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Folic acid-modified Laponite®-stabilized Fe3O4 nanoparticles for targeted T2-weighted MR imaging of tumor
Applied Clay Science 186 (2020) 105447
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Research Paper
Folic acid-modified Laponite®-stabilized Fe3O4 nanoparticles for targeted T2-weighted MR imaging of tumor
T
Ling Dinga,b,1, Ruizhi Wangc,d,e,1, Yong Hua, Fanli Xua, Ni Zhanga, Xueyan Caoa, Xiaolin Wangc,d, ⁎ Xiangyang Shia, Rui Guoa, a
College of Chemistry, Chemical Engineering and Biotechnology, State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University, Shanghai 201620, People's Republic of China b Aix-Marseille University, CNRS, Centre Interdisciplinaire de Nanoscience de Marseille (CINaM), Equipe Labellisée Ligue Contre le Cancer, 13288 Marseille, France c Department of Interventional Radiology, Zhongshan Hospital, Fudan University, Shanghai 200032, People's Republic of China d Department of Radiology, Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, People's Republic of China e Department of Radiology, Huadong Hospital, Fudan University, Shanghai, 20040, People's Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Laponite Iron oxide NPs MR imaging Folic acid PAMAM dendrimer
In this study, a targeted T2-weighted MR imaging agent based on laponite (LAP) was constructed for cancer cells overexpressing folate receptors. Firstly, LAP stabilized Fe3O4 NPs are synthesized by a controlled co-precipitation route, and then amphiphilic copolymer poly(lactic acid)-poly(ethylene glycol) (PLA-PEG-COOH) is assembled on the surface of LAP/Fe3O4 NPs to provide additional stability, followed by the conjugate of folic acid modified generation 2 polyamidoamine dendrimer (G2-FA) via EDC coupling chemistry. The formed LAP/Fe3O4PLA-PEG-G2-FA NPs can display good colloidal stability and an enhanced r2 relaxivity of 327.6 mM−1 s−1. In vitro experiments demonstrate that the hybrid nanoparticles are cytocompatible in the studied concentration range, and could specific target cancer cells with FA receptors overexpression. Finally, the xenografted tumor model experiment verifies that LAP/Fe3O4-PLA-PEG-G2-FA NPs can significantly reduce the MR signal of tumor site by specific accumulation, and be metabolized from the mice within 24 h. Therefore, LAP/Fe3O4-PLA-PEGG2-FA NPs with good biocompatibility, a high r2 relaxivity, and FA targetability show a huge potential as a targeted T2-weighted MR imaging contrast for the early diagnosis of tumors overexpressing FA receptors.
1. Introduction Magnetic resonance imaging (MRI) is one of most powerful noninvasive imaging techniques to provide 3D images of soft tissues with high spatial and temporal resolution. For accurate diagnosis of early tumor, contrast agents were always employed to improve the sensitivity and reliability of MRI by increasing the contrast effect of tumors from the normal tissues under external magnetic. Among different kinds of MR contrast agents, superparamagnetic iron oxide nanoparticles (Fe3O4 NPs) have attracted enduring interests due to their good biocompatibility, high saturation magnetization and passive tumor targeting by enhanced permeability and retention (EPR) effect (Arami et al., 2015; Hu et al., 2018a; Jabalera et al., 2019; Wahsner et al., 2019). However, the tendency to aggregate and the lack of active tumor-targeting property of Fe3O4 NPs are the main challenges in constructing high performance contrast agents to locate early tumor lesions.
Till now, various kinds of strategies are used to improve their colloidal stability (Cai et al., 2013; Chen et al., 2019; German et al., 2015; Hu et al., 2015; Nandwana et al., 2018; Zhu et al., 2015), and different targeting agents are modified on surface to enhance their specificity to tumor, such as folic acid (FA) (Li et al., 2013; Luong et al., 2017; Shi et al., 2008), hyaluronic acid (HA)(Li et al., 2014), arginine-glycineaspartic acid (RGD) peptide (Hu et al., 2015), and antibodies (Hadjipanayis et al., 2010). For example, Li et al. reported a polyethyleneimine (PEI)-mediated approach to synthesis HA modified Fe3O4-PEI NPs, which displayed a good colloidal stability and cytocompatibility, specific uptake and imaging of cancer cells overexpressing CD44 receptors (Li et al., 2014). Luong et al. used folic acid modified polyamidoamine (PAMAM) dendrimers to decorate Fe3O4 NPs, and demonstrated that the targeted NPs showed high accumulation at the folate receptor overexpressed cancer cells than nontargeted ones (Luong et al., 2017). However, probably due to the aggregation of
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Corresponding author. E-mail address: [email protected] (R. Guo). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.clay.2020.105447 Received 15 October 2019; Received in revised form 3 January 2020; Accepted 11 January 2020 0169-1317/ © 2020 Elsevier B.V. All rights reserved.
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dendrimer (G2-FA) via EDC coupling chemistry (Scheme 1). Various techniques would be applied to characterize the structure, morphology and physic-chemical properties of LAP/Fe3O4-PLA-PEG-G2-FA NPs. Moreover, a human cervical carcinoma cell line (HeLa cells) overexpressing FA receptors was used to evaluate the MR imaging performance in vitro and the HeLa xenografted tumor model was established for in vivo assessment. As far as we know, it is the first study reporting on the modification of LAP-Fe3O4 NPs as a promising targeted contrast agent for in vivo T2-weighted MR imaging of tumors.
iron oxide NPs during modification or the barrier effect of polymer shells, those contrast agents usually exhibited a decreased r2 relaxivity, which may restrict their performance for accurate MR imaging. Recently, 2-dimensional inorganic nanomaterials have inspired considerable interest in the field of biomedicine (Cao et al., 2018; Cheng et al., 2019). As a kind of synthesized nanoclay with good colloidal stability, biocompatible and biodegradability, Laponite® (LAP) has been widely applied in the delivery of therapeutic agents and functional nanoparticles by the virtue of its distinctive layered structure (Liu et al., 2019; Tomás et al., 2018). For instance, LAP could load anticancer drug doxorubicin with high encapsulating efficiency, release drugs in a pH-sensitive profile, and exhibit a better therapeutic efficiency than free drug (Wang et al., 2013; Wu et al., 2014). LAP stabilized gold nanoparticles are demonstrated to be a potential CT contrast agent with good stability and high X-ray attenuation coefficients (Zhuang et al., 2017). Especially in constructing MR imaging agents, LAP could not only provide additional colloidal stability for iron oxide NPs, but also improve the r2 relaxivity significantly by suppressing the magnetic interparticle interactions (Tzitzios et al., 2010). In our previous work, LAP-Fe3O4 NPs displayed a high r2 relaxivity as 475.9 mM−1 s−1, which is 2 folds higher than that of Fe3O4 NPs (Ding et al., 2016). However, the rapid clearance by the reticuloendothelial system (RES) and the non-specificity of LAP/Fe3O4 NPs may limit the dose available for the early diagnosis of tumors. Since PEG chains could mitigate the interaction between NPs and macrophages (Perry et al., 2012; Wang et al., 2014b), a biocompatible amphiphilic block copolymer polylactic acid-polyethylene glycol (PLA-PEG) could be assembled on the surface of LAP to escape the RES clearance and prolong the blood circulation time. Moreover, the active carboxyl group at the end of PLA-PEG could be further modified with targeting agents to endow the specific targeting capability of nanoparticles. In our previous study, folic acid (FA) was used as an effective targeting ligand to modify PAMAM dendrimer, and FA-conjugated PAMAM dendrimers were demonstrated to achieve high binding and internalization into cancer cells overexpressing folate receptors (Li et al., 2013; Liu et al., 2013; Wang et al., 2014a). Therefore, the assemble of PEG-PLA and decoration of FA-PAMAM dendrimer on the surface of LAP/Fe3O4 NPs would be a promising strategy to improve their in vivo stability and achieve active targeting of cancer overexpressing FAR. Therefore, in this study, Fe3O4 NPs were synthesized in the presence of LAP with a controlled co-precipitation method, and then amphiphilic PLA-PEG-COOH were assembled on the surface of LAP/Fe3O4 NPs to provide additional stability and active carboxyl groups, followed by the conjugate of folic acid modified generation 2 polyamidoamine
2. Experimental 2.1. Materials LAPONITE® (Na+0.7[(Si8Mg5.5Li0.3)O20(OH)4] −0.7, LAP) was purchased from Zhejiang Institute of Geology and Mineral Resources (Hangzhou, China). Folic acid, ferric chloride hexahydrate (FeCl3·6H2O > 99%), ferrous chloride tetrahydrate (FeCl2·4H2O > 99%), sodium hydroxide and hydrochloric acid (HCl = 37%) were obtained from Aldrich. PLA-PEG-COOH with a molecular weight of 8000 Da (3000 Da for PLA and 5000 Da for PEG) was purchased from Jinan Daigang Biological Technology Co, Ltd. (Jinan, China). Amine-terminated PAMAM dendrimers of generation 2 (G2) were purchased from Dendritech (Midland, MI). HeLa cells (a human cervical carcinoma cell line) were obtained from Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). Perls stain was obtained from Beijing Leagene Biotechnology Co., Ltd. (Beijing, China). Perlsstain A1 and Perls stain A2 was mixed equivalently. Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Hangzhou Jinuo Biomedical Technology Co., Ltd. (Hangzhou, China). All chemicals purchased were used without further purification. Water with a resistivity of 18.2 MΩ cm purified by using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) was used. 2.2. Preparation of LAP/Fe3O4-PLA-PEG-G2-FA NPs LAP/Fe3O4 NPs were firstly synthesized according to our previous work by a controlled co-precipitation method (Ding et al., 2016). In brief, 15 mL aqueous solution (containing 0.089 mL HCl) of FeCl3·6H2O (0.721 g) and FeCl2·4H2O (0.265 g) was added into the LAP solution (10 mg mL−1, 50 mL) magnetic stirring, and then 10 mL of NaOH (2.0 g) aqueous solution was added into the above mixture under highspeed stirring. After 2 h of reaction at 80 °C under N2 atmosphere, the
Scheme 1. Schematic illustration of the synthesis of LAP/Fe3O4-PLA-PEG-G2-FA. 2
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LFAR). Unless otherwise stated, the term of HeLa cells always represents the HeLa-HFAR cells. CCK-8 assay was used to quantify the viability of HeLa cells after treated with LAP/Fe3O4-PLA-PEG-G2-FA NPs at different concentrations. Briefly, HeLa cells were seeded in 96-well plates at a density of 1 × 104 cells/well with 200 μL of fresh DMEM at the day before the experiment. After 24 h, the medium was replaced by 200 μL fresh medium containing PBS (control) or LAP/Fe3O4-PLA-PEG-G2-FA NPs with different Fe concentrations (0.25, 0.5, 1.0, 1.5 and 2.0 mM). After 24 h incubation, the medium was discarded and the cells were washed with PBS 3 times, followed by addition of 200 μL fresh DMEM containing 20 μL CCK-8. After incubation of the cells at 37 °C for another 4 h, they were measured the absorbance by Thermo Scientific Multiskan MK3 ELISA reader (Thermo Scientific, Hudson, NH) at 450 nm in each well. The standard deviation of 5 wells of each sample were recorded. To further confirm the cytotoxicity of LAP/Fe3O4-PLA-PEG-G2-FA NPs, HeLa cells were treated with PBS or LAP/Fe3O4-PLA-PEG-G2-FA NPs at Fe concentration of 0.2, 0.4, 0.8, 1.5, and 2.0 mM, respectively. The cell morphology was observed by a Leica DM IL LED inverted phase contrast microscope with a magnification of 200 × for each sample. TEM observation was applied to visually examine the internalization of LAP/Fe3O4-PLA-PEG-G2-FA NPs in HeLa cells according to protocols described in the literature (Ding et al., 2016).
reaction mixture was purified by magnetic separation to obtain LAP/ Fe3O4 NPs. Then LAP/Fe3O4 solution (6.4 mL, 20 mg mL−1) was dropwise added into PLA-PEG-COOH (20 mL, 6 mg mL−1) water/ acetone (1:1) solution. After stirring for 24 h, LAP/Fe3O4-PLA-PEGCOOH solution was purified by dialyzing the mixture against phosphate buffered saline (PBS) and water (2 L each time, 3 times each day) for 3 days with a 14,000 MWCO dialysis membrane. G2-FA was synthesized according to our previous work (Liu et al., 2013). Briefly, FA (26.84 mg) was dissolved in DMSO (5 mL), and then EDC (10.8 mg) and NHS (6.5 mg) was added under magnetic stirring. After 3 h of reaction, the activated FA solution was added to 5 mL G2 (39.6 mg) DMSO solution under vigorous stirring for 3 days. Thereafter, the reaction mixture was extensively dialyzed against water with a 2000 MWCO dialysis membrane for 3 days to remove excess reactants. Finally, LAP/Fe3O4-PLA-PEG-COOH NPs solution obtained above (198 mg, 4 mL) was mixed with EDC (2.98 mg, 1 mL) and NHS (1.79 mg, 1 mL) under vigorous magnetic stirring for 3 h to activate the carboxyl group. Then, the G2-FA aqueous solution (28 mg, 5 mL) was dropwise added into the above solution under magnetic stirring for 3 days. The mixture was dialysis with water for 3 days to obtain LAP/ Fe3O4-PLA-PEG-G2-FA. 2.3. Characterization 1 H NMR spectra were measured on a Bruker AV400 NMR spectrometer. Samples were dissolved in deuterated D2O before measurement. The FTIR spectra was acquired using a Nicolet Nexus 670FTIR (NicoletThermo) spectrometer. The spectra of all samples were recorded in a transmission mode with the wavenumbers in the range of 400–4000 cm−1. Before analysis, the dry samples mixed with KBr crystals were pressed to form ground pellets. The iron concentrations of samples were analyzed by using a Leeman Prodigy ICP-OES system (Hudson, NH). UV/Vis spectroscopy was obtained using a Lambda 25 UV/Vis spectrophotometer (Perkin-Elmer, USA). The organic component of the samples was quantified by thermogravimetric analysis (TGA) using a TG 209 F1 (NETZSCH Instruments Co., Ltd., Selb/ Bavaria, Germany) thermogravimetric analyzer. The samples were heated from 25 °C to 900 °C at a rate of 10 °C/min under nitrogen atmosphere. Zeta potential and dynamic light scattering (DLS) measurements were carried out using a Malvern Zetasizer Nano ZS model ZEN3600 (Worcestershire, U.K.) equipped with a standard 633 nm laser. Transmission electron microscopy (TEM) was carried out with a JEOL 2010 analytical electron microscope (Tokyo, Japan) operating at 200 kV to characterize the morphology and size of the NPs. Before performing the measurements, the samples were prepared by putting a drop of diluted NP suspension (6 μL) onto a carbon-coated copper grid and dried in air. For each sample, at least 200 particles in different TEM images were randomly selected and measured by using ImageJ software to calculate the size distribution of NPs. T2 relaxometry was performed by a 0.5-T NMI20-Analyst NMR Analyzing and Imaging system (Shanghai Niumag Corporation, China). The samples were diluted in water with Fe concentrations in the range of 0.005–0.08 mM. The instrumental parameters were set at point resolution of 156 mm × 156 mm, section thickness of 0.6 mm, TR of 4000 ms, TE of 60 ms, and number of excitations of 1. The r2 relaxivity was calculated by a linear fit of the inverse T2 (1/T2) relaxation time as a function of Fe concentration.
2.5. Cellular uptake study ICP-OES was carried out to investigate the cellular uptake of LAP/ Fe3O4-PLA-PEG-G2-FA NPs by HeLa-HFAR or HeLa-LFAR cells. Both cells were seeded into 12-well plates at a density of 2 × 105 cells/well. After incubation at 37 °C and in 5% CO2 atmosphere overnight, the medium was replaced with fresh medium containing NPs at different Fe concentrations (0.05–0.2 mM). After 4 h incubation, the medium was discarded carefully and the cells were washed with PBS for 5 times, trypsinized, centrifuged, and counted by Handheld Automated Cell Counter (Millipore, Billerica, MA). The remaining cells were lysed using aqua Regia solution (1.0 mL) for 2 days, and then the Fe uptake in cells was quantified by ICP-OES. Prussian blue staining of LAP/Fe3O4-PLA-PEG-G2-FA NPs in HeLaLFAR or HeLa-HFAR cells was performed to visually examine the cellular Fe uptake. Similar to the protocol described above, HeLa cells were treated by LAP/Fe3O4-PLA-PEG-G2-FA NPs with Fe concentrations of 0.05 mM and 0.1 mM. After 4 h, the cells were washed for 3 times with PBS, fixed with glutaraldehyde solution (2.5%) at 4 °C for 15 min, and stained with Prussian blue reagent at 37 °C for 30 min. The cells were then imaged by Leica DM IL LED inverted phase contrast microscope with a magnification of 200 × for each sample. 2.6. In vitro MR imaging of cancer cells In brief, HeLa-HFAR or HeLa-LFAR cells were seeded into 6-well plates at a density of 2 × 106 cells/well with 2 mL of fresh DMEM and incubated at 37 °C and 5% CO2 overnight to bring the cells to confluence. Then the medium was replaced by LAP/Fe3O4-PLA-PEG-G2-FA NPs contained medium at different concentrations of Fe (0.1, 0.2, 0.4 mM, respectively) and incubated for 4 h. Afterwards, the cells were washed 5 times with PBS, trypsinized, centrifuged, and resuspended in 1 mL PBS (containing 0.5% agarose). T2-weighted MR imaging of each sample was carried out using a 1.5 T Signal HDxt super conductor clinical MR system (GE Medical Systems, Milwaukee, WI) under the following conditions: point resolution = 156 mm × 156 mm, section thickness = 0.6 mm, TR = 2700 ms, TE = 69 ms, and number of excitation = 1.
2.4. In vitro cytotoxicity assay and cellular uptake HeLa cells overexpressing FA receptors were cultured and passaged in 25-cm2 plates with DMEM supplemented with 10% FBS and 1% penicillin/streptomycin under 37 °C and 5% CO2. HeLa cells grown in FA-free medium expressed high-level FA receptors (denoted as HeLaHFAR), while the cells pre-treated with FA-containing medium (2.5 mM) for 5 h expressed low-level FA receptors (denoted as HeLa-
2.7. In vivo targeted MR imaging All animal experiments were approved by institutional committee 3
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COOH and LAP/Fe3O4-PLA-PEG-G2-FA displayed huge weight losses of 66.3% and 73.3% in the range of 200 to 900 °C, respectively. This confirmed that both PLA-PEG-COOH and G2-FA were successfully modified on LAP/Fe3O4 NPs as design. Finally, the morphology and size distribution of LAP/Fe3O4-PLA-PEG-G2-FA NPs were observed by TEM (Fig. 1c and d). The synthesized Fe3O4 NPs could be easily identified due to their high contrast under TEM observation, and displayed a mean size of 9.8 ± 1.7 nm, indicating that surface modification did not alter the size and nanocrystal structure of iron oxide. And it is interesting that LAP/Fe3O4-PLA-PEG-G2-FA NPs displayed a more dispersed state than LAP/Fe3O4 NPs (Fig. S3), probably attributing to the protective effect of polymers on surface. In sum, folic acid modified LAP/Fe3O4-PLA-PEG-G2-FA NPs were successfully synthesized. The hydrodynamic size and surface potential of LAP/Fe3O4-PLAPEG-G2-FA were further studied by DLS as shown in Table 1. The significant increase of surface potential from −34.9 mV of pristine LAP to −18.0 mV of LAP/Fe3O4, was mainly attributed to the offsetting of negative charge on LAP by positively charged Fe3O4. LAP/Fe3O4-PLAPEG-COOH displayed a surface potential of −10.9 mV by the shield of PLG-PEG, and further conjugate of G2-FA may reverse the Zeta potential to 19.8 mV because of the conjugate of positive G2-FA on surface. These results further verified the successful synthesis of LAP/Fe3O4PLA-PEG-FA. In addition, due to the modification of PEG chain, the hydrodynamic diameter of LAP/Fe3O4-PLA-PEG-COOH and LAP/ Fe3O4-PLA-PEG-FA increased to 271.1 nm and 332.1 nm, respectively, and keep below 500 nm when dispersed in PBS and culture medium (Fig. S4).
for animal care according to the policy of the National Ministry of Health. HeLa cells (1.5 × 106/mouse) were subcutaneously injected into the back of male 6-week-old BALB/c nude mice (15–20 g, Shanghai Slac Laboratory Animal Center, Shanghai, China) to establish the xenografted tumor model. When the tumor volume reached 0.5–0.8 cm in diameter (3 week post-injection), the mice were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg) and divided into 2 groups: For Group 1, the mice was intravenously injected with LAP/Fe3O4-PLA-PEG-G2-FA NPs; Group 2, the mice was intratumorally pre-injected with free FA (2 mM, 0.1 mL PBS) for 30 min, and then LAP/Fe3O4-PLA-PEG-G2-FA NPs (Fe: 1 mg mL−1, 150 μL PBS) was intravenously injected. For T2-weighted MR imaging, each mouse was imaged by 1.5 T Signal HDxt superconductor clinical MR system coupled with a custom-built rodent receiver coil (Chenguang Med Tech, Shanghai, China). Two-dimensional (2D) spin-echo MR images were obtained before and at the time points of 0, 0.5, 1.5 and 3 h post-injection of the LAP/Fe3O4-PLA-PEG-G2-FA NPs with the conditions similar to those used for in vitro MR imaging of cancer cells. 2.8. In vivo biodistribution To assess the biodistribution of LAP/Fe3O4-PLA-PEG-G2-FA NPs, the tumor-bearing BALB/c nude mice were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg). After intravenous injection of LAP/Fe3O4-PLA-PEG-G2-FA NPs ([Fe] =2 mg mL−1, 0.2 mL PBS), the mice were sacrificed at 1.5, 3, 12, and 24 h post injection, and the heart, liver, spleen, lung, kidney, and tumor were harvested, weighed and digested by aquaregia for 2 days. The tumor-bearing mice before treatment were used as control, and the Fe concentrations in tumor and organs were simultaneously determined by ICP-AES.
3.2. MR imaging property of LAP/Fe3O4-PLA-PEG-G2-FA solution The potential of LAP/Fe3O4-PLA-PEG-G2-FA as T2-weighted MR contrast agents was explored by measuring the MR image and transverse relaxation time (T2) of solutions (Fig. 2). With the increase of Fe concentration, the color MR image of NPs solutions turned from red to blue, indicating the decrease of MR signal by the negative contrast effect of LAP/Fe3O4-PLA-PEG-G2-FA. To quantitatively evaluate the MR imaging effect, the r2 relaxivity of LAP/Fe3O4-PLA-PEG-G2-FA was measured to be 327.6 mM−1 s−1 in terms of Fe. Although it is slightly lower than that of LAP/Fe3O4 due to the less accessibility of water protons to Fe3O4 surfaces by polymer modification (Ding et al., 2016), it is still significantly higher than individual Fe3O4 NPs (247.6 mM−1 s−1), and the commercial Feridex® and Rsovist® products as well (Wahsner et al., 2019). Therefore, LAP/Fe3O4-PLA-PEG-G2-FA could act as a high-performance T2-weighted MR contrast agent, especially considering their specific targeting capability to cancer cells overexpressing folate receptors.
2.9. Statistical analysis The one-way analysis of variance (ANOVA) statistical method was performed to evaluate the experimental data. A value of 0.05 was selected as the significance level and the data were indicated with (*) for p < .05, (**) for p < .01, and (***) for p < .001, respectively. 3. Results and discussion 3.1. Synthesis and Characterization of LAP/Fe3O4-PLA-PEG-G2-FA NPs In this study, folic acid modified LAP-stabilized Fe3O4 nanoparticles were synthesized by the assembly of PLA-PEG-COOH and conjugate G2FA on the surface of LAP/Fe3O4 NPs. The intermediate product and the formed LAP/Fe3O4-PLA-PEG-G2-FA were characterized by various techniques. Firstly, the successful conjugation of folic acid on G2 was confirmed by 1H NMR spectroscopy, and the number of FA modified onto each G2 was estimated to be 1.5 according to NMR integration (Fig. S1). Then the formation of LAP/Fe3O4-PLA-PEG-G2-FA NPs was qualitatively verified by FTIR spectroscopy (Fig. 1a). As shown in all samples, the obvious peak around 600 cm−1 belongs to the FeeO vibration of iron oxide (Ding et al., 2016). The successful modification of PLA-PEG-COOH is demonstrated by the new peaks at 2896 cm−1 of CeH stretching, 1755 cm−1 of C]O stretching, 1458 cm−1 of CH2 bending, and 1103 cm−1 of CeO stretching in the spectra of LAP/ Fe3O4-PLA-PEG-COOH (Zhuang et al., 2017). For LAP/Fe3O4-PLA-PEGG2-FA, the FT-IR spectra showed an intensity increase of peaks at 3500–3300 cm−1 and 1650 cm−1 probably due to NeH stretching and bending. And in their UV–vis spectra (Fig. S2), the appearance of the featured absorbance of FA at 280 nm clearly demonstrated the successful conjugation of G2-FA (Lin et al., 2009). In addition, the stepwise synthesis of LAP/Fe3O4-PLA-PEG-G2-FA NPs was verified by TGA (Fig. 1b). Similar to pristine LAP (Wu et al., 2014), LAP/Fe3O4 NPs only showed a slight weight loss of 9.0% under 200 °C due to the vaporization of interlayered water molecules, while LAP/Fe3O4-PLA-PEG-
3.3. In vitro cytotoxicity and cellular targeted uptake of LAP/Fe3O4-PLAPEG-G2-FA NPs In this study, HeLa cells with FA receptors over-expressed were chosen as model cells, named as HeLa-HFAR. HeLa cells expressing lowlevel of FAR were set by incubating HeLa cells in FA-containing medium (2.5 mM) for 5 h to block the FA receptors on surface, denoted as HeLa-LFAR (Hu et al., 2016; Hu et al., 2018b; Li et al., 2016). To investigate the biocompatibility of LAP/Fe3O4-PLA-PEG-G2-FA NPs, the viability of HeLa-HFAR incubated with NPs at different Fe concentration from 0.25 to 2.0 mM for 24 h were measured by CCK8 assays (Fig. 3a). Compared with cells treated with PBS, HeLa cells incubated with LAP/Fe3O4-PLA-PEG-G2-FA NPs at different concentrations displayed a similar viability about 99%, indicating their excellent cytocompatibility. This was also demonstrated by the morphology observation of cells treated with LAP/Fe3O4-PLA-PEG-G2-FA NPs in Fig. S5, which showed no significant change of cell morphology in comparison with PBS group. To verify the targeting capability of LAP/Fe3O4-PLA-PEG-G2-FA 4
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Fig. 1. (a) FT-IR spectra and (b) TGA curves of LAP/Fe3O4, LAP/Fe3O4-PLA-PEG-COOH, LAP/Fe3O4-PLA-PEG-G2-FA. (c)TEM micrograph and (d) the size distribution histogram of LAP/Fe3O4-PLA-PEG-G2-FA. Table 1 ζ-potential and hydrodynamic size of LAP, LAP/Fe3O4, LAP/Fe3O4-PLA-PEGCOOH, and LAP/Fe3O4-PLA-PEG-G2-FA. Materials
Zeta potential (mV)
Hydrodynamic size (nm)
Polydispersity index (PDI)
LAP LAP/Fe3O4 LAP/Fe3O4-PLAPEG-COOH LAP/Fe3O4-PLAPEG-G2-FA
−34.9 ± 1.5 −18.0 ± 1.3 −10.9 ± 0.9
113.1 ± 1.4 201.4 ± 0.2 271.1 ± 3.6
0.18 ± 0.01 0.28 ± 0.01 0.31 ± 0.04
+19.8 ± 0.4
332.1 ± 6.4
0.39 ± 0.03
NPs to HeLa cells with high expression of folate receptors, the Fe concentration in HeLa cells were measured by ICP after incubated with LAP/Fe3O4-PLA-PEG-G2-FA NPs at different concentrations for 4 h (Fig. 3b). It is clear that with the increasing concentration of LAP/ Fe3O4-PLA-PEG-G2-FA NPs, the intracellular Fe concentration in both HeLa-HFAR and HeLa-LFAR cells enhanced, indicating the cellular uptake of NPs. It is worth noting that HeLa-LFAR cells exhibited a significantly lower intracellular Fe concentration than HeLa-HFAR cells when incubated with the same concentration of NPs (p < .001), indicating that the block of FA receptors on cell surface by free FA could hinder the uptake of NPs. This result demonstrated that LAP/Fe3O4PLA-PEG-G2-FA NPs could specially target and accumulate at cancer cells overexpressing folate receptors via FA-mediated endocytosis. Moreover, the targeting and effective uptake of LAP/Fe3O4-PLAPEG-G2-FA NPs could be directly observed by Prussian blue staining (Fig. 3c), since Potassium ferrocyanide would change into blue dots after combining with intracellular Fe3+ and the depth of color depends on the concentration of Fe. Compared with the colorless PBS control group, blue dots inside cells were clearly observed after treated with LAP/Fe3O4-PLA-PEG-G2-FA NPs, and the color gradually changed darker as the increase of Fe concentration. More importantly, at the same NP concentration, HeLa-HFAR cells displayed much more blue dots than HeLa-LFAR cells, indicating that LAP/Fe3O4-PLA-PEG-G2-FA
Fig. 2. (a) Color T2-weighted MR images and (b) linear fitting of 1/T2 of LAP/ Fe3O4-PLA-PEG-G2-FA at Fe concentration of 0.01, 0.02, 0.04, 0.08, and 0.16 mM.
NPs could effectively target HeLa cells overexpressing FA receptors and be internalized efficiently. Additionally, the intracellular upake and localization of LAP/Fe3O4-PLA-PEG-G2-FA was visually tracked by TEM (Fig. S6). After 4 h of incubation, LAP/Fe3O4-PLA-PEG-G2-FA NPs could be taken up and trapped predominantly in the cytoplasm of HeLa cells. As a result, LAP/Fe3O4-PLA-PEG-G2-FA possessed good 5
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Fig. 3. (a) CCK8 viability assay of HeLa cells treated with LAP-Fe3O4-PLA-PEG-G2-FA NPs at Fe concentrations of 0–2.0 mM mL−1 for 24 h. (b) Cellular Fe concentration and (c) Prussian blue staining of HeLa-HFAR and HeLa-LFAR cells treated with PBS and LAP/Fe3O4-PLA-PEG-G2-FA NPs at different Fe concentrations for 4 h. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.5. In vivo MR imaging of tumors
biocompatibility and could target to cancer cells overexpressing folate receptors via FA-mediated endocytosis.
A xenografted HeLa tumor model was established, and LAP/Fe3O4PLA-PEG-G2-FA NPs were intravenously injected into mice to estimate the in vivo MR imaging effect. To assess the targeting effect of LAP/ Fe3O4-PLA-PEG-G2-FA, FA block group (FA + NPs) was set by intratumoral injection of FA (2 mM, 0.1 mL) solution 30 min before the administration of NPs in order to block the FAR expression on HeLa cells. Clearly, after the injection of LAP/Fe3O4-PLA-PEG-G2-FA NPs, the color of tumor area turned from red to yellow (NPs group) or green (FA + NPs group) within 1.5 h (Fig. 5a), indicating that the decrease of MR signal possibly resulted from the gradual accumulation of NPs at tumor sites. Moreover, compared with the FA + NPs group, more green color could be found in the tumor MR images of NPs group, demonstrating the enhanced decrease of MR signal intensity and the higher concentration of NPs at tumor. After 3 h postinjection, the tumor MR signal began to recover, which is likely because of the metabolism of NPs from tumor to other tissues and organs. The MR signal intensity result in Fig. 5b revealed a similar decreasing trend. More importantly, the tumor MR signal intensity of FA + NPs group is significantly higher than that of NPs group (p < .05). Specifically, at 1.5 h post-injection, the MR signal intensity of tumor in NPs group could reduce over 27%, while that in FA + NPs group only decreased 10%. This demonstrated that large amount of LAP/Fe3O4-PLA-PEG-G2-FA NPs could accumulate
3.4. Targeted MR imaging of cancer cells The in vitro MR imaging performance of LAP/Fe3O4-PLA-PEG-G2-FA NPs was evaluated by collecting the MR imaging of HeLa cells after incubated with NPs at different Fe concentration for 4 h (Fig. 4a). With the increase of NP concentration, the MR images of both HeLa-HFAR and HeLa-LFAR cells changed from red to deep blue, illustrating the gradual reduction of MR signal. And the decrease of MR signal intensity in HeLa-HFAR cells is much more obvious than that of HeLa-LFAR cells when treated with the same concentration of NPs. Then the MR signal intensity of different groups were quantitatively evaluated in Fig. 4b. The MR signal intensity decreased dramatically with the increase of NP concentration, indicating the enhanced cellular uptake of NPs. Importantly, the signal value of HeLa-HFAR cells is significantly lower than that of HeLa-LFAR cells at all concentrations (p < .01). This may be attributed to the specific targeting and more uptake of LAP/Fe3O4PLA-PEG-G2-FA NPs by HeLa-HFAR cells via FA-mediated endocytosis. Therefore, LAP/Fe3O4-PLA-PEG-G2-FA NPs could act as a targeted T2weighted MR contrast agent for cancer cells with high expression of folate receptors. 6
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Fig. 6. The biodistribution of Fe in the major organs and tumor at different time intervals after intravenous injection of LAP/Fe3O4-PLA-PEG-G2-FA NPs (Fe: 2 mg mL−1, 0.2 mL).
at tumor overexpressing FA receptors by both passive EPR effect and active FAR-mediated targeting pathway during body circulation. Therefore, LAP/Fe3O4-PLA-PEG-G2-FA NPs have the potential to be an effective contrast agent for in vivo targeted tumor MR imaging. 3.6. In vivo biodistribution Fig. 4. (a) Color T2-weighted MR images and (b) MR signal intensity analysis of cells after treated with LAP/Fe3O4-PLA-PEG-G2-FA NPs at an Fe concentration of 0, 0.1, 0.2, and 0.4 mM for 4 h, respectively. 1 and 2 represented HeLa-LFAR cells and HeLa-HFAR cells respectively.
In order to evaluate the metabolism of LAP/Fe3O4-PLA-PEG-G2-FA NPs, the Fe concentration at heart, liver, spleen, lung, kidney, and tumor were measured by ICP-OES at 0 h, 1.5 h, 3 h, 12 h, and 24 h (Fig. 6). Notably, tumor displayed a significant increase of Fe concentration even at 12 h postinjection of LAP/Fe3O4-PLA-PEG-G2-FA NPs (p < .05), while LAP/Fe3O4 would suffer from the limitation of rapid clearance within 12 h. This result indicated that LAP/Fe3O4-PLAPEG-G2-FA NPs could target and accumulate at tumor with a high concentration for a relatively long time, which is valuable for the accurate diagnosis of MR imaging. Moreover, the Fe concentration at major organs increased in the first 3 h postinjection of LAP/Fe3O4-PLAPEG-G2-FA NPs, and the Fe content in liver, spleen and lung increased significantly probably due to the uptake of NPs by RES. Interestingly, the Fe concentration in lung and spleen was much lower than unmodified LAP/Fe3O4 (Fig. S7), suggesting that the modification of PEG on surface could weaken the MPS clearance and thereby enhance the dose available for the tumor site (Hu et al., 2013). During 12–24 h, the Fe concentration in major organs began to gradually reduce to the preinjection level, indicating the metabolism of NPs. Therefore, as MR contrast agents, LAP/Fe3O4-PLA-PEG-G2-FA NPs could be not only accumulate at the tumor sites to enhance the MR imaging contrast of tumor, but also be excreted from the body efficiently within 24 h. 4. Conclusion In conclusion, a targeted LAP-based MR imaging agent was synthesized for cancer cells overexpressing folate receptors. The formed LAP/Fe3O4-PLA-PEG-G2-FA NPs possessed excellent colloidal stability, good biocompatibility, and an enhanced r2 relaxivity of 327.6 mM−1 s−1 for MR imaging. Moreover, they could specifically target cancer cells with high expression of FA receptors, and uptake efficiently by cancer cells via the FA-medicated endocytosis. In vivo experiments verified that LAP/Fe3O4-PLA-PEG-G2-FA NPs can specifically accumulate at tumor site, reduce their MR signal significantly, and be metabolized from body within 24 h. In sum, the synthesized LAP/ Fe3O4-PLA-PEG-G2-FA NPs have a huge potential as targeted T2-
Fig. 5. (a) In vivo T2-weighted MR images and (b) MR signal intensity of tumor after intravenous injection of LAP/Fe3O4-PLA-PEG-G2-FA (Fe: 1 mgmL−1, 150 μL) for 0 h, 0.5 h, 1.5 h, and 3 h (Group 2: NPs group). For Group 1: FA + NPs group, FA (2 mM, 0.1 mL) was intratumorally injected for 30 min before the administration of NPs.
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weighted MR imaging contrast for the early diagnosis of tumor overexpressing folate receptors.
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Acknowledgements This study is financially supported by National Natural Science Foundation of China (21785031 and 81761148028), Natural Science Foundation of Shanghai (17ZR1401200), and the Talent Development Fund of Shanghai (2019115). Credit author statement Ling Ding and Ruizhi Wang contribute equally to this article. Ling Ding, Xiangyang Shi and Rui Guo designed the experiments. Ling Ding, Ruizhi Wang, and Yong Hu performed the experiments. Ni Zhang and Fanli Xu contributed to discussion of experiments. Xiaolin Wang and Xueyan Cao contributed to the imaging and cell experiments. Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.clay.2020.105447. References Arami, H., Khandhar, A., Liggitt, D., Krishnan, K.M., 2015. In vivo delivery, pharmacokinetics, biodistribution and toxicity of iron oxide nanoparticles. Chem. Soc. Rev. 44, 8576–8607. Cai, H., An, X., Cui, J., Li, J., Wen, S., Li, K., Shen, M., Zheng, L., Zhang, G., Shi, X., 2013. Facile hydrothermal synthesis and surface functionalization of polyethyleneiminecoated iron oxide nanoparticles for biomedical applications. ACS Appl. Mater. Interfaces 5, 1722–1731. Cao, Z., Zhang, L., Liang, K., Cheong, S., Boyer, C., Gooding, J.J., Chen, Y., Gu, Z., 2018. Biodegradable 2D Fe–Al hydroxide for nanocatalytic tumor-dynamic therapy with tumor specificity. Adv. Sci. 5, 1801155. Chen, Z.Y., Peng, Y.T., Xie, X.X., Feng, Y., Li, T.T., Li, S., Qin, X., Yang, H., Wu, C.H., Zheng, C., Zhu, J., You, F.M., Liu, Y.Y., 2019. Dendrimer-functionalized superparamagnetic nanobeacons for real-time detection and depletion of HSP90 alpha mRNA and MR imaging. Theranostics 9, 5784–5796. Cheng, L., Wang, X., Gong, F., Liu, T., Liu, Z., 2019. 2D nanomaterials for cancer theranostic applications. advanced materials n/a, 1902333. Ding, L., Hu, Y., Luo, Y., Zhu, J., Wu, Y., Yu, Z., Cao, X., Peng, C., Shi, X., Guo, R., 2016. LAPONITE (R)-stabilized iron oxide nanoparticles for in vivo MR imaging of tumors. Biomat. Sci. 4, 474–482. German, S.V., Navolokin, N.A., Kuznetsova, N.R., Zuev, V.V., Inozemtseva, O.A., Anis’kov, A.A., Volkova, E.K., Bucharskaya, A.B., Maslyakova, G.N., Fakhrullin, R.F., Terentyuk, G.S., Vodovozova, E.L., Gorin, D.A., 2015. Liposomes loaded with hydrophilic magnetite nanoparticles: Preparation and application as contrast agents for magnetic resonance imaging. Colloids Surf. B: Biointerfaces 135, 109–115. Hadjipanayis, C.G., Machaidze, R., Kaluzova, M., Wang, L., Schuette, A.J., Chen, H., Wu, X., Mao, H., 2010. EGFRvIII antibody-conjugated iron oxide nanoparticles for magnetic Resonance imaging-guided convection-enhanced delivery and targeted therapy of glioblastoma. Cancer Res. 70, 6303–6312. Hu, H., Dai, A., Sun, J., Li, X., Gao, F., Wu, L., Fang, Y., Yang, H., An, L., Wu, H., Yang, S., 2013. Aptamer-conjugated Mn3O4@SiO2 core–shell nanoprobes for targeted magnetic resonance imaging. Nanoscale 5, 10447–10454. Hu, Y., Li, J.C., Yang, J., Wei, P., Luo, Y., Ding, L., Sun, W.J., Zhang, G.X., Shi, X.Y., Shen, M.W., 2015. Facile synthesis of RGD peptide-modified iron oxide nanoparticles with ultrahigh relaxivity for targeted MR imaging of tumors. Biomat. Sci. 3, 721–732. Hu, Y., Wang, R., Wang, S., Ding, L., Li, J., Luo, Y., Wang, X., Shen, M., Shi, X., 2016.
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Research Paper
Folic acid-modified Laponite®-stabilized Fe3O4 nanoparticles for targeted T2-weighted MR imaging of tumor
T
Ling Dinga,b,1, Ruizhi Wangc,d,e,1, Yong Hua, Fanli Xua, Ni Zhanga, Xueyan Caoa, Xiaolin Wangc,d, ⁎ Xiangyang Shia, Rui Guoa, a
College of Chemistry, Chemical Engineering and Biotechnology, State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University, Shanghai 201620, People's Republic of China b Aix-Marseille University, CNRS, Centre Interdisciplinaire de Nanoscience de Marseille (CINaM), Equipe Labellisée Ligue Contre le Cancer, 13288 Marseille, France c Department of Interventional Radiology, Zhongshan Hospital, Fudan University, Shanghai 200032, People's Republic of China d Department of Radiology, Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, People's Republic of China e Department of Radiology, Huadong Hospital, Fudan University, Shanghai, 20040, People's Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Laponite Iron oxide NPs MR imaging Folic acid PAMAM dendrimer
In this study, a targeted T2-weighted MR imaging agent based on laponite (LAP) was constructed for cancer cells overexpressing folate receptors. Firstly, LAP stabilized Fe3O4 NPs are synthesized by a controlled co-precipitation route, and then amphiphilic copolymer poly(lactic acid)-poly(ethylene glycol) (PLA-PEG-COOH) is assembled on the surface of LAP/Fe3O4 NPs to provide additional stability, followed by the conjugate of folic acid modified generation 2 polyamidoamine dendrimer (G2-FA) via EDC coupling chemistry. The formed LAP/Fe3O4PLA-PEG-G2-FA NPs can display good colloidal stability and an enhanced r2 relaxivity of 327.6 mM−1 s−1. In vitro experiments demonstrate that the hybrid nanoparticles are cytocompatible in the studied concentration range, and could specific target cancer cells with FA receptors overexpression. Finally, the xenografted tumor model experiment verifies that LAP/Fe3O4-PLA-PEG-G2-FA NPs can significantly reduce the MR signal of tumor site by specific accumulation, and be metabolized from the mice within 24 h. Therefore, LAP/Fe3O4-PLA-PEGG2-FA NPs with good biocompatibility, a high r2 relaxivity, and FA targetability show a huge potential as a targeted T2-weighted MR imaging contrast for the early diagnosis of tumors overexpressing FA receptors.
1. Introduction Magnetic resonance imaging (MRI) is one of most powerful noninvasive imaging techniques to provide 3D images of soft tissues with high spatial and temporal resolution. For accurate diagnosis of early tumor, contrast agents were always employed to improve the sensitivity and reliability of MRI by increasing the contrast effect of tumors from the normal tissues under external magnetic. Among different kinds of MR contrast agents, superparamagnetic iron oxide nanoparticles (Fe3O4 NPs) have attracted enduring interests due to their good biocompatibility, high saturation magnetization and passive tumor targeting by enhanced permeability and retention (EPR) effect (Arami et al., 2015; Hu et al., 2018a; Jabalera et al., 2019; Wahsner et al., 2019). However, the tendency to aggregate and the lack of active tumor-targeting property of Fe3O4 NPs are the main challenges in constructing high performance contrast agents to locate early tumor lesions.
Till now, various kinds of strategies are used to improve their colloidal stability (Cai et al., 2013; Chen et al., 2019; German et al., 2015; Hu et al., 2015; Nandwana et al., 2018; Zhu et al., 2015), and different targeting agents are modified on surface to enhance their specificity to tumor, such as folic acid (FA) (Li et al., 2013; Luong et al., 2017; Shi et al., 2008), hyaluronic acid (HA)(Li et al., 2014), arginine-glycineaspartic acid (RGD) peptide (Hu et al., 2015), and antibodies (Hadjipanayis et al., 2010). For example, Li et al. reported a polyethyleneimine (PEI)-mediated approach to synthesis HA modified Fe3O4-PEI NPs, which displayed a good colloidal stability and cytocompatibility, specific uptake and imaging of cancer cells overexpressing CD44 receptors (Li et al., 2014). Luong et al. used folic acid modified polyamidoamine (PAMAM) dendrimers to decorate Fe3O4 NPs, and demonstrated that the targeted NPs showed high accumulation at the folate receptor overexpressed cancer cells than nontargeted ones (Luong et al., 2017). However, probably due to the aggregation of
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Corresponding author. E-mail address: [email protected] (R. Guo). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.clay.2020.105447 Received 15 October 2019; Received in revised form 3 January 2020; Accepted 11 January 2020 0169-1317/ © 2020 Elsevier B.V. All rights reserved.
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dendrimer (G2-FA) via EDC coupling chemistry (Scheme 1). Various techniques would be applied to characterize the structure, morphology and physic-chemical properties of LAP/Fe3O4-PLA-PEG-G2-FA NPs. Moreover, a human cervical carcinoma cell line (HeLa cells) overexpressing FA receptors was used to evaluate the MR imaging performance in vitro and the HeLa xenografted tumor model was established for in vivo assessment. As far as we know, it is the first study reporting on the modification of LAP-Fe3O4 NPs as a promising targeted contrast agent for in vivo T2-weighted MR imaging of tumors.
iron oxide NPs during modification or the barrier effect of polymer shells, those contrast agents usually exhibited a decreased r2 relaxivity, which may restrict their performance for accurate MR imaging. Recently, 2-dimensional inorganic nanomaterials have inspired considerable interest in the field of biomedicine (Cao et al., 2018; Cheng et al., 2019). As a kind of synthesized nanoclay with good colloidal stability, biocompatible and biodegradability, Laponite® (LAP) has been widely applied in the delivery of therapeutic agents and functional nanoparticles by the virtue of its distinctive layered structure (Liu et al., 2019; Tomás et al., 2018). For instance, LAP could load anticancer drug doxorubicin with high encapsulating efficiency, release drugs in a pH-sensitive profile, and exhibit a better therapeutic efficiency than free drug (Wang et al., 2013; Wu et al., 2014). LAP stabilized gold nanoparticles are demonstrated to be a potential CT contrast agent with good stability and high X-ray attenuation coefficients (Zhuang et al., 2017). Especially in constructing MR imaging agents, LAP could not only provide additional colloidal stability for iron oxide NPs, but also improve the r2 relaxivity significantly by suppressing the magnetic interparticle interactions (Tzitzios et al., 2010). In our previous work, LAP-Fe3O4 NPs displayed a high r2 relaxivity as 475.9 mM−1 s−1, which is 2 folds higher than that of Fe3O4 NPs (Ding et al., 2016). However, the rapid clearance by the reticuloendothelial system (RES) and the non-specificity of LAP/Fe3O4 NPs may limit the dose available for the early diagnosis of tumors. Since PEG chains could mitigate the interaction between NPs and macrophages (Perry et al., 2012; Wang et al., 2014b), a biocompatible amphiphilic block copolymer polylactic acid-polyethylene glycol (PLA-PEG) could be assembled on the surface of LAP to escape the RES clearance and prolong the blood circulation time. Moreover, the active carboxyl group at the end of PLA-PEG could be further modified with targeting agents to endow the specific targeting capability of nanoparticles. In our previous study, folic acid (FA) was used as an effective targeting ligand to modify PAMAM dendrimer, and FA-conjugated PAMAM dendrimers were demonstrated to achieve high binding and internalization into cancer cells overexpressing folate receptors (Li et al., 2013; Liu et al., 2013; Wang et al., 2014a). Therefore, the assemble of PEG-PLA and decoration of FA-PAMAM dendrimer on the surface of LAP/Fe3O4 NPs would be a promising strategy to improve their in vivo stability and achieve active targeting of cancer overexpressing FAR. Therefore, in this study, Fe3O4 NPs were synthesized in the presence of LAP with a controlled co-precipitation method, and then amphiphilic PLA-PEG-COOH were assembled on the surface of LAP/Fe3O4 NPs to provide additional stability and active carboxyl groups, followed by the conjugate of folic acid modified generation 2 polyamidoamine
2. Experimental 2.1. Materials LAPONITE® (Na+0.7[(Si8Mg5.5Li0.3)O20(OH)4] −0.7, LAP) was purchased from Zhejiang Institute of Geology and Mineral Resources (Hangzhou, China). Folic acid, ferric chloride hexahydrate (FeCl3·6H2O > 99%), ferrous chloride tetrahydrate (FeCl2·4H2O > 99%), sodium hydroxide and hydrochloric acid (HCl = 37%) were obtained from Aldrich. PLA-PEG-COOH with a molecular weight of 8000 Da (3000 Da for PLA and 5000 Da for PEG) was purchased from Jinan Daigang Biological Technology Co, Ltd. (Jinan, China). Amine-terminated PAMAM dendrimers of generation 2 (G2) were purchased from Dendritech (Midland, MI). HeLa cells (a human cervical carcinoma cell line) were obtained from Institute of Biochemistry and Cell Biology, the Chinese Academy of Sciences (Shanghai, China). Perls stain was obtained from Beijing Leagene Biotechnology Co., Ltd. (Beijing, China). Perlsstain A1 and Perls stain A2 was mixed equivalently. Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Hangzhou Jinuo Biomedical Technology Co., Ltd. (Hangzhou, China). All chemicals purchased were used without further purification. Water with a resistivity of 18.2 MΩ cm purified by using a Milli-Q Plus 185 water purification system (Millipore, Bedford, MA) was used. 2.2. Preparation of LAP/Fe3O4-PLA-PEG-G2-FA NPs LAP/Fe3O4 NPs were firstly synthesized according to our previous work by a controlled co-precipitation method (Ding et al., 2016). In brief, 15 mL aqueous solution (containing 0.089 mL HCl) of FeCl3·6H2O (0.721 g) and FeCl2·4H2O (0.265 g) was added into the LAP solution (10 mg mL−1, 50 mL) magnetic stirring, and then 10 mL of NaOH (2.0 g) aqueous solution was added into the above mixture under highspeed stirring. After 2 h of reaction at 80 °C under N2 atmosphere, the
Scheme 1. Schematic illustration of the synthesis of LAP/Fe3O4-PLA-PEG-G2-FA. 2
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LFAR). Unless otherwise stated, the term of HeLa cells always represents the HeLa-HFAR cells. CCK-8 assay was used to quantify the viability of HeLa cells after treated with LAP/Fe3O4-PLA-PEG-G2-FA NPs at different concentrations. Briefly, HeLa cells were seeded in 96-well plates at a density of 1 × 104 cells/well with 200 μL of fresh DMEM at the day before the experiment. After 24 h, the medium was replaced by 200 μL fresh medium containing PBS (control) or LAP/Fe3O4-PLA-PEG-G2-FA NPs with different Fe concentrations (0.25, 0.5, 1.0, 1.5 and 2.0 mM). After 24 h incubation, the medium was discarded and the cells were washed with PBS 3 times, followed by addition of 200 μL fresh DMEM containing 20 μL CCK-8. After incubation of the cells at 37 °C for another 4 h, they were measured the absorbance by Thermo Scientific Multiskan MK3 ELISA reader (Thermo Scientific, Hudson, NH) at 450 nm in each well. The standard deviation of 5 wells of each sample were recorded. To further confirm the cytotoxicity of LAP/Fe3O4-PLA-PEG-G2-FA NPs, HeLa cells were treated with PBS or LAP/Fe3O4-PLA-PEG-G2-FA NPs at Fe concentration of 0.2, 0.4, 0.8, 1.5, and 2.0 mM, respectively. The cell morphology was observed by a Leica DM IL LED inverted phase contrast microscope with a magnification of 200 × for each sample. TEM observation was applied to visually examine the internalization of LAP/Fe3O4-PLA-PEG-G2-FA NPs in HeLa cells according to protocols described in the literature (Ding et al., 2016).
reaction mixture was purified by magnetic separation to obtain LAP/ Fe3O4 NPs. Then LAP/Fe3O4 solution (6.4 mL, 20 mg mL−1) was dropwise added into PLA-PEG-COOH (20 mL, 6 mg mL−1) water/ acetone (1:1) solution. After stirring for 24 h, LAP/Fe3O4-PLA-PEGCOOH solution was purified by dialyzing the mixture against phosphate buffered saline (PBS) and water (2 L each time, 3 times each day) for 3 days with a 14,000 MWCO dialysis membrane. G2-FA was synthesized according to our previous work (Liu et al., 2013). Briefly, FA (26.84 mg) was dissolved in DMSO (5 mL), and then EDC (10.8 mg) and NHS (6.5 mg) was added under magnetic stirring. After 3 h of reaction, the activated FA solution was added to 5 mL G2 (39.6 mg) DMSO solution under vigorous stirring for 3 days. Thereafter, the reaction mixture was extensively dialyzed against water with a 2000 MWCO dialysis membrane for 3 days to remove excess reactants. Finally, LAP/Fe3O4-PLA-PEG-COOH NPs solution obtained above (198 mg, 4 mL) was mixed with EDC (2.98 mg, 1 mL) and NHS (1.79 mg, 1 mL) under vigorous magnetic stirring for 3 h to activate the carboxyl group. Then, the G2-FA aqueous solution (28 mg, 5 mL) was dropwise added into the above solution under magnetic stirring for 3 days. The mixture was dialysis with water for 3 days to obtain LAP/ Fe3O4-PLA-PEG-G2-FA. 2.3. Characterization 1 H NMR spectra were measured on a Bruker AV400 NMR spectrometer. Samples were dissolved in deuterated D2O before measurement. The FTIR spectra was acquired using a Nicolet Nexus 670FTIR (NicoletThermo) spectrometer. The spectra of all samples were recorded in a transmission mode with the wavenumbers in the range of 400–4000 cm−1. Before analysis, the dry samples mixed with KBr crystals were pressed to form ground pellets. The iron concentrations of samples were analyzed by using a Leeman Prodigy ICP-OES system (Hudson, NH). UV/Vis spectroscopy was obtained using a Lambda 25 UV/Vis spectrophotometer (Perkin-Elmer, USA). The organic component of the samples was quantified by thermogravimetric analysis (TGA) using a TG 209 F1 (NETZSCH Instruments Co., Ltd., Selb/ Bavaria, Germany) thermogravimetric analyzer. The samples were heated from 25 °C to 900 °C at a rate of 10 °C/min under nitrogen atmosphere. Zeta potential and dynamic light scattering (DLS) measurements were carried out using a Malvern Zetasizer Nano ZS model ZEN3600 (Worcestershire, U.K.) equipped with a standard 633 nm laser. Transmission electron microscopy (TEM) was carried out with a JEOL 2010 analytical electron microscope (Tokyo, Japan) operating at 200 kV to characterize the morphology and size of the NPs. Before performing the measurements, the samples were prepared by putting a drop of diluted NP suspension (6 μL) onto a carbon-coated copper grid and dried in air. For each sample, at least 200 particles in different TEM images were randomly selected and measured by using ImageJ software to calculate the size distribution of NPs. T2 relaxometry was performed by a 0.5-T NMI20-Analyst NMR Analyzing and Imaging system (Shanghai Niumag Corporation, China). The samples were diluted in water with Fe concentrations in the range of 0.005–0.08 mM. The instrumental parameters were set at point resolution of 156 mm × 156 mm, section thickness of 0.6 mm, TR of 4000 ms, TE of 60 ms, and number of excitations of 1. The r2 relaxivity was calculated by a linear fit of the inverse T2 (1/T2) relaxation time as a function of Fe concentration.
2.5. Cellular uptake study ICP-OES was carried out to investigate the cellular uptake of LAP/ Fe3O4-PLA-PEG-G2-FA NPs by HeLa-HFAR or HeLa-LFAR cells. Both cells were seeded into 12-well plates at a density of 2 × 105 cells/well. After incubation at 37 °C and in 5% CO2 atmosphere overnight, the medium was replaced with fresh medium containing NPs at different Fe concentrations (0.05–0.2 mM). After 4 h incubation, the medium was discarded carefully and the cells were washed with PBS for 5 times, trypsinized, centrifuged, and counted by Handheld Automated Cell Counter (Millipore, Billerica, MA). The remaining cells were lysed using aqua Regia solution (1.0 mL) for 2 days, and then the Fe uptake in cells was quantified by ICP-OES. Prussian blue staining of LAP/Fe3O4-PLA-PEG-G2-FA NPs in HeLaLFAR or HeLa-HFAR cells was performed to visually examine the cellular Fe uptake. Similar to the protocol described above, HeLa cells were treated by LAP/Fe3O4-PLA-PEG-G2-FA NPs with Fe concentrations of 0.05 mM and 0.1 mM. After 4 h, the cells were washed for 3 times with PBS, fixed with glutaraldehyde solution (2.5%) at 4 °C for 15 min, and stained with Prussian blue reagent at 37 °C for 30 min. The cells were then imaged by Leica DM IL LED inverted phase contrast microscope with a magnification of 200 × for each sample. 2.6. In vitro MR imaging of cancer cells In brief, HeLa-HFAR or HeLa-LFAR cells were seeded into 6-well plates at a density of 2 × 106 cells/well with 2 mL of fresh DMEM and incubated at 37 °C and 5% CO2 overnight to bring the cells to confluence. Then the medium was replaced by LAP/Fe3O4-PLA-PEG-G2-FA NPs contained medium at different concentrations of Fe (0.1, 0.2, 0.4 mM, respectively) and incubated for 4 h. Afterwards, the cells were washed 5 times with PBS, trypsinized, centrifuged, and resuspended in 1 mL PBS (containing 0.5% agarose). T2-weighted MR imaging of each sample was carried out using a 1.5 T Signal HDxt super conductor clinical MR system (GE Medical Systems, Milwaukee, WI) under the following conditions: point resolution = 156 mm × 156 mm, section thickness = 0.6 mm, TR = 2700 ms, TE = 69 ms, and number of excitation = 1.
2.4. In vitro cytotoxicity assay and cellular uptake HeLa cells overexpressing FA receptors were cultured and passaged in 25-cm2 plates with DMEM supplemented with 10% FBS and 1% penicillin/streptomycin under 37 °C and 5% CO2. HeLa cells grown in FA-free medium expressed high-level FA receptors (denoted as HeLaHFAR), while the cells pre-treated with FA-containing medium (2.5 mM) for 5 h expressed low-level FA receptors (denoted as HeLa-
2.7. In vivo targeted MR imaging All animal experiments were approved by institutional committee 3
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COOH and LAP/Fe3O4-PLA-PEG-G2-FA displayed huge weight losses of 66.3% and 73.3% in the range of 200 to 900 °C, respectively. This confirmed that both PLA-PEG-COOH and G2-FA were successfully modified on LAP/Fe3O4 NPs as design. Finally, the morphology and size distribution of LAP/Fe3O4-PLA-PEG-G2-FA NPs were observed by TEM (Fig. 1c and d). The synthesized Fe3O4 NPs could be easily identified due to their high contrast under TEM observation, and displayed a mean size of 9.8 ± 1.7 nm, indicating that surface modification did not alter the size and nanocrystal structure of iron oxide. And it is interesting that LAP/Fe3O4-PLA-PEG-G2-FA NPs displayed a more dispersed state than LAP/Fe3O4 NPs (Fig. S3), probably attributing to the protective effect of polymers on surface. In sum, folic acid modified LAP/Fe3O4-PLA-PEG-G2-FA NPs were successfully synthesized. The hydrodynamic size and surface potential of LAP/Fe3O4-PLAPEG-G2-FA were further studied by DLS as shown in Table 1. The significant increase of surface potential from −34.9 mV of pristine LAP to −18.0 mV of LAP/Fe3O4, was mainly attributed to the offsetting of negative charge on LAP by positively charged Fe3O4. LAP/Fe3O4-PLAPEG-COOH displayed a surface potential of −10.9 mV by the shield of PLG-PEG, and further conjugate of G2-FA may reverse the Zeta potential to 19.8 mV because of the conjugate of positive G2-FA on surface. These results further verified the successful synthesis of LAP/Fe3O4PLA-PEG-FA. In addition, due to the modification of PEG chain, the hydrodynamic diameter of LAP/Fe3O4-PLA-PEG-COOH and LAP/ Fe3O4-PLA-PEG-FA increased to 271.1 nm and 332.1 nm, respectively, and keep below 500 nm when dispersed in PBS and culture medium (Fig. S4).
for animal care according to the policy of the National Ministry of Health. HeLa cells (1.5 × 106/mouse) were subcutaneously injected into the back of male 6-week-old BALB/c nude mice (15–20 g, Shanghai Slac Laboratory Animal Center, Shanghai, China) to establish the xenografted tumor model. When the tumor volume reached 0.5–0.8 cm in diameter (3 week post-injection), the mice were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg) and divided into 2 groups: For Group 1, the mice was intravenously injected with LAP/Fe3O4-PLA-PEG-G2-FA NPs; Group 2, the mice was intratumorally pre-injected with free FA (2 mM, 0.1 mL PBS) for 30 min, and then LAP/Fe3O4-PLA-PEG-G2-FA NPs (Fe: 1 mg mL−1, 150 μL PBS) was intravenously injected. For T2-weighted MR imaging, each mouse was imaged by 1.5 T Signal HDxt superconductor clinical MR system coupled with a custom-built rodent receiver coil (Chenguang Med Tech, Shanghai, China). Two-dimensional (2D) spin-echo MR images were obtained before and at the time points of 0, 0.5, 1.5 and 3 h post-injection of the LAP/Fe3O4-PLA-PEG-G2-FA NPs with the conditions similar to those used for in vitro MR imaging of cancer cells. 2.8. In vivo biodistribution To assess the biodistribution of LAP/Fe3O4-PLA-PEG-G2-FA NPs, the tumor-bearing BALB/c nude mice were anesthetized by intraperitoneal injection of pentobarbital sodium (40 mg/kg). After intravenous injection of LAP/Fe3O4-PLA-PEG-G2-FA NPs ([Fe] =2 mg mL−1, 0.2 mL PBS), the mice were sacrificed at 1.5, 3, 12, and 24 h post injection, and the heart, liver, spleen, lung, kidney, and tumor were harvested, weighed and digested by aquaregia for 2 days. The tumor-bearing mice before treatment were used as control, and the Fe concentrations in tumor and organs were simultaneously determined by ICP-AES.
3.2. MR imaging property of LAP/Fe3O4-PLA-PEG-G2-FA solution The potential of LAP/Fe3O4-PLA-PEG-G2-FA as T2-weighted MR contrast agents was explored by measuring the MR image and transverse relaxation time (T2) of solutions (Fig. 2). With the increase of Fe concentration, the color MR image of NPs solutions turned from red to blue, indicating the decrease of MR signal by the negative contrast effect of LAP/Fe3O4-PLA-PEG-G2-FA. To quantitatively evaluate the MR imaging effect, the r2 relaxivity of LAP/Fe3O4-PLA-PEG-G2-FA was measured to be 327.6 mM−1 s−1 in terms of Fe. Although it is slightly lower than that of LAP/Fe3O4 due to the less accessibility of water protons to Fe3O4 surfaces by polymer modification (Ding et al., 2016), it is still significantly higher than individual Fe3O4 NPs (247.6 mM−1 s−1), and the commercial Feridex® and Rsovist® products as well (Wahsner et al., 2019). Therefore, LAP/Fe3O4-PLA-PEG-G2-FA could act as a high-performance T2-weighted MR contrast agent, especially considering their specific targeting capability to cancer cells overexpressing folate receptors.
2.9. Statistical analysis The one-way analysis of variance (ANOVA) statistical method was performed to evaluate the experimental data. A value of 0.05 was selected as the significance level and the data were indicated with (*) for p < .05, (**) for p < .01, and (***) for p < .001, respectively. 3. Results and discussion 3.1. Synthesis and Characterization of LAP/Fe3O4-PLA-PEG-G2-FA NPs In this study, folic acid modified LAP-stabilized Fe3O4 nanoparticles were synthesized by the assembly of PLA-PEG-COOH and conjugate G2FA on the surface of LAP/Fe3O4 NPs. The intermediate product and the formed LAP/Fe3O4-PLA-PEG-G2-FA were characterized by various techniques. Firstly, the successful conjugation of folic acid on G2 was confirmed by 1H NMR spectroscopy, and the number of FA modified onto each G2 was estimated to be 1.5 according to NMR integration (Fig. S1). Then the formation of LAP/Fe3O4-PLA-PEG-G2-FA NPs was qualitatively verified by FTIR spectroscopy (Fig. 1a). As shown in all samples, the obvious peak around 600 cm−1 belongs to the FeeO vibration of iron oxide (Ding et al., 2016). The successful modification of PLA-PEG-COOH is demonstrated by the new peaks at 2896 cm−1 of CeH stretching, 1755 cm−1 of C]O stretching, 1458 cm−1 of CH2 bending, and 1103 cm−1 of CeO stretching in the spectra of LAP/ Fe3O4-PLA-PEG-COOH (Zhuang et al., 2017). For LAP/Fe3O4-PLA-PEGG2-FA, the FT-IR spectra showed an intensity increase of peaks at 3500–3300 cm−1 and 1650 cm−1 probably due to NeH stretching and bending. And in their UV–vis spectra (Fig. S2), the appearance of the featured absorbance of FA at 280 nm clearly demonstrated the successful conjugation of G2-FA (Lin et al., 2009). In addition, the stepwise synthesis of LAP/Fe3O4-PLA-PEG-G2-FA NPs was verified by TGA (Fig. 1b). Similar to pristine LAP (Wu et al., 2014), LAP/Fe3O4 NPs only showed a slight weight loss of 9.0% under 200 °C due to the vaporization of interlayered water molecules, while LAP/Fe3O4-PLA-PEG-
3.3. In vitro cytotoxicity and cellular targeted uptake of LAP/Fe3O4-PLAPEG-G2-FA NPs In this study, HeLa cells with FA receptors over-expressed were chosen as model cells, named as HeLa-HFAR. HeLa cells expressing lowlevel of FAR were set by incubating HeLa cells in FA-containing medium (2.5 mM) for 5 h to block the FA receptors on surface, denoted as HeLa-LFAR (Hu et al., 2016; Hu et al., 2018b; Li et al., 2016). To investigate the biocompatibility of LAP/Fe3O4-PLA-PEG-G2-FA NPs, the viability of HeLa-HFAR incubated with NPs at different Fe concentration from 0.25 to 2.0 mM for 24 h were measured by CCK8 assays (Fig. 3a). Compared with cells treated with PBS, HeLa cells incubated with LAP/Fe3O4-PLA-PEG-G2-FA NPs at different concentrations displayed a similar viability about 99%, indicating their excellent cytocompatibility. This was also demonstrated by the morphology observation of cells treated with LAP/Fe3O4-PLA-PEG-G2-FA NPs in Fig. S5, which showed no significant change of cell morphology in comparison with PBS group. To verify the targeting capability of LAP/Fe3O4-PLA-PEG-G2-FA 4
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Fig. 1. (a) FT-IR spectra and (b) TGA curves of LAP/Fe3O4, LAP/Fe3O4-PLA-PEG-COOH, LAP/Fe3O4-PLA-PEG-G2-FA. (c)TEM micrograph and (d) the size distribution histogram of LAP/Fe3O4-PLA-PEG-G2-FA. Table 1 ζ-potential and hydrodynamic size of LAP, LAP/Fe3O4, LAP/Fe3O4-PLA-PEGCOOH, and LAP/Fe3O4-PLA-PEG-G2-FA. Materials
Zeta potential (mV)
Hydrodynamic size (nm)
Polydispersity index (PDI)
LAP LAP/Fe3O4 LAP/Fe3O4-PLAPEG-COOH LAP/Fe3O4-PLAPEG-G2-FA
−34.9 ± 1.5 −18.0 ± 1.3 −10.9 ± 0.9
113.1 ± 1.4 201.4 ± 0.2 271.1 ± 3.6
0.18 ± 0.01 0.28 ± 0.01 0.31 ± 0.04
+19.8 ± 0.4
332.1 ± 6.4
0.39 ± 0.03
NPs to HeLa cells with high expression of folate receptors, the Fe concentration in HeLa cells were measured by ICP after incubated with LAP/Fe3O4-PLA-PEG-G2-FA NPs at different concentrations for 4 h (Fig. 3b). It is clear that with the increasing concentration of LAP/ Fe3O4-PLA-PEG-G2-FA NPs, the intracellular Fe concentration in both HeLa-HFAR and HeLa-LFAR cells enhanced, indicating the cellular uptake of NPs. It is worth noting that HeLa-LFAR cells exhibited a significantly lower intracellular Fe concentration than HeLa-HFAR cells when incubated with the same concentration of NPs (p < .001), indicating that the block of FA receptors on cell surface by free FA could hinder the uptake of NPs. This result demonstrated that LAP/Fe3O4PLA-PEG-G2-FA NPs could specially target and accumulate at cancer cells overexpressing folate receptors via FA-mediated endocytosis. Moreover, the targeting and effective uptake of LAP/Fe3O4-PLAPEG-G2-FA NPs could be directly observed by Prussian blue staining (Fig. 3c), since Potassium ferrocyanide would change into blue dots after combining with intracellular Fe3+ and the depth of color depends on the concentration of Fe. Compared with the colorless PBS control group, blue dots inside cells were clearly observed after treated with LAP/Fe3O4-PLA-PEG-G2-FA NPs, and the color gradually changed darker as the increase of Fe concentration. More importantly, at the same NP concentration, HeLa-HFAR cells displayed much more blue dots than HeLa-LFAR cells, indicating that LAP/Fe3O4-PLA-PEG-G2-FA
Fig. 2. (a) Color T2-weighted MR images and (b) linear fitting of 1/T2 of LAP/ Fe3O4-PLA-PEG-G2-FA at Fe concentration of 0.01, 0.02, 0.04, 0.08, and 0.16 mM.
NPs could effectively target HeLa cells overexpressing FA receptors and be internalized efficiently. Additionally, the intracellular upake and localization of LAP/Fe3O4-PLA-PEG-G2-FA was visually tracked by TEM (Fig. S6). After 4 h of incubation, LAP/Fe3O4-PLA-PEG-G2-FA NPs could be taken up and trapped predominantly in the cytoplasm of HeLa cells. As a result, LAP/Fe3O4-PLA-PEG-G2-FA possessed good 5
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Fig. 3. (a) CCK8 viability assay of HeLa cells treated with LAP-Fe3O4-PLA-PEG-G2-FA NPs at Fe concentrations of 0–2.0 mM mL−1 for 24 h. (b) Cellular Fe concentration and (c) Prussian blue staining of HeLa-HFAR and HeLa-LFAR cells treated with PBS and LAP/Fe3O4-PLA-PEG-G2-FA NPs at different Fe concentrations for 4 h. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.5. In vivo MR imaging of tumors
biocompatibility and could target to cancer cells overexpressing folate receptors via FA-mediated endocytosis.
A xenografted HeLa tumor model was established, and LAP/Fe3O4PLA-PEG-G2-FA NPs were intravenously injected into mice to estimate the in vivo MR imaging effect. To assess the targeting effect of LAP/ Fe3O4-PLA-PEG-G2-FA, FA block group (FA + NPs) was set by intratumoral injection of FA (2 mM, 0.1 mL) solution 30 min before the administration of NPs in order to block the FAR expression on HeLa cells. Clearly, after the injection of LAP/Fe3O4-PLA-PEG-G2-FA NPs, the color of tumor area turned from red to yellow (NPs group) or green (FA + NPs group) within 1.5 h (Fig. 5a), indicating that the decrease of MR signal possibly resulted from the gradual accumulation of NPs at tumor sites. Moreover, compared with the FA + NPs group, more green color could be found in the tumor MR images of NPs group, demonstrating the enhanced decrease of MR signal intensity and the higher concentration of NPs at tumor. After 3 h postinjection, the tumor MR signal began to recover, which is likely because of the metabolism of NPs from tumor to other tissues and organs. The MR signal intensity result in Fig. 5b revealed a similar decreasing trend. More importantly, the tumor MR signal intensity of FA + NPs group is significantly higher than that of NPs group (p < .05). Specifically, at 1.5 h post-injection, the MR signal intensity of tumor in NPs group could reduce over 27%, while that in FA + NPs group only decreased 10%. This demonstrated that large amount of LAP/Fe3O4-PLA-PEG-G2-FA NPs could accumulate
3.4. Targeted MR imaging of cancer cells The in vitro MR imaging performance of LAP/Fe3O4-PLA-PEG-G2-FA NPs was evaluated by collecting the MR imaging of HeLa cells after incubated with NPs at different Fe concentration for 4 h (Fig. 4a). With the increase of NP concentration, the MR images of both HeLa-HFAR and HeLa-LFAR cells changed from red to deep blue, illustrating the gradual reduction of MR signal. And the decrease of MR signal intensity in HeLa-HFAR cells is much more obvious than that of HeLa-LFAR cells when treated with the same concentration of NPs. Then the MR signal intensity of different groups were quantitatively evaluated in Fig. 4b. The MR signal intensity decreased dramatically with the increase of NP concentration, indicating the enhanced cellular uptake of NPs. Importantly, the signal value of HeLa-HFAR cells is significantly lower than that of HeLa-LFAR cells at all concentrations (p < .01). This may be attributed to the specific targeting and more uptake of LAP/Fe3O4PLA-PEG-G2-FA NPs by HeLa-HFAR cells via FA-mediated endocytosis. Therefore, LAP/Fe3O4-PLA-PEG-G2-FA NPs could act as a targeted T2weighted MR contrast agent for cancer cells with high expression of folate receptors. 6
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Fig. 6. The biodistribution of Fe in the major organs and tumor at different time intervals after intravenous injection of LAP/Fe3O4-PLA-PEG-G2-FA NPs (Fe: 2 mg mL−1, 0.2 mL).
at tumor overexpressing FA receptors by both passive EPR effect and active FAR-mediated targeting pathway during body circulation. Therefore, LAP/Fe3O4-PLA-PEG-G2-FA NPs have the potential to be an effective contrast agent for in vivo targeted tumor MR imaging. 3.6. In vivo biodistribution Fig. 4. (a) Color T2-weighted MR images and (b) MR signal intensity analysis of cells after treated with LAP/Fe3O4-PLA-PEG-G2-FA NPs at an Fe concentration of 0, 0.1, 0.2, and 0.4 mM for 4 h, respectively. 1 and 2 represented HeLa-LFAR cells and HeLa-HFAR cells respectively.
In order to evaluate the metabolism of LAP/Fe3O4-PLA-PEG-G2-FA NPs, the Fe concentration at heart, liver, spleen, lung, kidney, and tumor were measured by ICP-OES at 0 h, 1.5 h, 3 h, 12 h, and 24 h (Fig. 6). Notably, tumor displayed a significant increase of Fe concentration even at 12 h postinjection of LAP/Fe3O4-PLA-PEG-G2-FA NPs (p < .05), while LAP/Fe3O4 would suffer from the limitation of rapid clearance within 12 h. This result indicated that LAP/Fe3O4-PLAPEG-G2-FA NPs could target and accumulate at tumor with a high concentration for a relatively long time, which is valuable for the accurate diagnosis of MR imaging. Moreover, the Fe concentration at major organs increased in the first 3 h postinjection of LAP/Fe3O4-PLAPEG-G2-FA NPs, and the Fe content in liver, spleen and lung increased significantly probably due to the uptake of NPs by RES. Interestingly, the Fe concentration in lung and spleen was much lower than unmodified LAP/Fe3O4 (Fig. S7), suggesting that the modification of PEG on surface could weaken the MPS clearance and thereby enhance the dose available for the tumor site (Hu et al., 2013). During 12–24 h, the Fe concentration in major organs began to gradually reduce to the preinjection level, indicating the metabolism of NPs. Therefore, as MR contrast agents, LAP/Fe3O4-PLA-PEG-G2-FA NPs could be not only accumulate at the tumor sites to enhance the MR imaging contrast of tumor, but also be excreted from the body efficiently within 24 h. 4. Conclusion In conclusion, a targeted LAP-based MR imaging agent was synthesized for cancer cells overexpressing folate receptors. The formed LAP/Fe3O4-PLA-PEG-G2-FA NPs possessed excellent colloidal stability, good biocompatibility, and an enhanced r2 relaxivity of 327.6 mM−1 s−1 for MR imaging. Moreover, they could specifically target cancer cells with high expression of FA receptors, and uptake efficiently by cancer cells via the FA-medicated endocytosis. In vivo experiments verified that LAP/Fe3O4-PLA-PEG-G2-FA NPs can specifically accumulate at tumor site, reduce their MR signal significantly, and be metabolized from body within 24 h. In sum, the synthesized LAP/ Fe3O4-PLA-PEG-G2-FA NPs have a huge potential as targeted T2-
Fig. 5. (a) In vivo T2-weighted MR images and (b) MR signal intensity of tumor after intravenous injection of LAP/Fe3O4-PLA-PEG-G2-FA (Fe: 1 mgmL−1, 150 μL) for 0 h, 0.5 h, 1.5 h, and 3 h (Group 2: NPs group). For Group 1: FA + NPs group, FA (2 mM, 0.1 mL) was intratumorally injected for 30 min before the administration of NPs.
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weighted MR imaging contrast for the early diagnosis of tumor overexpressing folate receptors.
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Acknowledgements This study is financially supported by National Natural Science Foundation of China (21785031 and 81761148028), Natural Science Foundation of Shanghai (17ZR1401200), and the Talent Development Fund of Shanghai (2019115). Credit author statement Ling Ding and Ruizhi Wang contribute equally to this article. Ling Ding, Xiangyang Shi and Rui Guo designed the experiments. Ling Ding, Ruizhi Wang, and Yong Hu performed the experiments. Ni Zhang and Fanli Xu contributed to discussion of experiments. Xiaolin Wang and Xueyan Cao contributed to the imaging and cell experiments. Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.clay.2020.105447. References Arami, H., Khandhar, A., Liggitt, D., Krishnan, K.M., 2015. In vivo delivery, pharmacokinetics, biodistribution and toxicity of iron oxide nanoparticles. Chem. Soc. Rev. 44, 8576–8607. Cai, H., An, X., Cui, J., Li, J., Wen, S., Li, K., Shen, M., Zheng, L., Zhang, G., Shi, X., 2013. Facile hydrothermal synthesis and surface functionalization of polyethyleneiminecoated iron oxide nanoparticles for biomedical applications. ACS Appl. Mater. Interfaces 5, 1722–1731. Cao, Z., Zhang, L., Liang, K., Cheong, S., Boyer, C., Gooding, J.J., Chen, Y., Gu, Z., 2018. Biodegradable 2D Fe–Al hydroxide for nanocatalytic tumor-dynamic therapy with tumor specificity. Adv. Sci. 5, 1801155. Chen, Z.Y., Peng, Y.T., Xie, X.X., Feng, Y., Li, T.T., Li, S., Qin, X., Yang, H., Wu, C.H., Zheng, C., Zhu, J., You, F.M., Liu, Y.Y., 2019. Dendrimer-functionalized superparamagnetic nanobeacons for real-time detection and depletion of HSP90 alpha mRNA and MR imaging. Theranostics 9, 5784–5796. Cheng, L., Wang, X., Gong, F., Liu, T., Liu, Z., 2019. 2D nanomaterials for cancer theranostic applications. advanced materials n/a, 1902333. Ding, L., Hu, Y., Luo, Y., Zhu, J., Wu, Y., Yu, Z., Cao, X., Peng, C., Shi, X., Guo, R., 2016. LAPONITE (R)-stabilized iron oxide nanoparticles for in vivo MR imaging of tumors. Biomat. Sci. 4, 474–482. German, S.V., Navolokin, N.A., Kuznetsova, N.R., Zuev, V.V., Inozemtseva, O.A., Anis’kov, A.A., Volkova, E.K., Bucharskaya, A.B., Maslyakova, G.N., Fakhrullin, R.F., Terentyuk, G.S., Vodovozova, E.L., Gorin, D.A., 2015. Liposomes loaded with hydrophilic magnetite nanoparticles: Preparation and application as contrast agents for magnetic resonance imaging. Colloids Surf. B: Biointerfaces 135, 109–115. Hadjipanayis, C.G., Machaidze, R., Kaluzova, M., Wang, L., Schuette, A.J., Chen, H., Wu, X., Mao, H., 2010. EGFRvIII antibody-conjugated iron oxide nanoparticles for magnetic Resonance imaging-guided convection-enhanced delivery and targeted therapy of glioblastoma. Cancer Res. 70, 6303–6312. Hu, H., Dai, A., Sun, J., Li, X., Gao, F., Wu, L., Fang, Y., Yang, H., An, L., Wu, H., Yang, S., 2013. Aptamer-conjugated Mn3O4@SiO2 core–shell nanoprobes for targeted magnetic resonance imaging. Nanoscale 5, 10447–10454. Hu, Y., Li, J.C., Yang, J., Wei, P., Luo, Y., Ding, L., Sun, W.J., Zhang, G.X., Shi, X.Y., Shen, M.W., 2015. Facile synthesis of RGD peptide-modified iron oxide nanoparticles with ultrahigh relaxivity for targeted MR imaging of tumors. Biomat. Sci. 3, 721–732. Hu, Y., Wang, R., Wang, S., Ding, L., Li, J., Luo, Y., Wang, X., Shen, M., Shi, X., 2016.
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