

- S4 - (CA-SPIONs-HYD-Dox) - nanoparticles processed electrohydraulically, coated with citric acid, and loaded with Doxorubicin at 0.50 mg/mL, 0.25 mg/mL, and 0.10 mg/mL (1:1 ratio), 0.83 mg/mL, 0.42 mg/mL, and 0.17 mg/mL (1:5 ratio), and 0.91 mg/mL, 0.45 mg/mL, and 0.18 mg/mL (1:10 ratio).

- SPIONs-FA – Folic acid conjugated nanoparticles at 0.45 mg/mL and 0.18 mg/mL

- SPIONs–HYD-FA - Electrohydraulically treated + Folic acid conjugated nanoparticles at 0.50 mg/mL and 0.20 mg/mL

- SPIONs-FA-Dox - Folic acid conjugated nanoparticles functionalized with Doxorubicin at 0.45 mg/mL and 0.18 mg/mL

- SPIONs-HYD-FA-Dox - Electrohydraulically treated + Folic acid conjugated nanoparticles functionalized with Doxorubicin at 0.45 mg/mL and 0.18 mg/mL

Table 1. column 1 - Designations Corresponding to Different Concentrations (w/v) and column 2 - Concentration (mg/mL).

Table 1. column 1 - Designations Corresponding to Different Concentrations (w/v) and column 2 - Concentration (mg/mL).

Designations (w/v)	Concentrations (mg/mL)
DOX–0.1%	1.84 mM
DOX–0.05%	0.92 mM
DOX–0.02%	0.37 mM
S0–0.1%	1.00 mg/mL
S0–0.05%	0.50 mg/mL
S0–0.02%	0.20 mg/mL
S1–0.1%	1.00 mg/mL
S1–0.05%	0.50 mg/mL
S1–0.02%	0.20 mg/mL
S2–0.1%	1.00 mg/mL
S2–0.05%	0.50 mg/mL
S2–0.02%	0.20 mg/mL
S3–1:1–0.1%	0.50 mg/mL
S3–1:1–0.05%	0.25 mg/mL
S3–1:1–0.02%	0.10 mg/mL
S3–1:5–0.10%	0.83 mg/mL
S3–1:5–0.05%	0.42 mg/mL
S3–1:5–0.02%	0.17 mg/mL
S3–1:10–0.10%	0.91 mg/mL
S3–1:10–0.05%	0.45 mg/mL
S3–1:10–0.02%	0.18 mg/mL
S4–1:1–0.1%	0.50 mg/mL
S4–1:1–0.05%	0.25 mg/mL
S4–1:1–0.02%	0.10 mg/mL
S4–1:5–0.1%	0.83 mg/mL
S4–1:5–0.05%	0.42 mg/mL
S4–1:5–0.02%	0.17 mg/mL
S4–1:10–0.1%	0.91 mg/mL
S4–1:10–0.05%	0.45 mg/mL
S4–1:10–0.02%	0.18 mg/mL

Test substances were added at the specified concentrations, and after incubation, 10μL of MTT solution was added and incubation was carried out for 3-4 hours at 37°C, finally 100μL MTT solvent was added and gentle pipetting was performed. The optical density was measured with an ELISA counter at a wavelength of 570 nm.

3.2.1. Effects of Nanoparticles on RM1 Cell Lines

At the initial stage of the research, the cytotoxic effect of citric acid coated nanoparticles was studied. Based on the obtained data, it can be concluded at a glance that the RM1 cell line is a much more stable line, and the effect of doxorubicin on them is expressed only by a small variation in the indicators of cell survival, although it is worth noting that the efficiency of both citric acid-coated and doxorubicin-loaded nanoparticles is similar to the indicators observed as a result of previous studies.

Figure 7. Effects of nanoparticles on RM1 cell line, (MTT assay - cytotoxicity assessed at 570 nm) where control - represents untreated tumor cells; (a) S0-0.1%– cells treated with 1.00 mg/mL concentrations of superparamagnetic iron oxide nanoparticles; S1-0.1% - cells treated with 1.00 mg/mL concentration of superparamagnetic iron oxide nanoparticles coated with citric acid; and, S2-0.2%- cells treated with 1.00 mg/mL concentration of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid. (b) S0-0.02%– cells treated with 0.20 mg/mL concentrations of superparamagnetic iron oxide nanoparticles; S1-0.02% - cells treated with 0.20 mg/mL concentration of superparamagnetic iron oxide nanoparticles coated with citric acid; and, S2-0.02% - cells treated with 0.20 mg/mL concentration of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid.

Figure 7. Effects of nanoparticles on RM1 cell line, (MTT assay - cytotoxicity assessed at 570 nm) where control - represents untreated tumor cells; (a) S0-0.1%– cells treated with 1.00 mg/mL concentrations of superparamagnetic iron oxide nanoparticles; S1-0.1% - cells treated with 1.00 mg/mL concentration of superparamagnetic iron oxide nanoparticles coated with citric acid; and, S2-0.2%- cells treated with 1.00 mg/mL concentration of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid. (b) S0-0.02%– cells treated with 0.20 mg/mL concentrations of superparamagnetic iron oxide nanoparticles; S1-0.02% - cells treated with 0.20 mg/mL concentration of superparamagnetic iron oxide nanoparticles coated with citric acid; and, S2-0.02% - cells treated with 0.20 mg/mL concentration of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid.

In this case, two different concentrations of nanoparticles were compared. As expected, nanoparticles coated with citric acid and loaded with chemotherapy drug are more effective. This pattern is also well expressed in time and as expected the cytotoxic effect of the samples on RM1 line tumor cells is much higher at the concentration of 1.00 mg/mL

Figure 8. Effects of nanoparticles on RM 1 cell line, (MTT assay - optical density assessed at 570 nm) where control represents untreated tumor cells; (a) DOX-0.02% - cells treated with doxorubicin at 0.37 mM concentrations; S3-1:10-0.02%- cells treated with 0.18 mg/mL concentrations of superparamagnetic iron oxide nanoparticles functionalized with doxorubicin and coated with citric acid; And, S4 -1:10-0.02%- cells treated with 0.18 mg/mL concentrations of electrohydraulic processed superparamagnetic iron oxide nanoparticles functionalized with doxorubicin and coated with citric acid. (b) DOX-0.1%- cells treated with doxorubicin at 1.84 mM, S3-1:1-0.1%- cells treated with 0.50 mg/mL concentrations of superparamagnetic iron oxide nanoparticles functionalized with doxorubicin and coated with citric acid; And, S4-1:1--0.1%- cells treated with 0.50 mg/mL concentrations of electrohydraulic processed superparamagnetic iron oxide nanoparticles functionalized with doxorubicin and coated with citric acid.

Figure 8. Effects of nanoparticles on RM 1 cell line, (MTT assay - optical density assessed at 570 nm) where control represents untreated tumor cells; (a) DOX-0.02% - cells treated with doxorubicin at 0.37 mM concentrations; S3-1:10-0.02%- cells treated with 0.18 mg/mL concentrations of superparamagnetic iron oxide nanoparticles functionalized with doxorubicin and coated with citric acid; And, S4 -1:10-0.02%- cells treated with 0.18 mg/mL concentrations of electrohydraulic processed superparamagnetic iron oxide nanoparticles functionalized with doxorubicin and coated with citric acid. (b) DOX-0.1%- cells treated with doxorubicin at 1.84 mM, S3-1:1-0.1%- cells treated with 0.50 mg/mL concentrations of superparamagnetic iron oxide nanoparticles functionalized with doxorubicin and coated with citric acid; And, S4-1:1--0.1%- cells treated with 0.50 mg/mL concentrations of electrohydraulic processed superparamagnetic iron oxide nanoparticles functionalized with doxorubicin and coated with citric acid.

3.2.2. Effects of Functionalized Nanoparticles on MEC1 Cell Lines

In the first stage of the research, the cytotoxic effect of nanoparticles coated with citric acid was determined. The obtained results emphasize the fact that the nanoparticles coated with citric acid have a certain cytotoxic effect on the MEC 1 cell line, and if they are added in the form where they are also loaded with chemotherapy drug, it is assumed that this nanosystem will significantly increase the effectiveness of the test substance. The effect is also well timed and, in most cases, only fades slightly as time goes on. It is also worth noting the effectiveness of the drug coated with citric acid, which is detected immediately after treatment, but then the initial effect is overcome by tumor cells. The presented graph shows the cytotoxic effect of the nanosystems on tumor cells after the exposure at relatively high 1.00 mg/mL and relatively low 0.20 mg/mL concentrations.

Figure 9. Effects of nanoparticles on MEC 1 cell line, (MTT assay - cytotoxicity assessed at 570nm) where control represents untreated tumor cells; (a) S0-0.02% – cells treated with 0.20 mg/mL concentrations of superparamagnetic iron oxide nanoparticles; S1-0.02% - cells treated with 0.20 mg/mL concentrations of superparamagnetic iron oxide nanoparticles coated with citric acid; And, S2-0.02% - cells treated with 0.20 mg/mL concentrations of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid. (b) S0-0.1%– cells treated with 1.00 mg/mL concentrations of superparamagnetic iron oxide nanoparticles; S1-0.1%- cells treated with the 1.00 mg/mL concentrations of superparamagnetic iron oxide nanoparticles coated with citric acid; And, S2-0.1%- cells treated with - 1.00 mg/mL concentrations of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid.

Figure 9. Effects of nanoparticles on MEC 1 cell line, (MTT assay - cytotoxicity assessed at 570nm) where control represents untreated tumor cells; (a) S0-0.02% – cells treated with 0.20 mg/mL concentrations of superparamagnetic iron oxide nanoparticles; S1-0.02% - cells treated with 0.20 mg/mL concentrations of superparamagnetic iron oxide nanoparticles coated with citric acid; And, S2-0.02% - cells treated with 0.20 mg/mL concentrations of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid. (b) S0-0.1%– cells treated with 1.00 mg/mL concentrations of superparamagnetic iron oxide nanoparticles; S1-0.1%- cells treated with the 1.00 mg/mL concentrations of superparamagnetic iron oxide nanoparticles coated with citric acid; And, S2-0.1%- cells treated with - 1.00 mg/mL concentrations of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid.

As for the cytotoxic effect of nanoparticles loaded with doxorubicin, two different concentrations were compared for conspicuous results. As expected, nanoparticles coated with citric acid are more effective after being loaded with chemotherapeutic drugs. The effect is also well expressed in time, and as expected, the cytotoxic effect of the nanosystems on tumor cells after the exposure is relatively high at the 1.00 mg/mL concentration.

Figure 10. Effects of nanoparticles on MEC 1 cell line, (MTT assay - optical density assessed at 570 nm) where control - represents untreated tumor cells; (a) DOX-0.02% - cells treated with doxorubicin 0.37 mg/mL concentration; S3-1:5-0.02% - cells treated with 0.17 mg/mL concentration of doxorubicin functionalized citric acid coated superparamagnetic iron oxide nanoparticles; And, S4-1:5-0.02% - cells treated with 0.17 mg/mL concentration of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid and loaded with doxorubicin (b) DOX-0.1% - cells treated with doxorubicin 1.84 mg/mL concentration; S3-1:1-0.1% - cells treated with 0.83 mg/mL concentration of doxorubicin functionalized citric acid coated superparamagnetic iron oxide nanoparticles; And, S4-1:1-0.1% - cells treated with 0.83 mg/mL concentration of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid and loaded with doxorubicin .

Figure 10. Effects of nanoparticles on MEC 1 cell line, (MTT assay - optical density assessed at 570 nm) where control - represents untreated tumor cells; (a) DOX-0.02% - cells treated with doxorubicin 0.37 mg/mL concentration; S3-1:5-0.02% - cells treated with 0.17 mg/mL concentration of doxorubicin functionalized citric acid coated superparamagnetic iron oxide nanoparticles; And, S4-1:5-0.02% - cells treated with 0.17 mg/mL concentration of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid and loaded with doxorubicin (b) DOX-0.1% - cells treated with doxorubicin 1.84 mg/mL concentration; S3-1:1-0.1% - cells treated with 0.83 mg/mL concentration of doxorubicin functionalized citric acid coated superparamagnetic iron oxide nanoparticles; And, S4-1:1-0.1% - cells treated with 0.83 mg/mL concentration of electrohydraulic processed superparamagnetic iron oxide nanoparticles coated with citric acid and loaded with doxorubicin .

The interesting data was obtained as a result of the research emphasized the effectiveness of nanoparticles. However, in order to conduct a complete study, it was desirable to evaluate the efficiency of nanosystems obtained in a different way. For this very reason, after confirming the cytotoxic efficiency of nanoparticles coated with citric acid, it became interesting to evaluate the effect of nanoparticles conjugated with folic acid. In particular, the nanosystem were loaded with doxorubicin and the cytotoxic effect on tumor cells was evaluated with the help of the same MTT immunofluorescence method. The cytotoxic effect of nanoparticles surrounded by folic acid and with number of modifications was determined. In this case, it is notable that we have got a slightly different picture.

Figure 11. Effect of nanoparticles on tumor cells of MEC1 line, (MTT analysis - estimated optical density at 570 nm), where Control - represents untreated cells; (a) Bare – cells treated with solutions of 0.50 mg/mL and 0.20 mg/mL concentration of bare superparamagnetic iron oxide nanoparticles; (b) SPIONs-FA- cells treated with 1:10 concentration of 0.45 mg/mL and 0.18 mg/mL solutions of superparamagnetic iron oxide nanoparticles conjugated with folic acid; and, (c) SPIONs-HYD-FA- cells treated with 0.50 mg/mL and 0.20 mg/mL concentration of electrohydraulic processed superparamagnetic iron oxide nanoparticles solutions conjugated with folic acid at 1:10 concentration.

Figure 11. Effect of nanoparticles on tumor cells of MEC1 line, (MTT analysis - estimated optical density at 570 nm), where Control - represents untreated cells; (a) Bare – cells treated with solutions of 0.50 mg/mL and 0.20 mg/mL concentration of bare superparamagnetic iron oxide nanoparticles; (b) SPIONs-FA- cells treated with 1:10 concentration of 0.45 mg/mL and 0.18 mg/mL solutions of superparamagnetic iron oxide nanoparticles conjugated with folic acid; and, (c) SPIONs-HYD-FA- cells treated with 0.50 mg/mL and 0.20 mg/mL concentration of electrohydraulic processed superparamagnetic iron oxide nanoparticles solutions conjugated with folic acid at 1:10 concentration.

In the graphs above, different effects are clearly expressed when nanoparticles are used in different concentrations. What is particularly noteworthy is the almost total disappearance of the antitumor effect of nanoparticles conjugated with folic acid after 48 hours of incubation, a similar effect was not observed in the case of samples coated with citric acid.

Figure 12. Effect of nanoparticles on tumor cells of MEC1 line, (MTT analysis - estimated optical density at 570 nm), where Control - represents untreated cells; SPIONs-FA-DOX - cells treated with 1:10 concentration of 0.45 mg/mL and 0.18 mg/mL solutions of superparamagnetic iron oxide nanoparticles loaded with doxorubicin and conjugated with folic acid; and SPIONs-HYD-FA-DOX- cells treated with 1:10 concentration of 0.45 mg/mL and 0.18 mg/mL solutions of electrohydraulic processed, folic acid conjugated and doxorubicin loaded superparamagnetic iron oxide nanoparticles; DOX - cells treated with 1:10 concentration of 0.45 mg/mL solution of doxorubicin.

Figure 12. Effect of nanoparticles on tumor cells of MEC1 line, (MTT analysis - estimated optical density at 570 nm), where Control - represents untreated cells; SPIONs-FA-DOX - cells treated with 1:10 concentration of 0.45 mg/mL and 0.18 mg/mL solutions of superparamagnetic iron oxide nanoparticles loaded with doxorubicin and conjugated with folic acid; and SPIONs-HYD-FA-DOX- cells treated with 1:10 concentration of 0.45 mg/mL and 0.18 mg/mL solutions of electrohydraulic processed, folic acid conjugated and doxorubicin loaded superparamagnetic iron oxide nanoparticles; DOX - cells treated with 1:10 concentration of 0.45 mg/mL solution of doxorubicin.

It can be clearly seen from the mentioned graphs that the use of the same concentration of doxorubicin that was loaded on the nanoparticles conjugated with folic acid (0.045 mg/mL) does not actually give a cytotoxic effect, both at low and high doses. Which is a remarkable fact. Based on the results of these last studies, it was considered necessary to evaluate the ability of both doxorubicin (because it has the ability to fluoresce) and also the ability of nanoparticles loaded with doxorubicin to penetrate cells using fluorescence microscopy.

3.2.3. Results of Fluorescence Microscopy Performed on MEC1 Cell Culture

In the next stage of the research, it was important to evaluate how efficiently doxorubicin-loaded nanoparticles reached the tumor cells. It was also of interest to see if there was any difference between the nanoparticles coated with citric acid and nanoparticles conjugated with folic acid.

Figure 13. MEC1 cells +DOX 0.05% concentration (0.045 mg/mL) (a) overlay (b) green laser.

Figure 13. MEC1 cells +DOX 0.05% concentration (0.045 mg/mL) (a) overlay (b) green laser.

Figure 14. MEC1 cells +SPIONs-FA-DOX-0.05% (0.045 mg/mL) (a) overlay (b) green laser.

Figure 14. MEC1 cells +SPIONs-FA-DOX-0.05% (0.045 mg/mL) (a) overlay (b) green laser.

Figure 15. MEC1 cells +SPIONs-CA-DOX-0.05% (0.025 mg/mL) (a) overlay (b) green laser.

Figure 15. MEC1 cells +SPIONs-CA-DOX-0.05% (0.025 mg/mL) (a) overlay (b) green laser.

As can be seen from the microscopy photos, when the drug enters the cells and the samples are irradiated with a green laser, doxorubicin fluoresces red, which makes it especially easy to detect the entry of the nanoparticles loaded with the chemotherapy drug into the cells.

4. Discussion

5. Conclusion

References

1. Huang, K.; Yao, J. Beam Theory of Thermal–Electro-Mechanical Coupling for Single-Wall Carbon Nanotubes. Nanomaterials 2021, 11, 923. [Google Scholar] [CrossRef] [PubMed]
2. Lage, T.; Rodrigues, R.O.; Catarino, S.; Gallo, J.; Bañobre-López, M.; Minas, G. Graphene-Based Magnetic Nanoparticles for Theranostics: An Overview for Their Potential in Clinical Application. Nanomaterials 2021, 11, 1073. [Google Scholar] [CrossRef] [PubMed]
3. Ramírez-Morales, M.A.; Goldt, A.E.; Kalachikova, P.M.; Ramirez, B.J.A.; Suzuki, M.; Zhigach, A.N.; Ben Salah, A.; Shurygina, L.I.; Shandakov, S.D.; Zatsepin, T.; et al. lbumin Stabilized Fe@C Core–Shell Nanoparticles as Candidates for Magnetic Hyperthermia Therapy. Nanomaterials 2022, 12, 2869. [Google Scholar] [CrossRef] [PubMed]
4. Kargol, A.; Malkinski, L.; Caruntu, G. Biomedical Applications of Multiferroic Nanoparticles. In Advanced Magnetic Materials; Malkinski, L., Ed.; InTech, 2012 ISBN 978-953-51-0637-1.
5. Rodrigues, R.O.; Sousa, P.C.; Gaspar, J.; Bañobre-López, M.; Lima, R.; Minas, G. Organ-on-a-Chip: A Preclinical Microfluidic Platform for the Progress of Nanomedicine. Small 2020, 16, 2003517. [Google Scholar] [CrossRef] [PubMed]
6. Wang, Y.; Zhao, Q.; Han, N.; Bai, L.; Li, J.; Liu, J.; Che, E.; Hu, L.; Zhang, Q.; Jiang, T.; et al. Mesoporous Silica Nanoparticles in Drug Delivery and Biomedical Applications. Nanomedicine Nanotechnol. Biol. Med. 2015, 11, 313–327. [Google Scholar] [CrossRef] [PubMed]
7. Jose, S.; Sowmya, S.; Cinu, T.A.; Aleykutty, N.A.; Thomas, S.; Souto, E.B. Surface Modified PLGA Nanoparticles for Brain Targeting of Bacoside-A. Eur. J. Pharm. Sci. 2014, 63, 29–35. [Google Scholar] [CrossRef] [PubMed]
8. Chaparro, C.I.P.; Simões, B.T.; Borges, J.P.; Castanho, M.A.R.B.; Soares, P.I.P.; Neves, V. A Promising Approach: Magnetic Nanosystems for Alzheimer’s Disease Theranostics. Pharmaceutics 2023, 15, 2316. [Google Scholar] [CrossRef] [PubMed]
9. Poudel, P.; Park, S. Recent Advances in the Treatment of Alzheimer’s Disease Using Nanoparticle-Based Drug Delivery Systems. Pharmaceutics 2022, 14, 835. [Google Scholar] [CrossRef] [PubMed]
10. Obaidat, I.M.; Narayanaswamy, V.; Alaabed, S.; Sambasivam, S.; Muralee Gopi, C.V.V. Principles of Magnetic Hyperthermia: A Focus on Using Multifunctional Hybrid Magnetic Nanoparticles. Magnetochemistry 2019, 5, 67. [Google Scholar] [CrossRef]
11. Ioannou, P.; Baliou, S.; Samonis, G. Nanotechnology in the Diagnosis and Treatment of Antibiotic-Resistant Infections. Antibiotics 2024, 13, 121. [Google Scholar] [CrossRef] [PubMed]
12. Palzer, J.; Eckstein, L.; Slabu, I.; Reisen, O.; Neumann, U.P.; Roeth, A.A. Iron Oxide Nanoparticle-Based Hyperthermia as a Treatment Option in Various Gastrointestinal Malignancies. Nanomaterials 2021, 11, 3013. [Google Scholar] [CrossRef] [PubMed]
13. Mu, X.; Li, J.; Yan, S.; Zhang, H.; Zhang, W.; Zhang, F.; Jiang, J. siRNA Delivery with Stem Cell Membrane-Coated Magnetic Nanoparticles for Imaging-Guided Photothermal Therapy and Gene Therapy. ACS Biomater. Sci. Eng. 2018, 4, 3895–3905. [Google Scholar] [CrossRef] [PubMed]
14. Chowdhury, P.; Roberts, A.M.; Khan, S.; Hafeez, B.B.; Chauhan, S.C.; Jaggi, M.; Yallapu, M.M. Magnetic Nanoformulations for Prostate Cancer. Drug Discov. Today 2017, 22, 1233–1241. [Google Scholar] [CrossRef] [PubMed]
15. Bao, Y.; Sherwood, J.A.; Sun, Z. Magnetic Iron Oxide Nanoparticles as T 1 Contrast Agents for Magnetic Resonance Imaging. J. Mater. Chem. C 2018, 6, 1280–1290. [Google Scholar] [CrossRef]
16. Hillmen, P.; Pitchford, A.; Bloor, A.; Broom, A.; Young, M.; Kennedy, B.; Walewska, R.; Furtado, M.; Preston, G.; Neilson, J.R.; et al. Ibrutinib and Rituximab versus Fludarabine, Cyclophosphamide, and Rituximab for Patients with Previously Untreated Chronic Lymphocytic Leukaemia (FLAIR): Interim Analysis of a Multicentre, Open-Label, Randomised, Phase 3 Trial. Lancet Oncol. 2023, 24, 535–552. [Google Scholar] [CrossRef] [PubMed]
17. Russell, E.; Dunne, V.; Russell, B.; Mohamud, H.; Ghita, M.; McMahon, S.J.; Butterworth, K.T.; Schettino, G.; McGarry, C.K.; Prise, K.M. Impact of Superparamagnetic Iron Oxide Nanoparticles on in Vitro and in Vivo Radiosensitisation of Cancer Cells. Radiat. Oncol. 2021, 16, 104. [Google Scholar] [CrossRef] [PubMed]
18. Markhulia, J.; Kekutia, S.; Mikelashvili, V.; Saneblidze, L.; Tsertsvadze, T.; Maisuradze, N.; Leladze, N.; Czigány, Z.; Almásy, L. Synthesis, Characterization, and In Vitro Cytotoxicity Evaluation of Doxorubicin-Loaded Magnetite Nanoparticles on Triple-Negative Breast Cancer Cell Lines. Pharmaceutics 2023, 15, 1758. [Google Scholar] [CrossRef] [PubMed]
19. Stacchini, A.; Aragno, M.; Vallario, A.; Alfarano, A.; Circosta, P.; Gottardi, D.; Faldella, A.; Rege-Cambrin, G.; Thunberg, U.; Nilsson, K.; et al. MEC1 and MEC2: Two New Cell Lines Derived from B-Chronic Lymphocytic Leukaemia in Prolymphocytoid Transformation. Leuk. Res. 1999, 23, 127–136. [Google Scholar] [CrossRef] [PubMed]