Biosafety evaluation of Janus Fe₃O₄-TiO₂ nanoparticles in Sprague Dawley rats after intravenous injection

Figures & data

Figure 1 Images of Fe₃O₄-TiO₂ NPs and TiO₂ NPs captured by TEM and HRTEM.

Notes: (A) TEM of Fe₃O₄-TiO₂ NPs; (B) HRTEM of Fe₃O₄-TiO₂ NPs; (C) TEM of TiO₂ NPs.

Abbreviations: HRTEM, high-resolution TEM; NPs, nanoparticles; TEM, transmission electron microscopy.

Figure 2 Fe₃O₄-TiO₂ NPs captured by X-ray diffraction.

Abbreviation: NPs, nanoparticles.

Table 1 Organ weight:body weight coefficients of rats after 30 days’ exposure to Fe₃O₄-TiO₂ NPs or TiO₂ NPs through intravenous injection

Parameters	Control (mg/kg)	TiO2 NPs (mg/kg)			Fe3O4-TiO2 NPs (mg/kg)
		5	15	30	5	15	30
Heart	3.28±0.35	3.29±0.21	3.37±0.33	3.45±0.30	3.32±0.16	3.58±0.38	3.23±0.25
Liver	31.80±1.45	33.30±1.48	32.10±2.98	34.40±2.46	34.70±1.73	35.50±1.55*	33.60±1.67
Spleen	1.95±0.25	2.31±0.42	2.13±0.37	2.17±0.11	2.20±0.57	2.79±0.88*	2.12±0.16
Lung	3.79±0.40	3.42±0.42	3.69±0.48	3.37±0.16	3.35±0.47	3.80±0.62	3.20±0.28
Kidney	6.45±0.28	6.81±0.67	6.86±0.74	6.63±0.35	6.38±0.41	6.69±0.31	6.13±0.51
Brain	4.23±0.61	4.23±0.66	4.60±0.80	4.78±0.43	4.46±0.50	4.94±0.33	4.59±0.15
Testis	6.13±1.02	7.12±0.48	6.85±0.58	6.99±0.50	6.49±0.40	6.91±0.79	6.85±0.36

Note: Results given as means ± SD (*P<0.05 compared with control).

Abbreviation: NPs, nanoparticles.

Table 2 Hematological analysis after different treatments

Parameters	Control	TiO2 NPs (mg/kg)			Fe3O4-TiO2 NPs (mg/kg)
		5	15	30	5	15	30
WBCs (×10^9/L)	10.72±2.56	7.38±1.35^a	8.76±1.72	8.24±1.76	8.78±1.29	8.79±2.21	11.66±2.28
Neutrophils (%)	14.86±7.15	17.07±7.20	19.42±9.34	20.10±7.47	23.23±4.82	16.96±5.83	22.36±6.58
Lymphocytes (%)	83.56±2.83	79.53±8.50	84.40±3.78	81.36±6.27	76.45±5.19	80.17±3.05	77.84±5.82
Monocytes (%)	2.22±1.60	1.97±0.97	3.38±1.58	2.88±0.64	1.68±2.20	3.31±1.73	2.90±2.68
Eosinophils (%)	0.88±0.26	0.53±0.26	0.94±0.29	2.25±3.87	0.87±0.51	0.79±0.32	0.92±0.37
Basophils (%)	0.10±0.07	0.12±0.15	0.04±0.05	0.08±0.04	0.05±0.05	0.09±0.04	0.06±0.05
Neutrophils (%)	1.53±0.90	1.26±0.56	1.68±0.74	1.59±0.47	2.08±0.61	1.43±0.46	2.66±1.01
Lymphocytes (%)	8.36±0.79	5.79±1.42^a	7.30±1.41	6.61±1.51	6.49±0.74	7.02±1.94	8.84±1.52
Monocytes (%)	0.22±0.16	0.16±0.11	0.29±0.13	0.24±0.10	0.14±0.19	0.32±0.20	0.35±0.37
Eosinophils (%)	0.09±0.03	0.03±0.02	0.08±0.03	0.21±0.36	0.08±0.05	0.07±0.04	0.10±0.03
RBCs (×10^12/L)	7.51±0.14	7.18±0.57	7.46±0.44	7.45±0.24	7.26±0.90	7.11±0.67	7.01±0.68
Hb (g/L)	141.80±5.12	138.83±10.07	138.04±5.60	140.80±7.40	138.17±14.12	132.57±11.00	133.20±15.47
Hematocrit	0.44±0.01	0.43±0.03	0.43±0.02	0.44±0.02	0.43±0.03	0.42±0.03	0.42±0.04
MCV (fL)	58.64±1.68	59.70±1.73	57.92±2.00	58.50±1.20	59.78±3.76	58.39±2.62	60.22±1.68
MCH (pg)	18.86±0.71	19.38±0.56	18.56±0.73	18.92±0.63	19.12±0.62	18.66±0.60	18.94±0.59
MCHC (g/L)	321.60±4.88	324.83±3.97	320.80±4.44	323.00±4.30	319.83±9.95	319.57±5.71	314.80±11.43
RDW-CV (%)	13.26±0.63	13.37±0.45	13.34±0.44	13.16±0.34	14.32±3.38	14.00±1.04	14.98±2.93
Platelet count (×10^9/L)	751.20±239.82	630.67±159.65	736.00±104.9	545.60±261.68	586.00±189.9	727.57±84.60	756.60±74.29
PDW-CV (%)	14.94±0.05	14.90±0.09	14.88±0.08	14.96±0.13	14.95±0.08	14.81±0.11	14.88±0.18
MPV (fL)	6.88±0.08	6.83±0.08	6.86±0.18	6.86±0.25	6.78±0.12	6.73±0.11	6.76±0.25
Plateletcrit (%)	0.57±0.18	0.48±0.13	0.56±0.17	0.42±0.20	0.44±0.14	0.54±0.06	0.57±0.06

Notes: Results given as means ± SD (^aP<0.05 compared with control).

Abbreviations: Hb, hemoglobin; MCH, mean corpuscular Hb; MCHC, MCH concentration; MCV, mean corpuscular volume; MPV, mean platelet volume; NPs, nanoparticles; PDW-CV, platelet-distribution width coefficient of variation; RBCs, red BCs; RDW-CV, RBC-distribution-width coefficient of variation; WBCs, white blood cells.

Table 3 Biochemical analysis of liver, renal, and lipid profiles

Parameters	Control	TiO2 NPs (mg/kg)			Fe3O4-TiO2 NPs (mg/kg)
		5	15	30	5	15	30
Liver profile
TBil (µmol/L)	1.37±0.46	1.28±0.63	0.90±0.37	1.82±0.91	1.18±0.61	1.96±0.74	1.64±1.03
TP (g/L)	51.68±1.50	51.47±0.68	51.23±2.2	52.20±1.35	51.32±2.80	50.41±1.8	49.40±1.40
Alb (g/L)	26.85±1.04	27.03±0.19	26.25±1.43	27.52±0.79	26.68±1.34	25.41±1.22	25.7±1.22
Glob (g/L)	24.83±0.74	24.43±0.58	24.98±1.04	24.68±1.04	24.63±1.58	25.00±2.69	23.70±0.60
ALT (U/L)	50.83±5.78	47.00±5.40	46.33±5.05	36.40±2.19^b	42.00±4.52^a	44.86±5.08	37.6±6.11^b
AST (U/L)	134.00±29.40	99.00±15.92^b	110.17±9.58	83.00±11.38^b	94.00±11.28^b	95.00±14.10^b	91.20±6.34^b
AST/ALT	2.62±0.42	2.11±0.23^a	2.40±0.28	2.29±0.37	2.24±0.09	2.12±0.27^a	2.47±0.37
ALP (U/L)	289.83±28.30	297.67±70.11	247.83±42.40	274.80±61.65	273.33±52.34	318.00±84.06	224.80±30.03
Renal profile
Urea nitrogen (mmol/L)	7.15±0.36	6.49±0.74	5.71±0.45^b	6.03±0.61^b	6.62±0.31	6.18±0.57^a	6.31±0.57
Creatinine (µmol/L)	35.56±7.00	28.27±3.82^a	28.65±5.47	28.28±3.10	29.85±5.12	29.69±3.15	30.80±3.02
UrcA (µmmol/L)	166.17±64.13	115.33±12.29	124.00±19.12	119.60±13.92	105.67±32.44^b	99.57±14.37^b	98.80±22.63^b
Lipid profile
Chol (mmol/L)	1.35±0.16	1.50±0.06	1.33±0.20	1.39±0.16	1.33±0.11	1.32±0.16	1.59±0.31
TG (mmol/L)	0.90±0.29	0.77±0.27	0.80±0.14	0.78±0.28	1.08±0.28	0.94±0.24	1.06±0.29
HDL-C (mmol/L)	0.47±0.05	0.49±0.03	0.44±0.08	0.47±0.07	0.45±0.03	0.48±0.08	0.51±0.13
LDL-C (mmol/L)	0.43±0.04	0.52±0.05^a	0.46±0.08	0.46±0.05	0.45±0.04	0.46±0.05	0.57±0.10^b
Glucose (mmol/L)	12.87±2.71	10.08±1.70^a	11.10±0.62	10.85±1.05	11.58±1.19	11.34±1.38	11.34±1.33
Cardiac profile
CK (U/L)	3,589.80±	1,881.20±	3,019.30±	2,096.00±	2,588.00±	2,252.70±	2,145.00±
	1,758.30	897.60^a	328.05	617.80	1,100.70	873.01	377.16
CK-MB (U/L)	645.83±149.36	499.00±82.98	526.17±72.96	410.20±66.94^b	410.67±96.97^b	397.71±99.94^b	419.60±92.29^b
Amy (U)	2,457.67± 162.98	2,422.33± 112.64	2,333.17± 237.82	2,427.60± 255.11	2,384.33± 150.82	2,353.14± 145.86	2,443.80± 76.40
GPDA (U/L)	74.33±11.84	74.67±8.24	73.33±14.56	78.20±11.08	77.83±12.70	78.00±14.21	67.40±17.39
Electrolyte profile
K (mmol/L)	9.41±0.54	8.63±0.46	8.55±0.55	7.89±0.36	8.87±2.36	7.66±0.53^a	8.48±1.33
Cl (mmol/L)	98.52±0.84	100.90±0.63	99.95±1.18	100.58±1.11	100.97±1.24	100.70±3.14	101.42±2.12^a
Ca (mmol/L)	2.46±0.08	2.52±0.01	2.51±0.07	2.54±0.08	2.53±0.09	2.49±0.06	2.49±0.09
Mg (mmol/L)	0.90±0.08	0.83±0.04	0.87±0.05	0.86±0.05	0.87±0.05	0.86±0.07	0.84±0.05
P (mmol/L)	2.51±0.18	2.16±0.14^a	2.43±0.22	2.37±0.22	2.07±0.20^b	2.23±0.24	2.09±0.29^a

Notes: Results given as means ± SD (^aP<0.05, ^bP<0.01 compared with control).

Abbreviations: Alb, albumin; ALP, alkaline phosphatase; ALT, alanine aminotransferase; Amy, amylase; AST, aspartate transaminase; Chol, total cholesterol; CK, creatine kinase; CK-MB, creatinine kinase, Glob, globulin; GPDA, glycylproline dipeptidyl aminopeptidase; HDL-C, high-density-lipoprotein cholesterol; LDL-C, low-density-lipoprotein cholesterol; MB isoenzyme; TBil, total bilirubin; TG, triglycerides; TP, total protein; UrcA, uric acid.

Figure 3 Ti content in tissue of rats after treatment with Fe₃O₄-TiO₂ NPs and TiO₂ NPs.

Note: **P<0.01, ***P<0.001 compared with control.

Abbreviations: NPs, nanoparticles; Con, control.

Figure 4 Fe content in tissue of rats after treatment with Fe₃O₄-TiO₂ NPs or TiO₂ NPs.

Abbreviations: NPs, nanoparticles; Con, control.

Figure 5 Photomicrography of liver histopathology.

Notes: Images (magnification 10×) represent histopathological sections of the liver. Arrows represent small focal-like inflammatory cell infiltration around the hepatic portal (HP) area in the 5 mg/kg TiO₂ NP group and around the hepatic vein (HV) in the 15 mg/kg TiO₂ NP group, and lymphocyte aggregation in the 30 mg/kg TiO₂ NP group. Dark arrows represent liver hepatocyte (H) granule degeneration and small stove liver-cell shrinkage in both Fe₃O₄-TiO₂ NP and TiO₂ NP groups at 30 mg/kg.

Abbreviations: NPs, nanoparticles; Con, control; S, sinus; K, Kupffer cells.

Figure 6 Photomicrography of lung histopathology.

Notes: Images (magnification 10×) represent histopathological sections of the lung. Dark arrow in the 15 mg/kg TiO₂ NP group represents edema and exudation around bronchi and a large amount of inflammatory cell infiltration. Dark arrows in the 30 mg/kg TiO₂ NP group represent a large number of inflammatory infiltration around bronchi.

Abbreviations: NP, nanoparticle; Con, control; A, alveolar space; B, bronchioles; H, histiocytes; AS, alveolar septa; V, veins.

Figure 7 Photomicrography of spleen histopathology.

Note: Images (magnification 10×) represent histopathological sections of the spleen.

Abbreviations: NPs, nanoparticles; Con, control; RP, red pulp; WP, white pulp; T, trabeculae; CA, central artery; GC, germinal center.

Figure 8 Photomicrography of kidney histopathology.

Notes: Images (magnification 40×) represent histopathological sections of kidneys. No significant pathological changes were observed in the hearts in either Fe₃O₄-TiO₂ NPs or TiO₂ NPs treated animals (data not shown).

Abbreviations: NPs, nanoparticles; Con, control; G, glomerular; T, tubular epithelial cells.

Figure 9 Signal protein expression in liver tissue.

Notes: (A) Protein-expression levels of p38, p-p38, JNK, p-JNK, HO1, ERK, c-Jun, cleaved caspase 3, Bcl2, Bax, p50, p53, Nrf2, Akt, and GAPDH; (B) relative protein expression (normalized to GAPDH). *P<0.05, **P<0.01, ***P<0.001 compared with control.

Abbreviations: NPs, nanoparticles; Con, control.