In Vivo Biodistribution and Toxicity of Highly Soluble PEG-Coated Boron Nitride in Mice
- Bo Liu†1,
- Wei Qi†2Email author,
- Longlong Tian1,
- Zhan Li3Email author,
- Guoying Miao4,
- Wenzhen An5,
- Dan Liu1,
- Jing Lin1,
- Xiaoyong Zhang6 and
- Wangsuo Wu1Email author
© Liu et al. 2015
Received: 13 August 2015
Accepted: 23 November 2015
Published: 10 December 2015
The boron nitride (BN) nanoparticles, as the structural analogues of graphene, are the potential biomedicine materials because of the excellent biocompatibility, but their solubility and biosafety are the biggest obstacle for the clinic application. Here, we first synthesized the highly soluble BN nanoparticles coated by PEG (BN-PEG) with smaller size (~10 nm), then studied their biodistribution in vivo through radioisotope (Tc99mO4 −) labeling, and the results showed that BN-PEG nanoparticles mainly accumulated in the liver, lung, and spleen with the less uptake by the brain. Moreover, the pathological changes induced by BN-PEG could be significantly observed in the sections of the liver, lung, spleen, and heart, which can be also supported by the test of biochemical indexes in serum. More importantly, we first observed the biodistribution of BN-PEG in the heart tissues with high toxicity, which would give a warning about the cardiovascular disease, and provide some opportunities for the drug delivery and treatment.
The two-dimensional (2D) materials are a single-atom-layer materials with some special properties [1–3]. At present, the most famous 2D material is the carbon 2D nanomaterials namely graphene because of its excellent physicochemical properties [4–6]. Moreover, the boron nitrides, known as white graphene , have received more and more attention. BNs are structural analogues of graphene in which C atoms are replaced by alternating B and N atoms. Moreover, thanks to the special chemical and physical characteristics of the BN nanoparticles, many researchers have found a large number of applications in the field of nanotechnology, and several studies have shown their possible exploitation in the biomedical domain, such as nanocarriers  and nanotransducers . However, before the clinical application, it is very necessary and important for biomaterials of BN to investigate biosafety in vivo and in vitro. Biosafety investigations include toxicity in vivo and cytotoxicity in vitro; to clarify toxicity in vivo, it was essential a prerequisite to research its behavior and fate in the living things. Unfortunately, so far no one has explored the detailed investigations about behavior and toxicity of BN in vivo.
Considering that the pristine BN materials are hardly used in the biomedical domain due to the profound chemical inertness of insoluble BN structures , so, a layer of hydrophilic material such as polyethylene glycol (PEG) was often coated on the surface of pristine BN for improving the solubility and enhancing the biological compatibility . Moreover, the PEG that was thought as a safe and innocuous biomaterial is widely used to improve the biological compatibility and water solubility of various nanoparticles [12–14]. Furthermore, the coating of PEG would not alter the structure of BN and remain the property and character of BN, which would increase the application prospects of BN materials . Thereby, Weng et al.  successfully prepared the coating of porous BN materials with PEG, which preformed the lower cytotoxicity. However, the cytotoxicity of the porous BN may be different to that of entire BN. Therefore, it would be very imperative to study the behavior and toxicity of entire structure of BN after coating with PEG.
In current works, we found that some PEG-coated BN (BN-PEG) nanoparticles with smaller sizes (~10 nm) and quantum effects could be easily prepared through the treatment of PEG coating, but lower fluorescence quantum yield (~7.8 %) would be not conducive to detect signals from biological samples. Although a fluorescent labeling and imaging may be a popular method to study BN-PEG in vivo, but the potential quenching and other intrinsic limitations of fluorescence imaging could cause nonquantitative and less accurate biodistribution results . In comparison, the pharmacokinetics and biodistribution studies based on 99mTc-labeled, BN-PEG provides more reliable and quantitative information for the in vivo behaviors of biocompatibility functionalized BN . Therefore, here, we prepared the BN-PEG nanoparticles with high water solubility, and then 99mTcO4 − was used to label complexes, and studied the biodistribution and toxicity of BN-PEG in mice in vivo.
Synthesis and Characterization of PEG-Coated BN
A solution of 6-arm-polyethyleneglycol-amine (Sunbio Inc.) (3 mg/mL) was mixed to the BN sheets (0.5 mg/mL) (BN were purchased from Baoding Zhong Pu Rui Ta technology. LTD), and the mixture was stirred at 200 °C for 4 days under a steady nitrogen flow. After cooling the reaction mixture to a room temperature, then H2O (~60 mL) was added for extraction . The solution was sonicated and centrifuged (~5000g, 20 min), and the supernatants were collected and dialyzed about 1 week by dialysis membrane (MD25) to remove free PEG. Finally, the solution was dried at 50 °C in vacuum oven, and the solid product was the BN-PEG. And then, the product was characterized by TEM (Tecnai G2 F30), XRD (XRD-600) and fluorescence lifetime and steady-state spectroscopy (FLS; 920).
99mTc Labeling BN-PEG
The Biodistribution of BN-PEG
Kunming mice initially weighing 15 to 18 g were provided by the Laboratory Centre for Medical Science, Lanzhou University, Gansu, China. All animals were housed in individual cages in a temperature-controlled (21 to 22 °C) and light-controlled (turned on from 0800 h to 2000 h) environment and were fed food and tap water ad libitum. All animal protocols were in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC) and approved by Institutional Animal Care and Use Committees of Gansu Province Medical Animal Center and Lanzhou University Animal Committees Guideline (China). The mice were organized randomly into four groups (six mice/group). The mice were injected intravenously with 20 mg/kg bw of 99mTc-BN-PEG solution (pH = 7.26, CNaCl = 0.9 %), and then killed at 1, 6, 16, and 24 h after injection. Tissues from the heart, lung, liver, spleen, kidney, stomach, and brain were immediately dissected, and the blood was also collected. Each tissue was wrapped in foil, weighed and counted for 99mTc. Data were corrected for physical decay of radioactivity. The distribution of the tissues was presented in percent injected dose per gram of wet tissue (%ID/g), which could be calculated by the percent injected dose (tissue activity/total activity dose) per gram of the wet tissue.
Blood Analysis and Histology Examinations
CRP, ANP, BUN, and TB kits were purchased from Shanghai Heng Hitter Trade Co., Ltd. and then kept in the refrigerator at 4 °C. Approximately 0.2 mL of BN-PEG (20 mg/kg bw) was injected intravenously to the experimental mice (six mice). Approximately 0.2 mL of saline solution and PEG (20 mg/kg bw) was administered to the mice from the control groups. All mice were then killed. Approximately 2 mL of blood was collected to obtain the plasma, and then centrifuged for 10 min to collect the serum. The serum contents of CRP, ANP, BUN, and TB were then measured. At the same time, the liver, lung, spleen, kidney, and heart were harvested immediately. These tissues were fixed in 10 % buffered formalin and processed for routine histology with hematoxylin and eosin staining by the Centre for Medical Science, Lanzhou University (Lanzhou, China). Microscopic observation of tissues was performed with an Olympus Microphot-CX41 microscope coupled with a digital camera.
Results and Discussion
The Preparation and Characterization of BN-PEG
Biodistribution of BN-PEG Through Labeling of 99mTc
The Fig. 5b showed that BN-PEG was mainly distributed in the liver and spleen in 24 h but with lower distribution in the lung, interestingly, which is different with the biodistribution of other carbon nanoparticles [19, 20]. In our previous works [21–23], we reported that the distribution in the lung and stomach is the highest for the graphene oxide (GO), carbon nanotubes, and nano-diamond mainly. However, the distribution of BN-PEG has a good agreement with graphene oxide coated by PEG ; the BN and GO coated by PEG mainly accumulated in the reticuloendothelial system (RES) such as the liver and spleen (Fig. 5b), meaning that the coating of PEG affected and changed the behavior and fate of the nanoparticles in vivo. The high distribution of BN/GO-PEG in macrophage organs could be attributed to Kupffer cells which could devour the nanoparticles so as to decrease the tissues toxicity, but the high accumulation in the lung is owing to capture role of the pulmonary capillary bed on nanoparticles . After coating with PEG, the nanoparticles containing BN and graphene would pass through the pulmonary capillary bed, and entered into the liver and spleen with the development of the circulation of the blood; in here, most of them would be devoured by the Kupffer cells. The finding of the processes would be useful and important for us to protect and cure worker from harm of BN, and it would provide some new route to design the drug delivery system of BN.
Moreover, the peak of biodistribution of BN-PEG is at about 16 h post injections (Fig. 5b), but which is inconsistent with the dynamics of GOs (~2 h) , and this may be due to the structure difference of BN and GOs. We also found lower distribution of BN-PEG in the kidney and brain tissues (Fig. 5b). The kidney is the excretory system of nanoparticles through urine, and some papers have reported that the nanoparticles can be excreted by the urine with high biodistribution in the kidney, showing that the BN-PEG could be excreted with development of time, so the distribution in kidney increased slowly and reached the peak at 24 h (Fig. 5b). The biodistribution of BN-PEG in the brain was first reported for most of the nanoparticles in this work. It is generally accepted that the carbon nanoparticles and metal nanomaterials could not pass through blood-brain barrier, so it is very hard to deliver drug with these nanoparticles [25–27]. However, we found a degree of enrichment in brain with slow elimination, so we conferred that the BN-PEG would be able to pass through the blood-brain barrier. The result would be valuable to design the drug delivery system of BN-PEG for curing the disease in brain.
The Biochemical Characteristics in Serum
The BN is a poorly water-soluble nanoparticle, so the data of its in vivo biodistribution and safety is unclear up to now. Therefore, the highly soluble BN-PEG was prepared by an improvement of existing methods, and we, for the first time studied in vivo biodistribution and biotoxicity of BN-PEG by a radiolabeling method in mice. Both biodistribution measurements based on 99mTc labeling suggest that the BN-PEG mainly distributed in the liver, spleen, and lung, and observations from histological slices show that it could cause obvious tissue lesions in liver, spleen, lung, and heart. The heart damage is a novel and interesting finding for understanding and treating the toxicity of BN nanoparticles on mice; the test of biochemistry parameters including Cys-C, CREA, ALT, AST, TB, CRP, and BUN in serum further confirms the result. Therefore, the BN-PEG nanoparticles are the hazardous materials in vivo, and its biologic toxicity could be taken seriously.
This work was supported by the National Science Foundation of China NO. J1210001 and 21327801.
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