Preparation and Characterization of Fe₃O₄@MTX Magnetic Nanoparticles for Thermochemotherapy of Primary Central Nervous System Lymphoma in vitro and in vivo

Figures & data

Figure 1 Pattern diagram of Fe₃O₄@MTX MNPs synthesis process.

Notes：OA was chemisorbed on the surface of the Fe₃O₄ MNPs, MTX dispersed into the OA shell surrounding Fe₃O₄ MNPs, and the Pluronic F-127 that anchored at the OA-water interface conferred aqueous dispersity to the system.

Abbreviations: MNPs, magnetic nanoparticles; OA, oleic acid.

Figure 2 Characterization of Fe₃O₄@MTX composite magnetic nano drug-loading system. (A) Transmission electron microscopy image of Fe₃O₄ MNPs carrier; (B) Hydrodynamic diameter and particle size distribution of Fe₃O₄@MTX MNPs obtained by nanoparticle tracking analysis; (C) The hysteresis loops of Fe₃O₄ MNPs and Fe₃O₄@MTX MNPs (insert); (D) Zeta potential of Fe₃O₄ MNPs, OA-Fe₃O₄ MNPs, Pluronic-F127/OA Fe₃O₄ MNPs and Fe₃O₄@MTX MNPs.

Abbreviations: MNPs, magnetic nanoparticles; OA, oleic acid.

Table 1 Encapsulation Efficiency and Drug Loading Efficiency of MTX

Dosing Ratio (%W/W)	Loading Efficiency (%)	Encapsulation Efficiency (%)
5	2.15±0.1	43.9
10	5.55±0.17	58.7
15	8.37±0.15	60.9
20	9.32±0.21	51.2

Notes: n = 3, mean ± standard deviation.

Figure 3 Encapsulation efficiency and drug loading efficiency of MTX detected by UV-VIS-NIR spectrophotometer.

Note: When drug loading efficiency was 15%, drug encapsulation efficiency reached the maximum value of 60.9%.

Figure 4 Drug release curve of Fe₃O₄@MTX MNPs at different temperatures.

Note: The drug release occurred in two phases: an initial burst release within 12 hrs and a sustained release after that.

Abbreviation: MNPs, magnetic nanoparticles.

Figure 5 Heating capacity of MNPs at alternating magnetic field (A) Heating curve of Fe₃O₄ MNPs (0.5, 1.0, 1.5mg Fe/mL); (B) Heating curve of Fe₃O₄@MTX MNPs compared with Fe₃O₄ MNPs and ddH₂O (1.0mg Fe/mL).

Abbreviation: MNPs, magnetic nanoparticles.

Figure 6 Biocompatibility of Fe₃O₄ MNPs carrier (A) Inverted microscopy images of 3T3 cells incubated with Fe₃O₄ MNPs and Fe₃O₄@MTX MNPs; (B) CCK-8 assay of Fe₃O₄ MNPs and Fe₃O₄@MTX MNPs.

Abbreviations: MNPs, magnetic nanoparticles; CCK-8, cell counting kit-8.

Figure 7 Genotoxicity of Fe₃O₄ MNPs carrier.

Notes: n = 10. ^aP > 0.05, MN formation rates of Fe₃O₄ MNPs groups compared with negative control; ^bP < 0.05, MN formation rates between Fe₃O₄ MNPs groups and positive control.

Abbreviations: MN, micronucleus; PEC, polychromatic erythrocytes, MNPs, magnetic nanoparticles.

Table 2 Acute Toxicity Test for Detecting LD₅₀ of Fe₃O₄@MTX MNPs

Group	Dosage	n	Death Number
1	1.77	10	0
2	2.51	10	0
3	3.54	10	2
4	5.00	10	3
5	7.06	10	3
6	9.98	10	4
7	14.09	10	8
8	19.89	10	10

Notes: LD₅₀ of Fe₃O₄@MTX MNPs was 8.579 g/kg (95% CI: 6.300–12.333 g/kg).

Abbreviations: LD₅₀, median lethal dose; CI, confidence interval; MNPs, magnetic nanoparticles.

Figure 8 The labeling effect of Fe₃O₄@MTX MNPs on OCI-LY18 cells by Perls.

Note: Fe₃O₄@MTX MNPs could get into OCI-LY18 cells, 10×100 magnification.

Abbreviation: MNPs, magnetic nanoparticles.

Figure 9 Apoptosis rate of OCI-LY18 cells determined by flowcytometry.

Notes: Apoptosis rate: control group (7.21±3.68%), Fe₃O₄ MNPs group (13.87±1.36%), MTX group (31.35±3.02%), Fe₃O₄@MTX MNPs group (28.51±0.99%), Fe₃O₄ MNPs with hyperthermia group (17.22±2.71%), Fe₃O₄@MTX MNPs with hyperthermia group (52.00±5.50%). P<0.01, Fe₃O₄+H vs Control and FE₃O₄ groups; P<0.01, Fe₃O₄@MTX+H vs MTX and Fe₃O₄@MTX groups.

Abbreviation: MNPs, magnetic nanoparticles.

Figure 10 qPCR and Western blot analyses of OCI-LY18 cells after different treatments. (A) Transcription of Caspase-3, Bax and Bcl-2 genes were detected by qPCR; (B) Expression of Caspase-3, Bax and Bcl-2 proteins were detected by Western blot.

Notes: * P<0.05; ** P<0.01.

Figure 11 MRI detection of targeting and treatment effect. (A) Low signal region showed that MNPs could concentrated at tumor site after the application of a magnet; (B) MRI images after different treatment effects; (C) Volume of tumors in various treatment groups.

Notes: Low signal region indicated by the arrow in Figure 11A shows MNPs could target tumor tissues under the guidance of external magnetic field; High signal regions indicated by the arrows in Figure 11B show tumors after various treatments; * P<0.05.

Abbreviation: MNPs, magnetic nanoparticles.

Figure 12 Histopathologic changes in various treatment groups.

Notes: Hematoxylin & eosin staining, 10×100 magnification. The arrows illustrate tumor cells disappear at these areas.

Figure 13 qPCR and Western blot analyses of tumor tissues after different treatments. (A) Transcription of Caspase-3, Bax and Bcl-2 genes were detected by qPCR; (B) Expression of Caspase-3, Bax and Bcl-2 proteins were detected by Western blot; (C) Semi-quantitative results of Caspase-3, Bax and Bcl-2 proteins.

Notes: * P<0.05; ** P<0.01.