Core-shell NaGdF₄@CaCO₃ nanoparticles for enhanced magnetic resonance/ultrasonic dual-modal imaging via tumor acidic micro-enviroment triggering

Abstract

For cancer diagnosis, a paramount challenge still exists in the exploring of methods that can precisely discriminate tumor tissues from their surrounding healthy tissues with a high target-to-background signal ratio. Here, we report a NaGdF₄@CaCO₃-PEG core-shell nanoparticle which has the tumor acidic microenvironment enhanced imaging signals of ultrasound and magnetic resonance. Under the acidic conditions, the CaCO₃ shell will gradually dissolve which then facilitate the interaction of NaGdF₄ with the external aqueous environment to enhance water proton relaxation. Meanwhile, the CO₂ bubbles generated by the CaCO₃ dissolvement will generate strong elastic echo for US detection. The core-shell structure of NaGdF₄@CaCO₃-PEG can be observed by TEM, and its composition can be determined by STEM. The acid triggered generation of CO₂ bubbles and the enhancement of MRI signal could be demonstrated in vitro, and the excellent dual-modal magnetic resonance/ultrasonic cancer imaging abilities of NaGdF₄@CaCO₃-PEG could be also proved at the tumor site in vivo. The here described proof-of-concept nanoparticles with pH triggered magnetic resonance/ultrasonic dual-modal imaging enhancement, may serve as a useful guide to develop various molecular imaging strategies for cancer diagnosis in the future.

Introduction

Cancer is becoming to one of the most dreaded disease and still remains as a major threat to human life¹. The rising burden is ascribed to population growth, aging and an adoption of cancer-associated lifestyle choices including smoking, physical inactivity, etc^{1, 2}. Even worse, most patients were diagnosed at a later or advanced stage, which results in poor prognosis^{1, 3}. Therefore, early diagnosis of cancer is crucial for timely therapy to prevent the potential risk of cancer metastasis and improve the long-term survival. Many non-invasive biomedical imaging techniques have been applied in the diagnosis of cancer including magnetic resonance imaging (MRI)^4–9, ultrasound imaging (US)^{10, 11}, positron emission tomography (PET)^12–15, and computed X-ray tomography (CT)^16–19, etc. Among the clinically applied diagnostic modalities, MRI has a high potential to image the tissue pathological changes, as it could safely provide high spatial resolution information²⁰. On the other hand, as a noninvasive real-time imaging modality, US has serveal advantages such as high safety, low cost, and easy accessed by the public. However, the sensitivity of conventional MRI and US strategies heavily rely on contrast agents (CAs), such as the widely used paramagnetic gadolinium ions (Gd-(DTPA)]⁻² (Magnevist) and [Gd-(DOTA)]⁻¹ (Dotarem)^21–23, or gas-filled echogenic microbubbles^{24, 25}, that are always “on”, emitting constant signals regardless of their proximity or interaction with target tissues, cells, or environmental markers. As a result, a large volume of nonspecific signal which might lead to a poor signal-to-noise ratio, makes the anatomical features of interested tissue difficult to distinguish. A more attractive contrast agent whose signal should be switched from OFF to ON in response to specific biological stimulus, which will further maximize the signals of targets and minimize the background signals, which in turn could improve the sensitivity and specificity^26–29.

Several activatable CAs that respond to tumour-related factors, such as pH and redox potential, have been developed for tumour-specific MRI or US. For example, Mi et al. have reported a MRI contrast agent that rapidly amplify the magnetic resonance signals in response to pH via releasing confined Mn²⁺ ions from pH-sensitive calcium phosphate (CaP) nanoparticles to the aqueous environment³⁰. Min et al. developed the calcium carbonate (CaCO₃) nanopartciles which exhibited strong echogenic signals at tumoral acid pH by producing CO₂ bubbles and showed excellent echo persistence¹⁰. Even so, some intrinsic drawbacks of US and MRI still cannot be avoided, for example, US has a poor tissue discrimination ability while MRI cannot provide real-time images and usually time consuming. Thus, developing a sensitive dual modal imaging (US and MRI) agent would not only make them as favorable tools for precisely visualizing biological and physiological changes with high signal-to-noise ratio, but also would render synergistic efficacy to overcome their own inherent limitations³¹.

As a MRI contrast agent, various size of NaGdF₄ nanoparticles (NPs) with well-defined size distributions could be readily synthesized via pyrolysis methods^32–35. Their MRI performances have been demonstrated to increase with the decreasing of the NPs’ size, attributed to the increased number of surface Gd³⁺ ions relative to the core ions. Therefore, it will be very useful to synthesize ultrasmall NaGdF₄ NPs, e.g., smaller than 5 nm, to provide better MRI signals⁶. Furthermore, compared with the Gd-(DTPA) or Gd-(DOTA), the renal clearable Gd-based NPs would have better biosafty since it could not induce the excessive Gd³⁺ ion leakage to cause biological toxicity^{36, 37}, therefore it might be a more promising candidate for disease diagnosis. As for a new type of US contrast agent, CaCO₃ nanoparticles with rigid structure can penetrate into host tumoral environments for cancer imaging, while the frequently used gas-filled microbubbles suffer from inherent drawbacks, such as low stability, short half-life in blood and low penetration ability due to the large size, which is limited to the imaging of intravascular structures.

Herein, we designed a pH-responsive nanoparticle which could significantly enhance the contrast of MRI and US signals in tumor as illustrated in Fig. 1. The shell of CaCO₃ was deposited onto the core surface of NaGdF₄ nanoparticles through the microemulsion method; in addition, physicochemical properties of nanoscale systems, such as size, dispersibility, and toxicity were systematically analyzed; furthermore, the US and MRI imaging enhanced efficiency were both evaluated in vitro and in vivo, which clearly proved that our probe could be utilized for sensitive and specific tumor imaging with responding to extracellular acidic microenvironments.

Figure 1.

Schematic illustration of the synthesis of NaGdF₄@CaCO₃-PEG nanoparticle and its bioimaging application.

Results and Discussion

Characterization of NaGdF₄@CaCO₃-PEG

As shown in Fig. 1, monodispersed spherical NaGdF₄ nanocrystals were chosen as the morphology-deciding template to obtain magnetic nanoparticles, which were fabricated by thermal decomposition method using rare earth perchlorate as the RE precursor. From TEM images (Fig. 2A), the synthesized NaGdF₄ exhibit a very well particle size distribution around 8~10 nm, with a regular spherical morphology. A thin layer of dense CaCO₃ was deposited onto the surface of NaGdF₄ to form NaGdF₄@CaCO₃ core/shell nanoparticles by the well-known microemulsion method, which could further transfer the hydrophobic NaGdF₄ nanoparticle into aqueous soltion. In detail, the CaCl₂ aqueous solution was first added into the cyclohexane solution of hydrophobic NaGdF₄, while triton X-100, 1-hexanol were used as nonionic surfactants to create a stable water-in-oil emulsion after vigorous stirring. At this stage, the hydrophobic NaGdF₄ will transfer from oil phase to water droplets as the surfactants can self-assemble on these nanoparticles. Meanwhile, the hydrophilic groups (hydroxyl) of the surfactants has a well affinity with Ca²⁺ which could recruit these ions on the surfaces of the NaGdF₄ nanoparticles. After adding Na₂CO₃ aqueous solution, the precipitation reaction between Ca²⁺ and CO₃ ²⁻ therefore produces a layer of CaCO₃ on the NaGdF₄ nanoparticles. The obtained nanoparticles show a size of 10~12 nm, as well as with aspherical-like morphology. Then, PEG₈₀₀₀ was adsorbed on the surface of NaGdF₄@CaCO₃ nanoparticles through Van der Waals’ force, which could award good water dispersibility. Element mapping of NaGdF₄@CaCO₃-PEG with a thin or thick shell all shows the co-existence of Gd, F and Ca elements (Figure S1), which exhibit a much higher Ca signal compared to the core only NaGdF₄ nanoparticles. However, the Ca signal on the surface of NaGdF₄ is very weak on the outlayer of NaGdF₄ core due to the very small size of NaGdF₄ core (10 nm) and the very thin CaCO₃ coating layer (2 nm). Nevertheless, we still ensure that the CaCO₃ is on the shell because this layer is amorphous, while the NaGdF₄ core is highly crystal with noticeable lattice, as shown in the enlarged image inserted in Fig. 2B. In addition, the average size of the NaGdF₄ in cyclohexane was further determined to be 8 ± 2 nm by the DLS experiments (Figure S2), which is consistent with the TEM results. After depositon of CaCO₃, the average hydrodynamic size of the NaGdF₄@CaCO₃ and NaGdF₄@CaCO₃-PEG in water were increased to 100 ± 10 and 120 ± 15 nm, respectively, which may be due to the formation of a hydration layer on the surface of nanoparticles or the formation of small aggregations.

Figure 2.

Structural characterization of nanoparticles. NaGdF₄ dispersed in cyclohexane (A); NaGdF₄@CaCO₃ (B) and NaGdF₄@CaCO₃-PEG (C) dispersed in H₂O; and NaGdF₄@CaCO₃-PEG dispersed in PBS (pH 5.0).

Furthermore, the successful surface PEGlyation of NaGdF₄@CaCO₃ was also confirmed by FT-IR spectroscopy (Fig. 3). For analyzing the spectrum of PEG₈₀₀₀, the peaks at around 2917 cm⁻¹ and 1108 cm⁻¹ were observed corresponding to the CH₂ stretching and vibration groups. These two peaks could be also observed in the spectra of NaGdF₄@CaCO₃-PEG, in addition to the typical peaks of NaGdF₄@CaCO₃ at 502 cm⁻¹. These results suggest that the PEG₈₀₀₀ was successfully modified on the surface of NaGdF₄@CaCO₃. To make the CaCO₃ coating more evident form the IR spectra, we re-fabricated another NaGdF₄@CaCO₃ with a thicker shell via increasing the feeding amount of CaCl₂, and analysis their surface groups through FT-IR measurement. The characteristic IR absorption peaks of CaCO₃ at 1430, 876, 712 cm⁻¹ can be clearly presented in thick layered NaGdF₄@CaCO₃ (Figure S3).

Figure 3.

FT-IR spectra of NaGdF₄@CaCO₃ (a), PEG₈₀₀₀ (b) and NaGdF₄@CaCO₃-PEG (c).

Before further bio-imaging applications, it is necessary to determine the cytotoxicity of these nanoparticles. To assess the in vitro cytotoxicity of NaGdF₄@CaCO₃-PEG, the standard Cell Counting Kit (CCK-8) assay and live/dead staining were conducted by using LN3 and NIH3T3 cell lines. As shown in Figure S4, cell viability is not affected by the NaGdF₄@CaCO₃-PEG in the concentration ranged from 0 to 400 μg∙mL⁻¹, which suggested that the NaGdF₄@CaCO₃-PEG has low cytotoxicity so that it could be further applied for bio-imaging.

In Vitro Ultrasound Contrast Enhancement Ability of NaGdF₄@CaCO₃-PEG

First, the characteristics of CO₂ gas generation by NaGdF₄@CaCO₃-PEG was studied through detecting the bubble generation from the particles in PBS buffer with different pH values (pH 7.4, 7.0, 6.8 and 5.0). As shown in Fig. 4, initially, NaGdF₄@CaCO₃-PEG nanoparticles generate few bubbles at pH 7.4, 7.0 and 6.8, but significantly more bubble generation could be observed at pH 5.0. The degree of bubble generation continuously decreased at pH 5.0 over time, but the bubble generation was first increased and then decreased at pH 7.4, 7.0 and 6.8. The number of bubbles in pH 5.0 was counted to ca. 119, which was much higher than that in pH 7.4 (almost no bubbles), after 1 min incubation. However, with increasing the time, the number of bubbles was dramatically decreased at pH 5.0, and only 2 bubbles could be observed after 60 mins incubation (Figure S5). It is noteworthy that bubble generation at lower pH was higher than that at slightly stronger pH at all-time points. This result suggested that the NaGdF₄@CaCO₃-PEG might enhance US signal in the low pH environment by gas generation, since CaCO₃ could generate more CO₂ gas in an acidic environment by improved dissolution, which would be beneficial for imaging the tumor due to a little acidic microenvironment of the tumor micro-environment.

Figure 4.

Optical micrographs of CO₂-generation profiles of NaGdF₄@CaCO₃-PEG incubated in PBS at different pH conditions (pH 5.0, pH 6.8, pH 7.0 and pH 7.4) for 60 min.

Next, we measured the in vitro echogenic performance of the NaGdF₄@CaCO₃-PEG as a function of pH using an aqueous dispersion test at 37 °C. As shown in Fig. 5, the NaGdF₄@CaCO₃-PEG at pH 7.4 showed no significant contrast signals under a US field, most likely because the NaGdF₄@CaCO₃-PEG did not release sufficient CO₂ bubbles for echogenic reflection. This result is well consistent with the results of the pH-dependent CO₂ generation (Fig. 4). In contrast, US contrast images from the NaGdF₄@CaCO₃-PEG showed first increased and then decreased signal at all-time points at weakly acidic pH conditions (pH 7.0 and 6.8). This is a well understood phenomenon in which the enhanced echo intensity at weakly pH levels is ascribed to the relaxedly facilitated formation of CO₂ bubbles. Due to faster dissolution of CaCO₃ phases at pH 5.0, the echo improved immediately and faded away earlier than at higher pH. The excellent echo persistence at weak acidic pH can be ascribed to the relaxedly ionization of CaCO₃ solid phases. The contrast enhancement that derives from the NaGdF₄@CaCO₃-PEG at lower pH suggested that generation of CO₂ bubbles was responsible for echogenic US resonation. US imaging results were further quantitatively analyzed using region-of-interest (ROI) gray value quantification (Figure S6A). The gray value in pH 5.0 and pH 7.4 was respectively determined to be 139.2 ± 10.7 and 14.2 ± 1.2, after 30 min incubation. This result is in accordance with the CO₂ bubbles generation. Meanwhile, in order to further confirm that the US signal was derived from the dissolution of CaCO₃ at acidic solution rather than the NaGdF₄ itself, we tested US signal of the core-only (NaGdF₄) nanoparticle at pH 5.0 and 7.4, and the results are shown in Figure S7. NaGdF₄ nanoparticle showed no US signal at both pH 5.0 and 7.4, due to no CO₂ gas generation from NaGdF₄ nanoparticle. The feature of gas-generating at a broad range of weak acidic pH would be propitious to US imaging of tumors, which have tissue heterogeneity and diverse acidic pH levels.

Figure 5.

In vitro US images from NaGdF₄@CaCO₃-PEG at various pH (pH 5.0, pH 6.8, pH 7.0 and pH 7.4) conditions along with time.

pH-Dependent In Vitro MRI Properties of NaGdF₄@CaCO3-PEG

Since the doping of Gd³⁺-ions, NaGdF₄@CaCO₃-PEG could act as a T₁ MRI contrast agent as well. T₁-weighted MR images of the NaGdF₄@CaCO₃-PEG showed enhancing signal intensity when Gd³⁺ concentrations were increased. Firstly, the T₁-weighted MR image of the NaGdF₄@CaCO₃-PEG was studied at different pH conditions. As shown in Fig. 6, the NaGdF₄@CaCO₃-PEG at pH 7.4 showed no significant enhanced contrast signals under a MRI field, most likely because the shell of CaCO₃ did not decompose so that water molecular could not access to NaGdF₄ core. In contrast, MRI contrast images from the NaGdF₄@CaCO₃-PEG were slightly enhanced at weakly acidic pH conditions (pH 7.0, 6.8). It is noteworthy that we demonstrated strongest MRI contrast images at pH 5.0, which might due to the decomposition of the CaCO₃ shell so that water molecular could access to the NaGdF₄ core. To further demonstrate this mechanism, we compared the T₁-MRI of NaGdF₄@CaCO₃-PEG to that of core-only (NaGdF₄) nanoparticles at pH 7.4, and the results are shown in Figure S6. Of course, NaGdF₄ showed a much brighter MR image than NaGdF₄@CaCO₃-PEG at pH 7.4. With the increasing of incubation time, the contrast of MRI images gradually enhanced at pH 7.2 and 6.8, but almost no change at pH 7.4. MRI imaging results were then quantitatively analyzed using region-of-interest (ROI) gray value quantification (Figure S6B). The gray value in pH 5.0 and pH 7.4 was respectively determined to be 182.2 ± 10.1 and 79.9 ± 3.1, after 20 min incubation. These data are very consistent with the theory that the contrast of T₁ MRI imaging is closely related with water accessibility, and the enhancement of contrast associated with sufficient water contacting.

Figure 6.

In vitro MR images from NaGdF₄@CaCO₃-PEG at various pH (5.0, 6.8, 7.0 and 7.4) conditions along with the time.

In addition, the T₁ value of the NaGdF₄@CaCO₃-PEG was evaluated by using a 0.5 T MRI scanner to determine whether the pH can influence the T₁-weighted MR imaging performance (Fig. 7). As shown in Fig. 7A, the longitudinal (T₁) were measured at 0.5 T magnetic field based on the Gd concentration of NaGdF₄@CaCO₃-PEG at pH 7.4 and 5.0, and the relaxivities were determined to be 0.42 mM⁻¹ ∙s⁻¹ and 1.64 mM⁻¹ ·s⁻¹, respectively. At pH 5.0, it showed about 4 folds higher relaxivity than that at pH 7.4, which further proofed that the T₁-weighted MR imaging enhancement of the NaGdF₄@CaCO₃-PEG was influenced by pH levels. At the same time, Fig. 7B shows the magnetic resonance signal enhancing capability of the NaGdF₄@CaCO₃-PEG nanoparticles as a function of Gd concentration ranging from 1.25 to 12.5 mM. Compared with water (Gd, 0 mM), the measured T₁-weighted image contrast gradually increased with the increasing of Gd concentrations.

Figure 7.

T₁-Weighted MR images of various Gd³⁺ concentrations of NaGdF₄@CaCO₃-PEG (A); relaxation rate r₁ (1/T₁) against different Gd³⁺ concentrations of NaGdF₄@CaCO₃-PEG (B).

In Vivo US Imaging of Tumor with NaGdF₄@CaCO3-PEG

To verify the potential of NaGdF₄@CaCO₃-PEG for US imaging of tumor, we executed an intra-tumor injection of NaGdF₄@CaCO₃-PEG dispersion into LN3 tumor-xenograft-bearing nude mice and monitored the US images as a function of time (Fig. 8). After injection right away, we couldn’t acquire any contrast enhancment of the US signal. It is gratifying that enhancement of US signals was obtained after 1 h of injection, and this contrast enhancement maintained more than 2 h. In addition, this phenomenon is very consistent with the in vitro experiments. US imaging results were then quantitatively analyzed using region-of-interest (ROI) gray value quantification. As shown in Fig. 8B, the gray value of tumor site was increased from 32.5 ± 3.9 (0 h post-injection) to 53.1 ± 7.8 (1 h post-injection), and still maintained at 38.7 ± 1.35 after 2 h injection. Therefore, we can conclude that the NaGdF₄@CaCO₃-PEG could generate bubbles in tumor tissues then produce sufficient echogenic reflectivity under a US field.

Figure 8.

In vivo US imaging of the LN3 tumor (red dashed circles) by intratumoral injection of NaGdF₄@CaCO₃-PEG (A); the gray values of mice tumor (region of interest as indicated in A), p values were calculated using GraphPad Prism 6 (*p < 0.05, **p < 0.01, ***p < 0.001; n = 3).

In Vivo MR Imaging of Tumor with using NaGdF₄@CaCO₃-PEG

NaGdF₄@CaCO₃-PEG nanoparticles which have the advantages of highly efficient of T₁ contrast enhancement ability may hold great promise to serve as a novel MRI contrast enhancing agent. Therefore, we performed MRI study on tumor-xenograft-bearing nude mice at a 7.0 T clinical scanner by intratumor injection of NaGdF₄@CaCO₃-PEG with a dose of 200 μL of 2 mg∙mL⁻¹. Right after the injection, the MRI contrast enhancement was not found at tumor site; however, after one hour of intratumor injection, we were delighted to find contrast enhancement of MRI signal and the signal strength remained unchanged over time up to 3 h (Fig. 9A). T₁ imaging results were then quantitatively analyzed using region-of-interest (ROI) quantification. As shown in Fig. 9B, the gray value of tumor site was increased from 102 (0 h post-injection) to 153 (3 h post-injection). Overall, these results suggested that the NaGdF₄@CaCO₃-PEG could act as a potential MRI contrast enhancing agent for tumor imaging.

Figure 9.

In vivo MR imaging of the LN3 tumor (red dashed circles) by intratumoral injection of NaGdF₄@CaCO₃-PEG (A); the gray values of mice tumor (region of interest as indicated in A), p values were calculated using GraphPad Prism 6 (*p < 0.05, **p < 0.01, ***p < 0.001; n = 3).

Conclusions

In this work, a core-shell nanoparticle of NaGdF₄@CaCO₃-PEG was designed as an activatable MR/US dual-modal imaging contrast for cancer diagnosis, which is triggered by the acidic environment. The general idea behind this OFF/ON responsive MR imaging behavior consists of quenching the sphere Gd³⁺ relaxation effects by coating NaGdF₄ with a layer of hydrophobic CaCO₃ to limit water availability. At acidic aqueous solution, CaCO₃ was dissolved to generate CO₂ bubbles which is used to obtain US signal. Meanwhile, a strong MRI enhancement can be activated upon dissolution of CaCO₃ and release of the previously silenced NaGdF₄ into the aqueous solution. In vivo results demonstrated the strong dual-modal magnetic resonance/ultrasonic imaging abilities of NaGdF₄@CaCO₃-PEG at the tumor site with an acidic environment. We expect that this work may provide a new insight for strategies to design nanomaterials with responsive dual-modal imaging abilities.

Methods

Materials

Gd(CH₃CO₂)₃ and PEG₈₀₀₀ were purchased from Sigma Aldrich Co., Ltd. Acetone and cyclohexane were obtained from Sinopharm Chemical Reagent Co., Ltd. 1-Octadecene and Methanol was purchased from Alladin Company. Oleic acid was purchased from Alfa Aesar. Cell Counting Kit (CCK-8) was obtained from Dojindo laboratories. Penicillin-streptomycin, fetal bovine serum (FBS), and Dulbecco’s Modified Eagle Medium (DMEM) were purchased from Gibco BRL. All of these materials were used as received without further purification. Ultrapure water was used throughout.

Synthesis of NaGdF₄ nanoparticles

The synthesis of NaGdF₄ nanoparticles is similar to a previous publication about rare-earth up-conversion nanoparticles^{34, 38}. In brief, 24 mL of Gd(CH₃CO₂)₃ (0.2 M) was added to a 500 mL of two-necked round-bottomed flask containing 36 mL of oleic acid. When the mixture was heated to 150 °C for 30 min, 84 mL of 1-octadecene was added and then heated to 150 °C to form the Gd-oleate precursor solution. Then, the reaction system was cooled down to 60 °C naturally and a mixture of 8 mL NaOH (1 M) and 16 mL NH₄F (0.4 M) in methanol was added and stirred for 30 min. The resultant solution was heated at 110 °C for 30 min to remove the methanol, oxygen and water under argon atmosphere. Thereafter, the solution was heated to 300 °C and kept for 1.5 h before cooling down to room temperature. The as-prepared nanoparticles were precipitated by additional of ethanol, collected by centrifugation at 6000 rpm for 3 min, and washed with ethanol for several times. The NaGdF₄ nanoparticles are re-dispersed in 50 mL of cyclohexane for further application.

Synthesis of the core-shell NaGdF₄@CaCO₃-PEG nanoparticles

The core-shell NaGdF₄@CaCO₃ nanoparticles were prepared through a facile microemulsion method with using NaGdF₄ nanoparticles as templates³⁹. Briefly, 5 mL NaGdF₄ nanoparticles, 10 mL cyclohexane, 3.45 mL Triton X-100, and 3.2 mL 1-hexanol were added to a flask and mixed thoroughly, followed by the addition of a 30 mM solution of calcium chloride (800 μL) to form a well-dispersed water-in-oil emulsion. Then, the solution of sodium carbonate (40 μL, 2.92 M) was added. After keeping moderate stirring for 8 h of the mixture, the as-prepared NaGdF₄@CaCO₃ nanoparticles were collected by centrifugation and then re-dispersed in 10 mL of ultrapure water. Thereafter, 2 mL PEG₈₀₀₀ (0.2 M) aqueous solution was added and stirred for another 8 h, then the as-prepared nanoparticles were collected by centrifugation at 10000 rpm for 15 min, and re-dispersed in 20 mL of ultrapure water for further application.

Characterization of the NaGdF₄@CaCO₃-PEG nanoparticles

The morphology and the size of the obtained NaGdF₄@CaCO₃-PEG nanoparticles were performed on a Tecnai F20 transmission electron microscope (TEM, FEI Company, Hillsboro, OR) with an accelerating voltage of 200 kV⁴⁰. For the TEM experiment, the suspension of nanoparticles was dropped onto a carbon-coated copper grid, followed by drying naturally. Scanning transmission electron microscopy (STEM) was obtained using a JEM-2010 electron microscope (JEOL, Japan) to characterize the chemical composition of NaGdF₄@CaCO₃-PEG nanoparticles⁴⁰. Dynamic light scattering (DLS) experiments were recorded at 25 °C on a NanoZS (Malvern Instruments, UK) with a detection angle of 173°, and a 3 mW He–Ne laser operating at a wavelength of 633 nm. FT-IR spectra were performed on a Fourier transform infrared spectrometer (Perkin-Elmer, Spectrum-2000) over the spectral region of 4000 cm⁻¹ to 400 cm^{−1  41}.

Statistical analysis

All quantitative data were expressed as the mean ± standard deviation (SD). Graph Pad Prism version 6.0 was used for statistics analysis. Statistical analysis among different groups was performed using student T-Test. The P < 0.05 was considered as statistically significant.

Animals

All mice (4–5 weeks old; weighing: 18–22 g) were obtained from the Center for Animal Experiment of Fujian Medical University (License No: SCXKmin2012–0002), and housed at constant temperature (22 ± 2 °C) and 60% relative humidity, with a light/dark (hours) cycles of 12/12. All animal procedures were conducted in accordance with the approved guidelines of Animal Ethics Committee of Fujian Medical University.

Cytotoxicity assay

The LN3 and NIH3T3 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin streptomycin at 37 °C in a humidified atmosphere (5% CO₂). The in vitro cytotoxicity was investigated by the Cell Counting Kit (cck-8) assay. In detail, LN3 and NIH3T3 cells were cultured in a 96-well cell-culture plate at a density of 10⁴ (100 μL) cells per well for 24 or 48 h, respectively. Then, the medium was replaced with 100 μL fresh medium containing various concentrations of NPs. After 24 or 48 h incubation, the medium was removed. Then, 100 μL of fresh medium and 10 μL of CCK-8 were added and incubated for another 2 h. The absorbance was measured by a Bio-Rad Model-680 microplate reader at the wavelength of 450 nm. The cell viability (%) relative to control cells was calculated from following equation: ([Abs]_sample − [Abs]_blank)/([Abs]_control − [Abs]_blank) × 100%, where [Abs]_sample and [Abs]_control are the absorbance values of the cells with and without the treatment of nanocomplexes, respectively. The [Abs]_blank are the absorbance of CCK-8 itself at 450 nm. All experiments were investigated in sextuplicate. Results were presented as mean ± standard deviation (SD).

Visualization of CO₂ Bubble Generation from NaGdF₄@CaCO₃-PEG nanoparticles

To observe the bubble generation characteristic of NaGdF₄@CaCO₃-PEG, the aqueous dispersion was dropped on a glass slide, followed by drying naturally. Then, PBS buffer with various pH values from 5.0 to 7.4 was dropped on the samples, and the CO₂ bubble image from NaGdF₄@CaCO₃-PEG was obtained by an optical microscope at room temperature^{10, 11}.

In Vitro US Imaging at Various pH

In vitro US imaging of NaGdF₄@CaCO₃-PEG was performed in phosphate buffer solutions at various pH conditions (7.4, 7.0, 6.8, and 5.0)⁴¹. An optically transparent phantom gel plate, which was made by embedding a 500 μL Eppendorf tube in the agarose gel (3%, w/v) and then removing the tube after the phantom gel had cooled, was used for the in vitro experiments. Aqueous nanoparticle solutions (10 mg∙mL⁻¹) were prepared at various pH. US images were obtained using Vevo 2100 imaging system operated at 21 MHz of a static state using a contrast mode. The change of US intensity for each sample was measured up to 180 min, and the US intensity of the water as control was subtracted from the sample intensity for the normalization.

In Vitro MR Imaging at Various pH

In vitro MR imaging of NaGdF₄@CaCO₃-PEG was performed in phosphate buffer solutions at various pH conditions (7.4, 7.0, 6.8, and 5.0) using T₁ -weighted MRI on a 0.5 T NMI20-Analyst NMR system (Niumag Corporation, Shanghai, China) to evaluate the contrast-enhancement effect⁴².

Relaxivity and MRI phantom studies at 0.5 T magnetic field

A series of NaGdF₄@CaCO₃-PEG nanoparticle aqueous solutions with different Gd concentrations (12.5, 10, 7.5, 5.0, 2.5, and 1.25 mM) were prepared for MRI phantom and relaxivity studies. All experiments were performed on a 0.5 T NMI20-Analyst NMR system (Niumag Corporation, Shanghai, China)^42–44. The longitudinal relaxation times (T₁) were measured using an inversion recovery (IR) sequence. The longitudinal (r1) was determined from the slope of the plot of 1/T₁ against the Gd concentration (mM).

In vivo US imaging of Xenograft Tumor

To form a solid tumor in nude mice, the LN3 xenograft tumor was estabilished in 4-week-old male nude mice by injecting 10⁶ LN3 cells into the right thigh of mice. After injection, tumor-bearing nude mice were kept for 10~14 days to achieve a tumor size around 80 mm³. Then, 200 μL of normal saline (NS) containing NaGdF₄@CaCO₃-PEG (2 mg∙mL⁻¹) was injected by an intratumoral injection. After injection, the tumor was imaged with the Vevo 2100 imaging system.

In Vivo MR Imaging of Xenograft Tumor

For in vivo MRI measurements, LN3 tumor-bearing mice were intra-tumour injected with 200 μL of 2 mg∙mL⁻¹ NaGdF₄@CaCO₃-PEG. At different intervals (0–3 h), T₁-weighted MR images were observed using the rapid acquisition with relaxation enhancement sequence on 7.0 T small animal MRI scanner (Bruker Avance II 500WB spectrometer)⁴³. Imaging parameters are as follows: repetition time, 2500 ms; echo time, 35 ms; rare factor, 8; field of view = 30 × 30mm²; image size, 256 × 256; slice thickness,0.7mm; and number of average, 2.

Author Contributions

Zuwu Wei, Xiao Lin and Ming Wu designed the study, fabricated samples and wrote the manuscript. Bixing Zhao and Da Zhang provided chemical compounds and performed data analysis. Ruhui Lin, Gang Liu, and Yun Zhang helped with the MRI and US experiments. Xiaolong Liu and Jingfeng Liu designed the study, wrote the manuscript and contributed to discussion.

Competing Interests

The authors declare that they have no competing interests.