Yuan
Qiu‡
a,
Weiwen
Lin‡
b,
Lili
Wang‡
b,
Rui
Liu
a,
Jiangao
Xie
b,
Xin
Chen
a,
Feifei
Yang
a,
Guoming
Huang
*a and
Huanghao
Yang
*c
aCollege of Biological Science and Engineering, Fuzhou University, Fuzhou 350116, P. R. China. E-mail: gmhuang@fzu.edu.cn
bDepartment of Diagnostic Radiology, Union Hospital, Fujian Medical University, Fuzhou 350001, P. R. China
cMOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry, Fuzhou University, Fuzhou 350116, P. R. China. E-mail: hhyang@fzu.edu.cn
First published on 26th September 2019
In this work, the potential of FeP nanoparticles as a pH-responsive T1 contrast agent was investigated. The FeP nanoparticles have good biocompatibility and can significantly amplify T1 magnetic resonance signals in response to the acidic microenvironment of solid tumors, holding great promise in serving as an acid-activatable T1 contrast agent for tumor imaging.
The exploitation of highly specific and sensitive imaging contrast agents is of great importance for precise disease diagnosis.18 Activatable imaging contrast agents that can respond to biological stimulis (e.g., pH, redox potential, and enzyme) to produce contrast signals, have emerged as the next generation of molecular imaging probes.19–22 They can minimize the signal from nontarget background, therefore greatly improve the target-to-background ratio. Conventional T1 contrast agents such as Gd2O3 nanoparticles and MnO nanoparticles have been demonstrated that can afford effective T1 shortening effect to improve the visibility. However, these contrast agents continuously emit signals are “always on”, which fail to response to pathological parameters and hence lack in specificity and sensitivity. Activatable MRI contrast agents that only generate signals in response to a certain stimuli (e.g., physiological difference in pH in tumor microenvironment) thus are highly desirable, because they not only greatly enhance the specificity and sensitivity of disease diagnosis, but also potentially allow MRI to monitor biological processes.23–25 Herein, we report a novel pH-activatable T1 contrast agent based on FeP nanoparticles. We found that the as-synthesized FeP nanoparticles can respond to the acidic microenvironment of solid tumor to produce significant T1 contrast enhancement by releasing paramagnetic Fe ions. Furthermore, both in vitro and in vivo investigations indicate that the FeP nanoparticles have good biocompatibility that show no obvious cytotoxicity and harmful effects. Therefore, the FeP nanoparticles can potentially serve as an acid-responsive T1 MRI contrast agent for tumor imaging.
To investigate the pH-responsive T1 MRI performance of FeP nanoparticles, we dispersed the nanoparticles in buffers with different pH values and conducted the measurements. We first collected the T1-weighted phantom images (Fig. 2a). Significant brighten signals can be detected when FeP nanoparticles are dispersed in acidic buffers (pH 5.0 and pH 6.0), suggesting that FeP nanoparticles generate T1 contrast enhancement at acidic conditions. In contrast, no obvious brighten signals are measured at pH 7.4, demonstrating that FeP nanoparticles have little contrast enhancement effect under neutral conditions. We then measured the longitudinal relaxivity (r1) values of FeP nanoparticles (Fig. 2b). FeP nanoparticles have a relatively low r1 value (∼0.2 mM−1 s−1) at pH 7.4, and the value show little change over time, suggesting FeP nanoparticles have little T1 shortening effect under neutral conditions. In contrast, a gradual enhancement in r1 values can be observed when FeP nanoparticles are in acidic buffers. For example, the r1 value of FeP nanoparticles increases to 4.6 ± 0.2 mM−1 s−1 for pH 5.0 at 24 h. This value is close to that of commercial Gd-based MRI contrast agents such as Gd-DTPA and Gd-DOTA (4–5 mM−1 s−1 at 0.5 T).10,22,27 These results confirm that FeP nanoparticles can effectively shorten the T1 relaxation time of the surrounding water protons at acidic environments. To investigate this pH-responsive behavior of FeP nanoparticles, we further measured the release of Fe ions from FeP nanoparticles under different pH conditions by ICP-MS (Fig. S4†). FeP nanoparticles show very little release of Fe ions at pH 7.4 buffer. However, a significant increase in the release of Fe ions can be detected when FeP nanoparticles are in acidic environments. Paramagnetic Fe ions have the ability to shorten the T1 relaxation time of the water protons because of their high magnetic moment and long electron spin relaxation time. The pH-dependent release property makes FeP nanoparticles to be potential contrast agents for acid-triggered MRI. We further investigated the pH-responsive imaging ability of FeP nanoparticles in cells. MCF-7 cells were incubated with FeP nanoparticles and then were harvested at different time points for imaging. T1-weighted images show that the T1 signals of MCF-7 cells gradually enhance with the increase of incubation time (Fig. 2c). Cells can uptake nanomaterials via endocytosis and the nanomaterials are trapped in endosomes and lysosomes.28 The acidic environment of endosomes/lysosomes trigger FeP nanoparticles to release Fe ions, thus resulting in the T1 signal enhancement inside the cells.
We then investigated the in vivo acid-responsive MRI performance of FeP nanoparticles using MCF-7 tumor bearing mice as models. The biodistribution analysis confirms that FeP nanoparticles can effectively accumulate in tumor via enhanced permeability and retention (EPR) effect (Fig. S5†). T1-weighted images of the mice were collected before and after the injection of FeP nanoparticles at different time points. Gradual brightening signals can be observed in tumor areas after the injection of FeP nanoparticles (Fig. 3a). To further quantify the contrast enhancement, we calculated the signal-to-noise ratio (SNR) in tumor region, and defined the contrast enhancement as the change of SNR, where ΔSNR = (SNRpost − SNRpre)/SNRpre. The measured ΔSNR values are 56.0 ± 23.8%, 82.7 ± 13.6%, 26.5 ± 8.6% at 2 h, 8 h, 24 h after the injection, respectively (Fig. 3b). This time-dependent T1 signal change confirms that FeP nanoparticles can respond to acidic microenvironment of tumor, leading to the shortening effect of T1 relaxation in tumor area.
Biocompatibility is the key factor for a nanoparticle for biomedical applications. To investigate the biocompatibility of FeP nanoparticles, we first assessed the cytotoxicity of by tetrazolium-based colorimetric assay (MTT assay). FeP nanoparticles show no significant cytotoxicity on both MCF-7 and L02 cells after being incubated with these cells for 24 h, suggesting the little cytotoxicity of FeP nanoparticles (Fig. S6†). We then evaluated the in vivo toxicity of FeP nanoparticles in mice. The mice were injected with FeP nanoparticles, and after 14 days, haematoxylin and eosin (H&E) stained histological images of major organs were collected to study the systemic toxicity of FeP nanoparticles. All major organs including heart, liver, spleen, lung, and kidney, maintain their typical tissue structures and exhibit no appreciable organ damage or inflammatory lesion, indicating the long-term safety of FeP nanoparticles (Fig. 4a). Moreover, blood biochemistry and hematology analyses of the mice were also performed (Fig. 4b). Various serum biochemistry parameters including aspartate transaminase (ALT), alanine aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) maintain at similar levels as the controls and all fall within the normal reference intervals, suggesting that the injection of FeP nanoparticles does not affect the liver and kidney functions of mice. The hematology indices including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count (PLT) also show no significant physiological difference comparing to the control group and maintain at normal levels, further confirming the long-term biosafety of FeP nanoparticles.
Fig. 4 (a) H&E stained histological images and (b) blood biochemistry and hematology analyses (n = 5) of the mice collected at 14 days after the injection of FeP nanoparticles. |
Footnotes |
† Electronic supplementary information (ESI) available: Experimental and theoretical details, Fig. S1–S9. See DOI: 10.1039/c9ra06886d |
‡ These authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2019 |