Biocompatibility of Fe3O4@Au composite magnetic nanoparticles in vitro and in vivo

Figures & data

Table 1 RGR and toxicity grade conversion

Toxicity grade	RGR
Grade 0	≥1
Grade 1	0.75–0.99
Grade 2	0.50–0.74
Grade 3	0.25–0.49
Grade 4	0.01–0.24
Grade 5	0

Abbreviation: RGR, relative growth rate.

Figure 1 Characterization of Fe₃O₄@Au composite MNPs. (A) Transmission electron microscopy image; (B) scanning electron microscopy image and energy dispersive spectrometry (inset); (C) the average diameter of Fe₃O₄@Au composite MNPs and Fe₃O₄-MNPs (inset); (D) the zeta potential of Fe₃O₄@Au composite MNPs and Fe₃O₄-MNPs (inset); (E) ultraviolet-visible absorption spectra of Fe₃O₄@Au composite MNPs (curve b) and Fe₃O₄-MNPs (curve a); and (F) hysteresis loops of Fe₃O₄@Au composite MNPs (curve b) and Fe₃O₄-MNPs (curve a).

Abbreviation: MNPs, magnetic nanoparticles.

Figure 2 Morphology analysis of L929 cells incubated at different concentrations of Fe₃O₄@Au composite magnetic nanoparticles leaching liquor. (A) Negative control; (B) 25% leaching liquor; (C) 50% leaching liquor; (D) 75% leaching liquor; (E) 100% leaching liquor; and (F) positive control.

Note: Inverted microscopy, ×100 magnification.

Figure 3 The MTT results of Fe₃O₄@Au composite magnetic nanoparticles.

Notes: n = 8, mean ± standard deviation. The RGR of L929 cells incubated with 25%, 50%, 75%, and 100% leaching liquor were 0.977, 0.872, 0.849, and 0.826, respectively, which all classified as toxicity grade 1.

Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OD, optical density; RGR, relative growth rate.

Table 2 Hemolysis test of Fe₃O₄@Au composite magnetic nanoparticles

Groups	OD	HR (%)
Negative control	0.0143 ± 0.002	–
100 mg/mL Fe3O4@Au	0.0167 ± 0.003	0.278
75 mg/mL Fe3O4@Au	0.0163 ± 0.001	0.232
50 mg/mL Fe3O4@Au	0.0160 ± 0.004	0.197
Positive control	0.8762 ± 0.012	–

Note: n = 3, mean ± standard deviation.

Abbreviations: OD, optical density; HR, hemolysis rate.

Figure 4 The results of MN test of Fe₃O₄@Au composite magnetic nanoparticles.

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

Abbreviations: MN, micronucleus; PEC, polychromatic erythrocytes.

Table 3 The results of acute toxicity testing of Fe₃O₄@Au composite MNPs

Group	Dosage		n	Death number	Death rate (p)	Survival rate (q)	p × q
	g · kg^−1	Logarithm
1	1.77	0.249	10	0	0	1.0	0
2	2.51	0.399	10	0	0	1.0	0
3	3.54	0.549	10	2	0.2	0.8	0.16
4	5.00	0.699	10	2	0.2	0.8	0.16
5	7.06	0.849	10	4	0.4	0.6	0.24
6	9.98	0.999	10	5	0.5	0.5	0.25
7	14.09	1.149	10	7	0.7	0.3	0.21
8	19.89	1.299	10	10	1.0	0	0
		i = 0.15			∑p = 3.0

Notes: lg LD₅₀ = X_k – I(∑p – 0.5) = 1.299 – 0.15 (3.0 – 0.5) = 0.924; s_m = i × (∑pq/n)^1/2 = 0.15 × 0.36 = 0.054; lg LD₅₀ and its 95% CI were 0.924 ± 1.96 × 0.054 = 0.924 ± 0.106; LD₅₀ and its 95% CI were 8.39 g/kg (6.58–10.72 g/kg).

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

Figure 5 Effect of Fe₃O₄@Au composite MNPs administered via liver injection on body weight (kg) in beagle dogs.

Notes: n = 6, mean ± standard deviation. There was no significant difference in body weight values of beagle dogs between the experimental group and the control group at the five time points of before administration and 1, 2, 3, and 4 weeks after administration of Fe₃O₄@Au composite MNPs (P > 0.05).

Abbreviation: MNPs, magnetic nanoparticles.

Figure 6 Liver function of beagle dogs in 4-week toxicity study of Fe₃O₄@Au composite MNPs.

Notes: n = 6, mean ± standard deviation. ALT and AST of the experimental group administered with Fe₃O₄@Au composite MNPs showed no significant difference compared with the control group (P > 0.05).

Abbreviations: MNPs, magnetic nanoparticles; ALT, alanine aminotransferase; AST, aspartic acid aminotransferase.

Figure 7 Renal function of beagle dogs in 4-week toxicity study of Fe₃O₄@Au composite MNPs.

Notes: n = 6, mean ± standard deviation. BUN and Cr of the experimental group administered with Fe₃O₄@Au composite MNPs showed no significant difference compared with the control group (P > 0.05).

Abbreviations: MNPs, magnetic nanoparticles; BUN, blood urea nitrogen; Cr, creatinine.

Figure 8 Effect of Fe₃O₄@Au composite magnetic nanoparticles administered via liver injection on organ body weight indices of beagle dogs after 4 weeks.

Notes: n = 6, mean ± standard deviation. There was no significant difference in organ body weight indices including heart, liver, spleen, lung, kidney, and brain between the experimental group and the control group (P > 0.05).

Figure 9 Effect of Fe₃O₄@Au composite MNPs administered via liver injection on the tissue of beagle dogs after 4 weeks (hematoxylin-eosin staining, ×100 magnification). (A1) Little impact on the liver of the control group; (A2) Fe₃O₄@Au composite MNPs were taken up by cells around the needle passage in the liver of the experimental group, indicated by arrows; (B–F) no significant pathological changes were detected in the tissues including heart (B1 and B2), spleen (C1 and C2), lung (D1 and D2), kidney (E1 and E2), and brain (F1 and F2).

Abbreviation: MNPs, magnetic nanoparticles.

Table S1 Hematological values of beagle dogs in 4-week toxicity study of Fe₃O₄@Au composite MNPs

Group/parameter (units)	WBC (×10^9/L)	RBC (×10^12/L)	Hb (mg/mL)	PLT (×10^9/L)	Neu (×10^9/L)	Lymp (×10^9/L)
Before administration
Control	11.69 ± 1.80	7.17 ± 0.36	160.17 ± 14.73	298.83 ± 69.34	2.15 ± 1.47	9.00 ± 1.50
Experimental	9.65 ± 2.23	7.23 ± 0.27	168.00 ± 10.24	355.00 ± 103.54	2.35 ± 1.52	7.04 ± 0.83
1 week after administration
Control	11.45 ± 1.87	7.48 ± 0.44	171.00 ± 19.31	274.50 ± 43.08	2.50 ± 1.53	8.67 ± 0.70
Experimental	11.61 ± 3.56	7.18 ± 0.32	167.67 ± 13.85	307.17 ± 88.05	2.38 ± 0.82	9.31 ± 3.11
2 weeks after administration
Control	13.46 ± 2.04	7.52 ± 0.65	170.00 ± 21.82	240.67 ± 27.96	2.76 ± 2.03	10.06 ± 3.07
Experimental	12.01 ± 2.50	7.52 ± 0.45	176.67 ± 12.50	340.83 ± 113.71	2.78 ± 1.66	8.66 ± 2.28
3 weeks after administration
Control	10.88 ± 3.03	7.09 ± 0.86	161.33 ± 28.24	274.00 ± 53.23	2.61 ± 1.19	7.82 ± 2.14
Experimental	14.20 ± 3.13	7.30 ± 0.43	171.67 ± 11.91	291.83 ± 60.71	2.81 ± 1.48	10.99 ± 3.09
4 weeks after administration
Control	12.76 ± 1.79	7.15 ± 0.38	156.83 ± 19.95	261.67 ± 38.07	3.35 ± 1.55	8.57 ± 0.59
Experimental	11.49 ± 2.04	7.11 ± 0.50	165.17 ± 10.87	322.17 ± 77.87	2.88 ± 1.09	7.95 ± 1.37

Notes: n = 6, mean ± standard deviation. There was no significant difference in hematological values of beagle dogs between the experimental group and the control group at the five time points of before administration and 1, 2, 3, and 4 weeks after administration of Fe₃O₄@Au composite MNPs (P > 0.05).

Abbreviations: MNPs, magnetic nanoparticles; WBC, white blood cells; RBC, red blood cells; Hb, hemoglobin concentration; PLT, platelets; Neu, neutrophils; Lymp, lymphocytes.