The Journal of Toxicological Sciences
Online ISSN : 1880-3989
Print ISSN : 0388-1350
ISSN-L : 0388-1350
Original Article
Acute hepatotoxicity of multimodal targeted imaging contrast agent NaLuF4:Gd,Yb,Er-PEG/PEI-FA in mice
Yuan ChenXiaoxiao FeiChenqiao YeQinqing QianZhiqiu YeSiqi XieJiamin ChenMingliao ZhuNa RanMingsheng HouLin XuZhangsen Yu
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2019 Volume 44 Issue 9 Pages 621-632

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Abstract

In the past few decades, upconversion nanoparticles (abbreviated as UCNPs) have been more widely applied in the biomedical fields, such as in vitro and in vivo upconversion fluorescent bioimaging, photodynamic therapy, biological macromolecular detection, imaging mediated drug delivery and so on. But meanwhile, there is still not much research on the acute toxicity of upconversion nanoparticles in vivo, such as acute hepatotoxicity. In this work, we studied the in vivo biodistribution and acute hepatotoxicity of multimodal targeted contrast agent NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe, which were synthesized by the solvothermal method and modified with Polyethylene glycol (PEG), Polyetherimide (PEI), folic acid (FA) on the surface. The acute hepatotoxicity in mice was systematically assessed after tail vein injection of different concentration of UCNPs. The results showed that NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoparticles with an average diameter of 44.5 ± 10.4 nm, and three typical upconversion fluorescence emission bands at 520 nm, 540 nm and 660 nm under the excitation of 980 nm laser. In vivo distribution experiments results demonstrated that approximately 87% of UCNPs injected through the tail vein accumulate in the liver. In the acute hepatotoxicity test, the intravenously injection dose of UCNPs was 10, 40, 70 and 100 mg/kg, respectively. The body weight, blood routine, serum biochemistry, histomorphology and liver oxidative stress were detected and observed no significant acute hepatotoxicity damage under the injection dose of 100 mg/kg. In conclusion, NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobes are safe and reliable, and have potential applications in the field of tumor targeted multimodal imaging.

INTRODUCTION

As nanotechnology has advanced, a wide variety of nanomaterials have been extensively researched and applied in biomedical fields (Giner-Casares et al., 2016; Xing and Zhao, 2016; Simon-Yarza et al., 2018). In the past two decades, rare earth doped upconversion nanoparticles have become a new generation of fluorescent nanoprobes because of their specific superiorities, which refers to very low photo-bleaching, absence of auto-fluorescence from the biosome, high sensitivity, and large light penetration depth in biotissue and so on (González-Béjar et al., 2016; Chen et al., 2016; Pilch et al., 2017; Yu et al., 2017; Yu et al., 2018). The greatest advantage of UCNPs is that the excitation light, such as 980 nm, 808 nm near-infrared (NIR) laser, used is very less damaging to biological tissue (Zeng et al., 2016; Chan et al., 2018). Therefore, UCNPs can achieve depth biotissues fluorescence imaging with excellent performance. In addition, due to the versatility of lanthanide ions, multimodal imaging probes can be constructed by doping different types of rare earth ions in the same host, and simultaneously realized the functions of upconversion fluorescence, magnetic resonance (MR) and computer tomography (CT) imaging (Tang et al., 2014; Feng et al., 2017; Xue et al., 2017). Therefore, UCNPs apparently have great potential applications in biomedicine fields. However, in the current research, there is still little attention to the acute hepatotoxicity with rare earth doped UCNPs (Sun et al., 2015; Gnach et al., 2015).

Nanobiomaterials are called double-edged swords. While nanobiomaterials have powerful functions, their biological toxicity cannot be ignored. Toxicological studies of nanobiomaterials include pathways for nanomaterials to enter organisms, absorption, distribution, metabolism, excretion pathways, acute toxicity, and long-term toxicity in organisms (Dwivedi et al., 2018). Similarly, as UCNPs have been increasingly studied and applied in biomedical fields, their toxicological problems have begun to attract the attention of some researchers. For example, the Zhao group summarized the in vivo and in vitro toxicities of the representative inorganic nanoparticles (including UCNPs) used in biomedical imagings and discussed the origin of toxicity of inorganic nanomaterials (Wolfram et al., 2015). Li and Liu’s groups studied the cells phagocytosis and cytotoxicity of UCNPs, and their distribution, metabolism and excretion in vivo (Xiong et al., 2010; Cheng et al., 2011). These results indicate that the UCNPs have relatively low overall biotoxicity and can be applied to multimodal imaging and photochemotherapy in vitro cells and in vivo organisms. However, our previous study showed that UCNPs were mainly accumulated in the mononuclear phagocyte system, such as the liver and spleen, after injection into mice through the tail vein (Yu et al., 2018). Therefore, the local toxicity of UCNPs in the liver requires more intensive and detailed research. In recent years, studies on the acute hepatotoxicity of livers of UCNPs have been rarely reported.

In this paper, we report our synthesis of PEG, PEI and FA modified NaLuF4:Gd,Yb,Er-PEG/PEI-FA multifunctional nanoprobes. Their size, morphology, crystal phase and surface groups and the properties of multimodal imaging were characterized. In addition, the acute hepatotoxicity of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe was evaluated in depth through blood routine, blood biochemical indicators, liver tissue slices and oxidative stress indicators. The trimodal imaging properties of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe and their acute hepatotoxicity assay indicators are shown in Fig. 1. The NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe has a size of 44.5 ± 10.4 nm and has excellent upconversion fluorescence, MR and CT imaging properties. And the values of longitudinal (r1), transverse (r2) relaxivities and CT imaging contrast parameters are 1.56 mM-1s-1, 2.57 mM-1s-1, 17.52 HU•(g•L-1)-1. Experimental results based on ICP detection of lutetium ion content in the main organs of mice showed that approximately 87% of the nanoprobe accumulated in the liver. Moreover, our experiment results based on the body weight, blood biochemistry, liver tissue slices and liver tissue oxidative stress of mice demonstrated that NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe intravenous doses below 100 mg/kg had no obvious acute hepatotoxicity.

Fig. 1

Schematic illustration of the NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe multifunction and toxicology test indicators of acute hepatotoxicity.

MATERIALS AND METHODS

Chemicals and Reagents

LnCl3•6H2O (Ln= Lu, Gd, Yb, Er), Ammonium fluoride (NH4F) were purchased from Alfa Aesar. Most chemical materials ethylene glycol, Polyethyleneimine (PEI) 25,000 Dalton, PEG4000, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), Ethylene glycol (EG), sulfo-N-hydroxysuccinimide (sulfo-NHS) and Folic Acid were bought from Aladdin Reagent Co., Shanghai, China. SOD, GSH and MDA assay kits were purchased from Nanjing Jiancheng Bioengineering institute (Nanjing, China). Deionized water was used in the experiments throughout.

Synthesis of NaLuF4:Gd,Yb,Er-PEG/PEI

NaLuF4:Gd,Yb,Er-PEG/PEI (Lu:Gd:Yb:Er = 68:10:20:2) UCNPs were synthesized by the solvothermal method as reported previously with modifications (Wang and Liu, 2008). Firstly, 10 mL of deionized water was added to 30 mL of EG to obtain a mixed solution. Subsequently, 30 mL of above solution containing LuCl3•6H2O (0.68 mmol), GdCl3•6H2O (0.10 mmol), YbCl3•6H2O (0.20 mmol), ErCl3 (0.02 mmol), NaCl (0.146 g), PEG4000 (0.50 g), PEI (0.125 g) were added (solution A). NH4F (4.0 mmol) was added to the rest 10 mL of above mixed solution and dissolved at 50°C (solution B). And then, the solution B was added dropwise into the solution A, and the reaction solution was stirred at room temperature for 30 min. Finally, the reaction mixture was transferred into a 50 mL stainless Teflon-lined autoclave and reacted at 180°C for 2 hr. Afterwards, the reaction solution was naturally cooled down to room temperature, and the samples were centrifugated with 12000 g for 20 min at the condition of 4°C. The as-prepared samples were washed three times with mixture of ethanol and deionized water (Vethanol:Vwater=1:1). Finally, the NaLuF4:Gd,Yb,Er-PEG/PEI samples were re-dispersed into deionized water and stored in 4°C for using in the following experiment.

Synthesis of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobes

1.00 mg FA, 0.87 mg EDC and 1.23 mg sulfo-NHS were added to 10 mL of physiological saline solution (PBS) at room temperature for 4 hr. And then, 20 mg of NaLuF4:Gd,Yb,Er-PEG/PEI samples were dispersed in 10 mL of PBS and added dropwise to the above reaction solution. The mixture was continuously stirred for 20 hr at room temperature. After the coupling reaction was completed, the products were centrifuged at 12000 g for 20 min. And then, the obtained samples were washed three times with ethanol and deionized water (Vethanol:Vwater=1:1). Finally, the NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobes were dispersed in deionized water.

Characterization

Transmission electron microscopy (TEM) images of the nanoparticles were taken on a JEE-1011 transmission electron microscope operating at 100 kV. The particle size was determined by counting at least 300 nanoparticles per sample. Powder X-ray diffraction (XRD) patterns were performed on an X-ray diffractometer (Empyrean, PANalytical BV) under Cu Kα radiation (λ = 1.54056 Å). Upconversion fluorescence spectra were recorded on F7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) equipped with an external 0-5 W adjustable semiconductor laser at 980 nm (HI-Tech Optoelectronics Co. Ltd, Shanghai, China) as the excitation source. Computed Tomography (CT) images of the nanoparticles were taken by Siemens Somatom 64-slice CT system. The MR relaxivity and MR imaging of nanoparticles were measured on a MesoMR23-060H-I MR instrument with a magnetic field of 0.5 T (Niumag, Shanghai, China). Fourier transform infrared spectroscopy (FT-IR) spectra were recorded using a Nexus 670 spectrometer. Dynamic light scattering (DLS) and Zeta potential measurements were carried out at Zeta sizer Nanoseries (Nano-ZS, Malvern Instruments, Worcestershire, UK) and the nanoparticles were suspended in phosphate buffered saline (PBS) with pH 7.4 at 25°C. The concentration of rare earth ions was determined using an inductively coupled plasma (ICP) optical emission spectrometry produced by Prodigy XP (model Prodigy XP, Teledyne Leeman Lab, USA).

UCL, MR and CT imaging performance test

For upconversion fluorescence imaging, the 1.0 mg/mL NaLuF4:Yb,Er,Gd-PEG/PEI-FA nanoprobe dispersion was placed in a cuvette, and then the upconversion fluorescent photograph was taken using a digital camera under a 980 nm laser excitation. Upconversion fluorescence spectra were recorded on F7000 fluorescence spectrophotometer equipped with an external 0-5 W adjustable semiconductor laser at 980 nm as the excitation source.

The MR performance test of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe includes T1, T2 relaxation rate and MR imaging effects. First, a gradient concentration of the probe dispersion was prepared, and the concentration of Gd3+ in each sample was measured by ICP. The concentration of each sample in the experiment was 0, 0.05, 0.1, 0.2, 0.3, 0.6 mM, respectively. Then, set TE=20 ms, TR=4000 ms, and measure the T1 and T2 values of the samples at different concentrations according to the MRI analyzer. According to the relationship between the concentration of Gd3+ and the reciprocal of relaxation time 1/T1, 1/T2, the slope obtained by fitting is the longitudinal and transverse relaxation rates r1 and r2 of the probes. At the same time, different concentrations of samples were selected for MRI to obtain images of different concentrations of NaLuF4:Gd,Yb,Er-PEG/PEI-FA probes.

Meanwhile, in order to evaluate the efficacy of CT contrast imaging of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobes, a series of different concentrations of probes aqueous solutions 0, 1.0, 2.0, 4.0, 8.0 and 12.0 mg/mL in 1.5 mL Eppendorf tubes were prepared. And the CT images of probes by Siemens Somatom 64-slice CT system. X-ray attenuation values for probes were finally calculated in Hounsfield Units (HU) by SIEMENS CT software. The resulting HU values were recorded at different concentration and plotted vs. mass concentration of NaLuF4:Gd,Yb,Er-PEG/PEI-FA probes. And then the slope of this line provides the CT contrast effect parameter.

Animal treatment

A total of 36 female ICR mice (aged 4 weeks, weight ~20 ± 1.05 g mean ± SD), from Zhejiang Experimental Animal Center, were maintained under standard conditions (two mice per cage, constant temperature of 22°C ± 2°C, 50% ± 10% relative humidity, 12 hr L:12 hr D light cycles) with ad libitum access to water and standard mice chow and used for the experiment. Animals were acclimated to the environment for at least one week prior to the experiment. Mice were randomly divided into five groups with six in each group. The control group received normal saline via tail vein injection. And the acute hepatotoxicity groups were administered a single dose of 10, 40, 70, and 100 mg/kg with intravenous injection. Another six mice were used to test the biodistribution of NaLuF4:Gd,Yb,Er-PEG/PEI-FA probes in mice. All animal procedures described below were performed in accordance with protocol approved by the Animal Care Research Committee of Shaoxing University.

Body weight, mortality rate and coefficient of main organs

The body weight of the mice was recorded before the two days start of the experiment and then every two days thereafter. During the same time, we recorded the survival and mortality rate of mice and observed the mental state and food intake in mice for 18 days after dosing. Body weight of all mice was weighted before combining use of excess anesthetic and cervical dislocation of the mice. Then the mice were sacrificed and their organs were collected. Afterwards, the electronic balance was used to weight the organ weight. The organ coefficients of heart, liver, spleen, lung, and kidney to body weight were measured.

In vivo biodistribution

In order to investigate the biodistribution of the NaLuF4:Gd,Yb,Er-PEG/PEI-FA probes in mice, the mice were injected with 100 μL of probe solution (10 mg/mL) through the tail vein. After then, 24 hr and 18 d after the nanoprobe injection, the blood samples were collected from the eyeball, and the mice were sacrificed and the main organs were taken. Finally, the content of Lu3+ ions in the main organs of mice was accurately determined by ICP to determine the distribution of nanoprobes in mice.

Liver function and blood routine

The blood was collected from the eyeballs of mice. The serum was separated by centrifugation and used for studying various biochemical parameters. The blood was analyzed by blood analyzer (Sysmes XT-1800i, Sysmex Corporation, Kobe, Japan) and Hitachi 7600-110 autoanalyzer (Hitachi). The blood routine parameters mainly include red blood cell (RBC), white blood cell (WBC), hemoglobin (HGB) and blood platelet (PLT). The serum biochemistry test includes lactate dehydrogenase (LDH), alanine transaminase (ALT), total protein (TP), albumin (ALB), globulin (GLB), total protein (TP), aspartate transaminase (AST) and so on.

Oxidative stress analysis

As soon as dislocated mice were put to death, the liver was separated and removed. And then accurately weighed 0.1 g of liver tissue with an electronic balance, shred carefully by a tissue homogenizer, and simultaneously add normal saline at the ratio of 1 to 9. All steps should be in the 0-4°C low temperature conditions (ice box). 10% pre-prepared homogenate was used to balanced centrifuge which required the temperature of 4°C for 10 min at 2000 rpm. In the last step, took the supernatant and store it in the -80°C refrigerator for the determination of each index.

What we needed to determine the indicators of oxidative stress are superoxide (GSH), glutathione (SOD) and malondialdehyde (MDA), and these entire oxidative stress indexes were measurement by commercial kit. And all experiments are carried out in strict accordance with the instructions attached to the commercial kit.

Liver histopathology

After measuring the organ coefficient, the liver slices were made. The specimens of the removed liver tissue were fixed with buffered 10% formaldehyde for 24 hr and then dehydrated through graded ethanol dehydration, embedded in paraffin, sectioned to form 4 μm thick slices, and stained with haematoxylin and eosin (HE). And then, the tissue sections were observed under an optical microscope. According to the slice performance of liver tissue under the microscope, the degree of pathological damage was evaluated.

Statistical analysis

All the data were expressed as mean ± standard error of the mean (S.E.M.). Comparisons of the results between various experimentally treated groups and corresponding controls was evaluated by Dunnett’s test. A P-value < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Synthesis, characterization and performance of multifunctional NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe

The water soluble NaLuF4:Gd,Yb,Er-PEG/PEI nanoparticles were prepared by a modified solvothermal method according to a previous report (Huang et al., 2017). And then, EDC and sulfo-NHS were used as a coupling agent to couple the amino group at the end of the PEI with the carboxyl group of the folic acid to obtain a NaLuF4:Gd,Yb,Er-PEG/PEI -FA nanoprobe having a folate receptor targeting function. As shown in Fig. 2 (a), NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe were dispersed in deionized water without agglomeration phenomenon and approximately spherical shape. and the size distribution of nanoprobe is relatively wide, from 30 nm to 60 nm, with a mean diameter of 44.5 ± 10.4 nm (Fig. 2 (b)). The XRD pattern of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe is presented in Fig. 2 (c), and the standard pattern of NaLuF4 is also shown for comparison. In which we notice that all of the diffraction peaks of the obtained nanoprobe can correspond to the standard α-NaLuF4 XRD pattern. After modification with folic acid, the hydrodynamic diameter and zeta potential of NaLuF4:Gd,Yb,Er-PEG/PEI and NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe increased from 155.2 nm (Pdi = 0.069) to 175.6 nm (Pdi = 0.021) and changed from 17.4 mV to -6.8 mV, respectively (Fig. 2 (d)). This result was good indication that folic acid was successfully coupled to the nanoparticle surface.

Fig. 2

(a) TEM image of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe; (b) Mean diameter of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe; (c) XRD pattern of NaLuF4:Yb,Er,Gd-PEG/PEI-FA and the standard XRD pattern of α-NaLuF4 (JCPDS No. 27-0725); (d) DLS Size and Zeta potential of NaLuF4:Gd,Yb,Er-PEG/PEI and NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe.

Upconversion fluorescence, MR and CT trimodal imaging performance

Figure 3 shows the upconversion fluorescence spectrum and the schematic diagram of the NaLuF4:Yb,Er,Gd-PEG/PEI-FA nanoprobe structure. The inserted upconversion fluorescence photograph is the nanoprobe aqueous solution under the 980 nm laser excitation. As we can see, there were three characteristic fluorescence peaks at 500-530 nm, 530-570 nm and 630-700 nm band, respectively corresponding to Er3 + from the excited state 2H11 / 2, 4S3 / 2, 4F9 / 2 transition to the ground state 4I15/2. For magnetic resonance imaging, as shown in the Fig. 4 (a), we tested it in vitro model T1-weighted magnetic resonance imaging, and was performed with pure water as a blank control. We found that with the concentration of NaLuF4:Gd,Yb,Er-PEG/PEI-FA UCNPs increases, the MRI contrast effect is also improved. We measured the longitudinal relaxation time (T1) and transverse relaxation time (T2) of the samples, with 1/T1 (T2) as the ordinate and the concentration of Gd3+ as the abscissa. The slopes of the obtained fitting straight lines were r1 and r2. The results showed that the r1, r2 values of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe reached 1.56 mM-1s-1 and 2.57 mM-1s-1. And the r2/r1 ratio is 1.65, indicating that the UCNPs may be a good candidate as T1 MR contrast agent. As for CT imaging, with increasing the concentration of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe, the grayscale value of the CT imaging was increased, these results were depicted in the Fig. 4 (b). Furthermore, we performed CT imaging of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe and the results are still satisfactory. Lu3+, Gd3+ and Yb3+ ions doped in UCNPs have a higher density, strong absorption of X-ray. The CT value and concentration showed a good linear relationship, the slope of the fitting line was 17.52. At the concentration of 12 mg/mL, the CT value reached to 207.9 HU. By analyzing the above experimental results scrupulously, we can draw the conclusion that NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe has good upconversion fluorescence, CT and MR imaging contrast performance.

Fig. 3

Upconversion fluorescence spectrum and image of the collected NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe colloids under excitation at 980 nm laser. The inserted image is a schematic diagram of the structure of nanoprobe.

Fig. 4

(a) Plot of relaxation rates (1/T1 and 1/T2) over the Gd3+concentration and T1-weighted MR images of NaLuF4:Gd,Yb,Er-PEG/PEI-FA UCNPs in aqueous solution; (b) CT images and CT values of NaLuF4:Gd,Yb,Er-PEG/PEI-FA UCNPs with different mass concentrations.

Physiological changes of mice

Fluctuation in body weight is an effective indicator of the acute toxicity of nanomaterials. In this experiment, the body weight of mice was recorded every other day from before two days to after 18 days of administration. As shown in Fig. 5 (a), every group mice gained normal body weight within 4 days after the injection of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe. At 5-8 days, all groups’ mice lost weight. After 8 days, there was no significant difference in body weight between the control group and the treatment groups (p > 0.05). In addition, the survival rates of control and varying concentrations of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe groups were also detected. The results were shown in Fig. 5 (b), after intravenous administration of nanoprobe, the survival rates of the mice in each group on the 18 day were 100%, 83.3%, 83.3%, 100%, and 66.7%, respectively. These results indicated that the NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe has no effect on mouse body weight, but when the injection dose reached 100 mg/kg, that began to cause death in mice.

Fig. 5

Body weight (a) and survival rates (b) for mice exposed to varying concentrations of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe (n = 6, mean ± SD).

For investigating the in vivo biodistribution of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe, mice were intravenously injected with nanoprobe at a dose of 100 mg/kg, sacrificed at 24 hr and 18 days after injection, respectively. The blood, intestine, brain and other organs were collected. The in vivo biodistribution of nanoprobe was obtained by measuring Lu3+ content in each tissue. As can be seen from Fig. 6 (a), the nanoprobe content in the liver was highest at both time points compared to other tissues. Moreover, the Lu3+ concentration in the liver remained at 6.28 μg/g after 18 days of injection. These results shown that nanoprobe was easily accumulated in the liver and difficult to metabolize out of the body in a short time. Because of the long-term accumulation of nanoprobe in the liver of mice, a certain degree of lesions to the liver occurs. Figure 6 (b) showed the organ coefficients of heart, liver, spleen, lung and kidney in mice of each experimental group. It can be seen that, except for the liver, the organ coefficient of other organs has no correlation with the dose of the injected nanoprobe. There was no difference in the liver organ coefficient between the 10 mg/kg mice and the control group. However, the liver organ coefficients of the mice injected at a dose of 40 mg/kg/ and 70 mg/kg were significantly lower, indicating that the liver was damaged and atrophied after the NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe was exposed. The liver organ coefficient of mice injected at a dose of 100 mg/kg was slightly larger than that of the control group. This is related to the liver having congestion, edema or hypertrophy.

Fig. 6

In vivo Biodistribution in mice after 100 mg/kg intravenous administration after 24 hr and 18 d (a), organ coefficient for mice exposed to varying concentrations of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe (b) (n = 3, mean ± SD). Single asterisk (*) denotes a significant difference when compared with the control group (P < 0.05). Double asterisk (**) denotes a significant difference when compared with the control group (P < 0.01).

Blood routine and blood biochemistry analyses

In order to further evaluate the acute toxic damage caused to the liver of mice after exposure by nanoprobe, the blood routine and blood biochemical analyses were performed on the mice blood. In the blood routine test, important parameters such as white blood cells (WBC), percentage of lymphocyte (LYN), absolute lymphocyte value (LYMPH), red blood cells (RBC), hematocrit (HCT), red blood cell volume distribution width – standard deviation (RDW-SD), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), hemoglobin (HGB), and platelets (PLT) were selected for analysis. It can be seen from Fig. 7 (a) that there is a significant difference in all blood routine indexes between the different dose of exposure groups and the control group.

Fig. 7

Effect of different doses of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe on hematology (a) and blood biochemistry (b) data of mice. Error bars are based on the standard deviations of five samples (n = 3, mean ± SD).

The liver is the body’s most enzyme-rich organ. There are hundreds of enzymes in the liver cells that play an important role in the metabolism of and biotransformation of the whole body. When liver cells are damaged, the degree of liver damage can be evaluated by detecting changes in certain enzymes in the serum (Volkovova et al., 2015). Furthermore, for more accurately investigate the acute toxicity of the NaLuF4:Gd,Yb,Er-PEG/PEI-FA probe to the liver, in blood biochemical test, the main liver function parameters analyzed included: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLB), triglyceride (TRIG), total cholesterol (CHOL), glucose (GLU), cholinesterase (CHE), lactate dehydrogenase (LDH). The serum biochemistry parameters results are shown in Fig. 7 (b). CHE is synthesized by the liver, once the synthesis of the liver function went down; their concentration in the blood will also decrease. ALB is also synthesized by the liver, its reduction mainly seen in acute liver damage. The results showed that the content of ALB decreased slightly (P < 0.05) with the increase of dose, same as CHE, which indicated that the liver parenchyma cells were slightly damaged. LDH is a glycolytic enzyme widely found in myocardium, skeletal muscle, liver and kidney and other tissues. When these tissues are damaged, the cell membrane is damaged or the cell membrane permeability changes, and then LDH is released into the serum. In this work, compared with control group, the concentration of LDH is significantly increased in 40 mg/kg group (P < 0.05). And 10 mg/kg, 70 mg/kg were slightly increased, but it was not significant. ALT is the most sensitive marker of acute hepatocellular damage, once the liver cell necrosis, ALT will be released into the blood in large quantities, so that the content increased rapidly. AST is also one of the indicators to help detect liver damage. The results showed that compared with the control group, ALT and AST of each dose group did not increase, indicating that the damage of liver cells is not very serious. Other liver function parameters were within the normal range, and there were no significant differences in the exposure groups and the control group. In summary, biochemical indicators showed no significant damage to the liver by NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe.

Oxidative stress parameters

Maintaining the balance of oxidation and antioxidants in the body plays a crucial role with the liver achieving normal physiological function. However, this balance will be destroyed after exposure of the organism to the nanoprobe, thereby triggering an oxidative stress response in the liver tissue (Pisoschi and Pop, 2015). Therefore, the expressions of antioxidant and oxidant enzymes, including superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) were measured by a diagnostic commercially reagent kits according to the manufacturer’s introductions. As shown in Fig. 8, the content of GSH in the liver tissue of the mice was decreased with the increase of NaLuF4:Gd,Yb,Er-PEG/PEI-FA (P < 0.05). And the reduced glutathione is the main source of sulfhydryl group in most living cells. It plays an important role in maintaining the proper redox state of protein thiol, and is a key antioxidant in animal cells. With the decrease of GSH content in the liver tissue, the anti-oxidation ability of the liver was weakened. As for SOD, compared with the control group, 100 mg/kg group decreased significantly (P < 0.05). SOD plays a crucial role on the body’s oxidation and antioxidant balance; it can clear O2- and protect cells from damage. It is through catalyzing the disproportionate of superoxide anion, and produce hydrogen peroxide and oxygen, other group showed slightly lower than control group, but it was no significant. Because some fatty acids are gradually decomposed into a series of complex compounds, including MDA, Therefore, the level of lipid oxidation was measured by detecting the level of MDA. The results showed that the content of MDA in the liver tissue of this experiment increased slightly with the increase of NaLuF4:Gd,Yb,Er-PEG/PEI-FA, but was no significant. In conclusion, the decreased SOD and elevated GSH and MDA levels indicated that the exposure of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe response for the presence of oxidative stress, and this nanomaterials induces certain oxidative impairment but no serious damage.

Fig. 8

Detection of superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) levels in liver tissues of mice. Error bars are based on the standard deviations of five samples. Values represent mean SD (n = 3, mean ± SD).

Histological observation

From the point view of disease diagnosis, histopathological analysis is undoubtedly the gold standard for diagnosis, which no other inspection items can replace it. As shown in Fig. 9, by observing the main organs H&E stained sections of the mice, it was found that the heart, spleen of the mice in the different dose groups showed no obvious damage. In addition, in the experimental group with a nanoprobe injection dose of 70, 100 mg/kg, there was a slight thickening and congestion of the alveolar wall in the lung slice, but no alveolar effusion. And in the experimental group at a dose of 100 mg/kg, the renal tubules of the mice showed diffuse thickening.

Fig. 9

Histological analysis of heart, spleen, lung and kidney from mice injected with different doses of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe. No significant lesion or necrosis was observed in the main organs of the mice in the groups exposed to various nanoprobe concentrations (n = 3, mean ± SD).

However, the NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe accumulates excessively and for a long time in the liver. Therefore, the liver damage is more pronounced as the injection dose of nanoprobe increases. As shown in Fig. 10 (a-c), under the microscope, almost all of the hepatocytes of the liver slice in the control group and the experimental groups injected at a dose of 10, 40 mg/kg were normal. The hepatocytes are arranged in a line around the central vein, and sinus structure show clear, liver lobules were completed. When the injected dose of the nanoprobe is increased, the hepatocytes gradually showed diffuse edema, loose cytoplasm, translucent, and there is mild congestion. At the dose of 70 mg/kg, near the central venous area of liver cell edema was lighter than the outer (Fig. 10 (d)). On the contrary, at the dose of 100 mg/kg, near the central venous area of liver cell edema was more serious than the outer (Fig. 10 (e)). Hepatocyte edema is caused by a large number of water molecules entering the cell, after injection with nanoprobe, cell fluid and plasma homeostasis changed, resulting in edema. Liver congestion is the deposition of blood on the venous end of the hepatic lobule, and leading to the expansion of hepatic lobule central venous and hepatic sinusoids congestion. As can be seen from Fig. 10, as the injected dose increases, there is no irreversible damage to hepatocytes occurs. These experimental results indicated that the NaLuF4:Gd,Yb,Er-PEG/PEI-FA probe had a certain acute toxicity to the mice liver, but did not cause severe degeneration and necrosis of the liver.

Fig. 10

Histological analysis of liver from mice injected with different doses of NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe. And the injection dose were (a) control, (b) 10 mg/kg, (c) 40 mg/kg, (d) 70 mg/kg, and (e) 100 mg/kg (n = 3, mean ± SD).

In summary, we synthesized a high performance NaLuF4:Gd,Yb,Er-PEG/PEI-FA nanoprobe by solvothermal method to achieve multimodal targeted imaging in vivo. Furthermore, we evaluated the acute hepatotoxicity of nanoprobe in mice. Mice body weight, blood routine, serum biochemical parameters, liver tissue oxidative stress index and liver tissue slices results showed that the nanoprobe did not produce significant acute toxicity when injected at dose less than 100 mg/kg. The experimental results obtained in this study provide a toxicological basis for the application of rare earth-based multifunctional upconversion nano fluorescent probers in biomedical fields. Moreover, in order to further reduce the in vivo toxicity of rare earth based nanoprobes, we will further study its toxicological mechanism in the future.

ACKNOWLEDGMENT

We are grateful for the financial support from the College Students Science and Technology Innovation Project of Zhejiang Province, China (2017R428021), National College Students Innovation and Entrepreneurship Training Program, China (201710349011), National Natural Science Foundation of China (51402195), Educational Commission of Zhejiang Province, China (Y201839726, Y201225801), University Laboratory Research Project of Zhejiang Province, China (YB201722), Science and Technology Plan in Shaoxing City (2018C10011).

Conflict of interest

The authors declare that there is no conflict of interest.

REFERENCES
 
© 2019 The Japanese Society of Toxicology
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