Research Article www.acsami.org
Nanosized Ultrasound Enhanced-Contrast Agent for in Vivo Tumor Imaging via Intravenous Injection Manse Kim,† Jong Hyun Lee,† Se Eun Kim,‡ Seong Soo Kang,‡ and Giyoong Tae*,†,§ †
School of Materials Science and Engineering, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea College of Veterinary Medicine, Chonnam National University, Gwangju 61186, Republic of Korea § Center for Theragnosis, Biomedical Research Institute, KIST, Seoul 02792, Republic of Korea ‡
S Supporting Information *
ABSTRACT: To enhance the detection limit of ultrasound (US) imaging, ultrasound enhanced-contrast agents (UECAs) that can go preferentially to the target tissue such as a tumor and amplify the US signal have been developed. However, nanosized UECAs among various UECAs developed are very limited to clearly demonstrate proper ability for selective tumor detection by US imaging upon their intravenous injection. In this study, we prepared CaCO3 nanoparticles that were formed inside a flexible and biocompatible pluronic-based nanocarrier. This nanosized UECA was stable in serum-containing media and generated CO2, more preferentially at low pH; thus, it could be detected by US imaging. After intravenous injection into tumorbearing mice, this nanosized UECA showed a significant US contrast enhancement at the tumor site in 1 h, in contrast to no change in the liver, followed by a rapid clearance from the body in 24 h. Therefore, the present nanosized UECA could be applied as an effective diagnostic modality for in vivo tumor imaging by ultrasonography. KEYWORDS: ultrasound imaging, nanosized UECA, CaCO3, pluronic, nanocarrier agents (UECAs) have been employed,1,15,17 which include organic1,17,18 and inorganic materials.19,20 In general, UECAs are based on either microbubble, nanobubble, or hollow structure with gas desorption characteristics to improve the ultrasound contrast characteristic by the difference of ultrasonic properties between solid and nonsolid parts.1,15,18,21 Furthermore, particles that can generate gas in an aqueous environment by loading gas-forming materials such as bicarbonate compounds and calcium carbonate have also been developed as more efficient UECAs. 22−24 Gas-generating UECAs by hydrolysis of gas-generating side chain showed low stability in aqueous environment and a short gas-generating time.23 UECAs with calcium carbonate showed enhanced stability and US imaging by the accelerated gas generation at an acidic tumor environment upon intratumoral injection.24 Among various UECAs, nanosized UECAs not only have the similar ultrasound enhanced-contrast effect with microsized UECA but also possess enhanced tumor accumulation in vivo relative to microsized UECAs due to the enhanced permeability and retention (EPR) effect by their small size.25,26 Thus, nanosized UECAs are expected to detect the tumor sites upon
1. INTRODUCTION Ultrasound (US) imaging is a diagnostic imaging modality that uses high-frequency sound waves.1,2 US imaging is more advantageous than other diagnostic methods in terms of patient safety and comfort, even though other diagnostic methods can provide better imaging resolution than US imaging.3,4 For example, magnetic resonance imaging (MRI) can result in focal heating onto patient’s skin and tissues5−7 or can be dangerous for patients who have a cardiac pacemaker device or claustrophobia.4,8−10 Computed tomography (CT) imaging has an intrinsic risk from X-ray irradiation.11−14 Thus, standard regulations have been established to minimize these risks of the MR and CT imaging. However, US imaging is accepted as a very safe method, even for pregnant woman.4 In addition, US imaging is widely available in most hospitals due to its low cost, whereas access to MR or CT imaging is limited. US imaging can show the structure of tissues inside the body by reflection and refraction of ultrasound signal from the tissue structure based on different ultrasonic properties between the tissues.15,16 However, abnormal tissues such as tumors have ultrasonic properties rather similar to those of normal tissues, so it is difficult to distinguish the abnormal tissues from the normal tissues simply by US imaging alone.15 To improve the low-resolution of US imaging due to the similarity of ultrasonic properties between tissues, ultrasound enhanced-contrast © 2016 American Chemical Society
Received: February 20, 2016 Accepted: March 17, 2016 Published: March 24, 2016 8409
DOI: 10.1021/acsami.6b02115 ACS Appl. Mater. Interfaces 2016, 8, 8409−8418
Research Article
ACS Applied Materials & Interfaces
8W, Viber Lourmat, France) was applied for 15 min at 1.3 mW/cm2 to produce bare nanocarrier (bare NC) by photopolymerization among the DA-PF 127 micelles. Then, bare NC was purified by dialysis for 2 days in DIW and lyophilized to get a powder state of bare NC. 2.3. Preparation of Calcium Carbonate Loaded Nanocarrier. A calcium carbonate loaded nanocarrier (CaCO3AlgNC) was prepared by first loading alginate into the nanocarrier, as previously reported by us.32 In brief, 14 mg of powder state bare NC was dissolved in 1 mL of 10 mg/mL sodium alginate solution at 4 °C for 24 h with gentle shaking by using a rotary shaker (Barnstead International, Dubuque, IA, USA). Then, 1 mL of DIW was added into the alginate-loaded NC (AlgNC) solution, and the mixed solution was incubated at 37 °C over 30 min. To remove unloaded alginate into NC, the AlgNC solution was filtered by Nanosep centrifugal devices (MWCO = 300 kDa) at 12000 rpm and 37 °C for 10 min. After that, 1 mL of AlgNC solution was added dropwise into 1 mL of 20 mM CaCl2 solution at 37 °C under shaking at 500 rpm, and the mixed solution was incubated for 3 h. Then, the solution was filtered by 0.45 μm syringe filter and added dropwise into a 1.5 times volume of 20 mM Na2CO3 solution to induce the mineralization of calcium carbonate inside the AlgNC at 37 °C under shaking at 800 rpm for 15 h. Then, the final product was purified by using spin-filtration (11000 rpm and 37 °C for 10 min) and 0.45 μm syringe filter. Then, the purified solution was lyophilized by using a freeze-dryer. 2.4. Characterization of Calcium Carbonate Filled Nanocarrier. The size and surface charge of calcium carbonate filled nanocarrier (CaCO3AlgNC), alginate-loaded nanocarrier (AlgNC), and bare NC were measured by using an electrophoretic light scattering system (ELS-8000, Otsuka Electonics Co., Ltd., Tokyo, Japan) in triplicate. The morphology of CaCO3AlgNC was characterized by using a transmission electron microscope (TEM) (JEM-2100, JEOL, Tokyo, Japan) at a 200 kV accelerating voltage. To prepare the CaCO3AlgNC sample for TEM, 20 μL of 1 mg/mL CaCO3AlgNC was dropped on a 200-mesh carbon-coated copper grid, which was incubated at 37 °C overnight. The stability of CaCO3AlgNC was estimated by measuring the size of the sample at 37 °C. In brief, 1 mL of 1 mg/mL CaCO3AlgNC in cell culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin was incubated at 37 °C and 100 rpm. The size of CaCO3AlgNC was analyzed by using an electrophoretic lightscattering system at determined time points for 2 days in triplicate. The serum protein absorption onto CaCO3AlgNC after incubation in a serum containing medium was also characterized by absorption spectra, size, and surface charge of the CaCO3AlgNC. In short, 1 mg/ mL of CaCO3AlgNC in a serum containing cell culture medium (10% FBS) was incubated at 37 °C and 100 rpm for 24 h. After that, the CaCO3AlgNC was separated from the free serum by spin filtration. The presence of serum protein in the CaCO3AlgNC was analyzed by absorption spectra from 240 to 350 nm. Bare NC was used as a control. The size and surface charge of the particle after incubation were characterized by using an electrophoretic light-scattering system in triplicate and compared with the prior incubation state. To confirm the presence of CaCO3 in calcium carbonate filled nanocarrier, an FTIR spectrometer (SPECTRUM 2000, PerkinElmer, Boston, MA, USA) was used. The lyophilized bare NC, AlgNC, and CaCO3AlgNC were powdered by grinding and placed in a KBr film. IR spectra of each sample were measured at 0.2 cm−1 resolution with 20 scans from 400 to 4000 cm−1 wavenumber range. The structures of CaCO3AlgNC, AlgNC, and bare NC were analyzed by X-ray diffraction (XRD) (Rigaku RINT 2000, Rigaku Co., Tokyo, Japan) by Cu Kα radiation to assess the crystallinity of CaCO3AlgNC. First, to compare the structure with CaCO3AlgNC, CaCO3 was prepared by mixing 20 mM CaCl2 and 20 mM Na2CO3 with different incubation time (3 s reaction, 15 min, and overnight) with 800 rpm stirring at 37 °C and washed by DIW three times to remove unreacted components. After that, XRD patterns of each lyophilized samples were measured from 10 to 50 degree (°). A thermogravimetric analyzer (TGA) (TGA4000, PerkinElmer) was used to estimate the organic/inorganic mass ratio of calcium
systemic delivery. However, reports of appropriate UECAs for tumor diagnosis to clearly distinguish the tumor from other tissues by nanosized UECAs upon intravenous (iv) injection are very limited. For example, when the nanosized UECA was administered via iv injection, tumor detection by US imaging was not possible due to a low accumulation of UECA in the tumor tissue, whereas it could be detected by US imaging after only intratumoral injection of nanosized UECA.27 A very recent study reported the tumor accumulation of UECA via iv injection only after the attachment of a specific tumor-targeting ligand. Without the tumor-targeting ligand, no significant tumor accumulation of UECA was observed.28 In another case, UECA of ∼300 nm in size containing perfluorohexane showed enhanced-contrast signal not only in the tumor but also more significantly in the liver by US imaging after iv injection.29 Thus, the selective tumor detection without the targeting ligand by US imaging was not demonstrated. In this study, we have developed a nanosized UECA that can detect the tumor site by US imaging upon intravenous injection. A pluronic-based nanocarrier was used as a platform to synthesize nanosized UECA because of its good stability, easy and efficient loading of various materials, and tumor targeting, as previously reported.30,31 After alginate was loaded inside the nanocarrier,32 this nanoalginate gel was used to form calcium carbonate particles in the nanocarrier. Thus, a nanosized, calcium carbonate-filled nanocarrier was prepared and the characteristics as an ultrasound enhanced contrast were evaluated. Then, the tumor diagnosis by US imaging upon iv injection of this nano-UECA was demonstrated in vivo.
2. MATERIALS AND METHODS 2.1. Materials. Acryloyl chloride, trimethylamine, anhydrous toluene, calcium chloride, 1,9-dimethylmethylene blue (DMMB), agarose type V, sodium carbonate, sodium phosphate dibasic, sodium chloride, potassium chloride, potassium phosphate monobasic, anhydrous diethyl ether, and sodium alginate (A2158, ratio of mannuronic to gluronic acid = 1.67 from Macrocystits pyrifera, molecular weight = 50 kDa)33,34 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Citric acid monohydrate was bought from Junsei Chemical (Tokyo, Japan). Pluronic F127 (PF 127, PEO100PPO65-PEO100, molecular weight = 12.6 kDa) was donated from BASF (Seoul, Korea). Ethanol absolute was bought from Merck KgaA (Darmstadt, Germany). 4-(2-Hydroxyethoxy)phenyl-(2-hydroxy-2propyl) ketone (Irgacure 2959) was purchased from Ciba Specialty Chemicals (Basel, Switzerland). For purification of synthetic samples, 0.45 and 0.2 μm syringe filters were bought from Whatman International (Florham Park, NJ, USA). A Nanosep centrifugal device (MWCO = 300 kDa) was purchased from Pall Life Sciences (Ann Arbor, MI, USA). 2.2. Preparation of Pluronic-Based Nanocarrier as a NanoUECA Platform. A nanoparticle platform based on PF 127 was prepared by simple photo-cross-linking of diacrylated pluronic in an dilute environment, as previously reported.30,31,35 First, diacrylated PF 127 (DA-PF 127) was synthesized. Briefly, 5 g of dried PF 127 was mixed with 644.8 μL of acryloyl chloride and 744.6 μL of trimethylamine overnight in 100 mL of anhydrous toluene under argon. Then, the reacted polymers were collected by the precipitation of polymer in cold diethyl ether and purified by glass filter. Finally, the filtered polymer solution was dried under vacuum. The degree of acrylation of PF 127 was >98%, analyzed by 1H NMR spectroscopy (D2O, JNM-ECX-400P, JEOL, Japan) by comparing peak intensities from acryl protons (5.80−6.40 ppm) and methyl protons (1.10 ppm). A 10 wt % DA-PF 127 solution was dissolved in deionized water (DIW) and filtered by 0.2 μm syringe filter. Then, it was diluted to 0.77 wt % to form a micelle state of DA-PF 127 with the addition of a photoinitiator (Irgacure 2959, 0.057 wt %). UV irradiation (VL-4.LC, 8410
DOI: 10.1021/acsami.6b02115 ACS Appl. Mater. Interfaces 2016, 8, 8409−8418
Research Article
ACS Applied Materials & Interfaces carbonate in CaCO3AlgNC. The rate of temperature increase was fixed at 10 °C/min under N 2 atmosphere. Three milligrams of CaCO3AlgNC or bare NC was loaded in TGA and heated from 20 to 800 °C to compare the weight loss behaviors between CaCO3AlgNC and bare NC in triplicate. The amounts of alginate in CaCO3AlgNC were measured by using the DMMB assay. Briefly, 500 μL of a known concentration of alginate or unknown samples was mixed with 60 μL of 2.25 M citric acid at room temperature for 10 min. After that, 200 μL of the mixed solution placed in a 96-well plate was reacted with 20 μL of 0.3 mM DMMB for 15 min. Then, the amounts of alginate in the samples were calculated by the absorbance of the reacted solution at 525 nm in triplicate. 2.5. Cytotoxicity Measurement and Cellular Uptake of CaCO3AlgNC. To characterize the cytotoxicity of CaCO3AlgNC and compare with that of bare NC and AlgNC, the nanoparticles were dispersed in cell culture medium at several concentrations ranging from 0 to 1.25 mg/mL. As a positive control, 1 mg/mL of nanoparticles containing 5% ethanol (5% EtOH) was used. Squamous cell carcinoma (SCC7) cells from American Type Culture Collection (Rockville, MD, USA) were cultured in cell culture medium (RPMI 1640, Gibco, Grand Island, NY, USA) containing 10% FBS and 1% penicillin−streptomycin. The cells were seeded in a 96-well tissue culture plate at 4 × 103 cells/well and incubated at 37 °C for 24 h under a 5% CO2 atmosphere. After that, the cells were washed with PBS (pH 7.4) and treated with the prepared samples for 24 h at 37 °C. The treated cells were washed with PBS, and the cytotoxicity of the sample was measured by Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), which is based on the conversion of the reagent from a water-soluble tetrazolium salt to formazan dye in the presence of dehydrogenase and electron mediator in cell.36,37 To characterize the cellular uptake of CaCO3AlgNC, 5 × 104 SCC7 cells were spread onto gelatin-coated coverslips (12 mm) in a 24-well tissue culture plate and incubated for 24 h.31 After 24 h of incubation with CaCO3AlgNC having Cy5.5 (0.031 wt %) in cell culture medium, the cells were washed by PBS and fixed by 4% formaldehyde for 30 min. The fixed cells were washed by PBS, and 4,6-diamindino-2phenylindole (DAPI) was treated onto the cells to stain the nucleus of cells. The fluorescence image of Cy5.5 and DAPI was obtained by a confocal laser scanning microscope. The cellular uptake of CaCO3AlgNC into the SCC7 cells was also quantified by a BD FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). SCC7 cells (1 × 105) were spread onto a tissue culture plate and incubated for 24 h.31 Then, CaCO3AlgNC with Cy5.5 was added and incubated for 24 h. After removal of the supernatant and washing by PBS, the cells were detached by trypsin/EDTA and collected by centrifugation. Resuspended cells in PBS with 10% FBS were measured by the flow cytometer in triplicate. 2.6. In Vitro Ultrasound (US) Imaging of CaCO3AlgNC. To analyze the bubble generation characteristic of CaCO3AlgNC, CaCO3AlgNC samples were attached on a glass slide using a double-sided adhesive tape. Then, PBS buffer with various pH values from 6.5 to 7.4 was dropped on the samples, and the bubble generation was observed by optical microscopy. In addition, the amounts of CO2 generation from CaCO3AlgNC as well as AlgNC in PBS at different pH values was quantitatively analyzed by using an air quality meter (IAQ-Calc indoor air quality meter model 7535, TSI Inc., Shoreview, MN, USA). In brief, CaCO3AlgNC samples were placed in a 50 mL vial. The vial was sealed with a rubber stopper, and the air quality meter was connected to the sample-loaded vial by an injection needle. Then, PBS solution at pH 6.5 or 7.4 was dropped on the samples (20 mg/mL of CaCO3AlgNC), and the amount of CO2 generation was measured at predetermined time points. The CO2 generation from AlgNC without CaCO3 was also measured as a control in the same experimental condition. For characterizing the US imaging ability of CaCO3AlgNC in vitro, 1 wt % agarose gel was prepared with different pH buffers (pH 7.4 and 6.5 with PBS and pH 5.0 with acetate buffer) as a mold to contain the sample. Then, 20 μL of bare NC, AlgNC, or CaCO3AlgNC in PBS (pH 7.4) at 30 mg/mL was injected into the agarose gel at different
pH values. The injected agarose gels were immersed in the same buffer at 37 °C, and the US signal from the samples was detected by using an ultrasound diagnostic unit (LOGIQ7, GE Healthcare, Waukesha, WI, USA) with a 10 MHz linear transducer. The US signal of CaCO3AlgNC was also collected over time at the same setup. The intensity of the US signal was calculated on the basis of the PBS injection case by using an imaging program (Adobe Photoshop, Adobe system, San Jose, CA, USA) in triplicate. Then, the ultrasound contrast-enhanced ratio (contrast-enhanced ratio), defined as I/I0, where I0 is the US signal intensity with PBS injection and I is the US signal intensity in the region of interest (ROI, red dash circle in figures) of the sample, was calculated. 2.7. In Vivo Imaging of CaCO3AlgNC. To optically monitor the CaCO3AlgNC, Cy5.5 was attached to CaCO3AlgNC. First, an aminefunctionalized nanocarrier was synthesized, as previously reported,31 and then the same protocol was used to make a calcium carbonate filled nanocarrier. The amount of Cy5.5 in CaCO3AlgNC was ∼0.031 wt %, measured by fluorescence emission at 700 nm with 680 nm excitation, using a multiplate reader (Molecular Devices, SpectraMaxM2e, Trenton, NJ, USA). For animal experiments, 6-week-old nude mice (C3H/HeN, Oriental Bio Co., Seongnam, Korea) were used and handled according to the guidelines of the Animal Care and Use Committee of Gwangju Institute of Science and Technology (GIST) and the Animal Care and Use Committee of Chonnam National University. To form a solid tumor in nude mice, SCC7 cells were injected into the right thigh of the nude mice by using an insulin syringe injector at 1 × 106 cells per 50 μL of PBS. After injection, tumor-bearing nude mice were kept for 10−12 days to achieve a tumor size of around 80 mm3. Then, Cy5.5attached CaCO3AlgNC (2 mg per 100 μL of PBS (pH 7.4)) and AlgNC (2 mg per 100 μL of PBS (pH 7.4)) were injected into the tail vein of the tumor-bearing nude mice. After injections, US images of tumors in nude mice at determined time points (30 min and 1 h) were obtained by using the ultrasound diagnostic unit. To analyze the in vivo clearance of CaCO3AlgNC with time, 100 μL of 20 mg/mL Cy5.5 attached CaCO3AlgNC was injected into the tail vein of the tumor-bearing nude mice, when a tumor size of around 80 mm3 was achieved. After injection, near-infrared (NIR) fluorescence from the tumor was measured at 30 min and 1, 3, 6, 12, and 24 h by using an IVIS 100 imaging system (Xenogeny Corp., Alameda, CA, USA) with a Cy5.5 filter. Also, the main organs at 1 and 24 h postinjection of Cy5.5 attached CaCO3AlgNC via tail vein of the nude mice were extracted, and NIR fluorescence from the organs was measured by the IVIS to compare the presence of CaCO3AlgNC in the extracted organs between 1 and 24 h postinjection. As a control, the same amount of free Cy5.5 as the Cy5.5 attached CaCO3AlgNC was used. Furthermore, to confirm the presence of calcium carbonate in the tumor at 1 h after CaCO3AlgNC injection via tail vein, the extracted tumors were fixed by 1% formaldehyde and stained with Alizarin Red S.
3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of CaCO3AlgNC. To synthesize a nanosized gas generation system having ultrasound enhanced-contrast ability, CaCO3 was used in this study because of the good stability and ultrasound enhanced ability of CaCO3 in physiological environment.24,38 To make the CaCO3-loaded nanosystem, we first prepared a pluronic-based nanocarrier filled with Ca2+−alginate gel, as previously reported by us.32 Then, precaptured Ca2+ inside the nanocarrier was used to induce the formation of CaCO3 in the nanocarrier.39,40 First, temperature-sensitive, pluronic-based nanocarrier (bare NC) was prepared by photo-cross-linking DA-PF 127 in micelle state.30 Then, alginate was loaded into the bare NC by reversible volume change of bare NC with temperature change from 4 to 37 °C (AlgNC).30,32,41 After removal of free alginate by spin filtration (12000 rpm, 10 min, 37 °C), AlgNC solution was added dropwise into CaCl2 8411
DOI: 10.1021/acsami.6b02115 ACS Appl. Mater. Interfaces 2016, 8, 8409−8418
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ACS Applied Materials & Interfaces solution to form Ca2+−alginate gel inside the NC.32,40 Then, Na2CO3 solution was added to induce the formation of CaCO3 inside the nanocarrier (CaCO3AlgNC) by the reaction between Ca2+ and CO32− (Scheme 1).
temperature-induced size reduction at higher temperature, but additional formation of CaCO3 did not result in further change in size. As a result, we could obtain CaCO3-loaded nanocarrier of ∼160 nm at 37 °C, which is small enough to show the EPR effect. The formation of CaCO3 was confirmed by TEM (Figure 1A). The TEM image showed that many small CaCO3 nanoparticles filled the nanocarrier instead of a big single particle inside the nanocarrier. The measurements of surface charges of various nanocarriers showed that AlgNC and CaCO3AlgNC have surface charges similar to that of bare NC, which is significantly higher and close to neutral than a highly negative charged state of alginate (Figure 1B). This result indicated that Ca2+-induced alginate gel as well as CaCO3 particles were all formed and present inside the nanocarrier, but not on the surface of the nanocarrier. Thus, we could obtain relatively neutral and nanosized (∼160 nm at 37 °C) flexible nanocarrier filled with lots of CaCO3 particles. The stability of CaCO 3AlgNC was characterized in physiological environment. In both DIW and PBS (pH 7.4) at 37 °C, the size and surface charge of CaCO3AlgNC were ∼160 nm and −10 mV, respectively, at 37 °C (data not shown). Furthermore, the long-term stability of CaCO3AlgNC was also characterized by monitoring the size change over time in a cell culture medium containing 10% serum at 37 °C and 100 rpm (Figure S1A). After a small increase initially, the size of CaCO 3AlgNC did not change over 2 days. Thus, CaCO3AlgNC showed a good dispersion stability in physiological condition, which is important for intravenous injection and in vivo biodistribution of CaCO 3 AlgNC because biodistribution of nanoparticles is most significantly affected by the size of particles.42,43 In addition, neither CaCO3AlgNC nor bare NC showed noticeable protein absorption or deformation of size and surface charge of the particle after 24 h of incubation in a cell culture medium containing 10% serum at 37 °C and 100 rpm (Figure S1B−D)), followed by spin filtration to separate free serum. These nonfouling characteristics of nanocarriers must result from the hydrophilic, PEO part of pluronic that covers the surface of nanocarriers in an aqueous environment.44,45 As a result, CaCO3AlgNC had good stability with nonfouling property in a serum-containing environment. FTIR and XRD measurements were used to verify the presence of CaCO3 and qualitative analysis of CaCO3 in CaCO3AlgNC (Figure 2). In FTIR spectra (Figure 2A), CaCO3 itself showed a very strong peak at 1430 cm−1 as well as small peaks at 710 and 880 cm−1, which are characteristic peaks of pure CaCO3.46 Alginate showed peaks at 1370 and 1125 cm−1, which are not overlapped with bare NC, and AlgNC, alginate-loaded NC, showed the peaks of both alginate and bare NC. When 20 mM Ca2+ was added to AlgNC (Ca2+-added AlgNC), this alginate peak became weak and disappeared, which might be caused by the interaction between alginate and Ca2+. However, after the formation of CaCO3 in AlgNC had been induced, the characteristic peaks of alginate were recovered, presumably due to the reduced interaction between Ca2+ and alginate, and the characteristic peak of CaCO3 (∼1430 cm−1) was clearly shown in CaCO3AlgNC, although the intensity was weaker than that of pure CaCO3. The crystallinity of CaCO3 formed in the nanocarrier was measured and compared with that of pure CaCO3 prepared at various reaction times as well as other nanocarriers by XRD (Figure 2B). Bare NC and AlgNC showed similar XRD signals, which indicates that the XRD pattern of bare NC was not
Scheme 1. Preparation of CaCO3-Loaded Nanocarrier (CaCO3AlgNC)
The size and surface charge of CaCO3AlgNC were compared with those of bare NC and AlgNC (Figure 1). CaCO3AlgNC and AlgNC showed similar sizes at 25 and 37 °C, which were larger than that of bare NC (Figure 1A), whereas all nanocarriers were similar in size (∼400 nm) at 4 °C. Therefore, the filling of bare NC with alginate gel reduced the
Figure 1. (A) Comparison of size of CaCO3AlgNC with that of other nanocarriers and TEM image at 37 °C (scale bar = 200 nm). (B) Comparison of surface charge of CaCO3AlgNC with that of other nanocarriers and alginate at 37 °C. 8412
DOI: 10.1021/acsami.6b02115 ACS Appl. Mater. Interfaces 2016, 8, 8409−8418
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CaCO3AlgNC was 12 ± 2 wt % alginate, 47 ± 7 wt % nanocarrier, and 41 ± 5 wt % CaCO3. 3.2. In Vitro Ultrasound Enhanced-Contrast Ability of CaCO3AlgNC. First, the characteristic of CO2 gas generation by CaCO3AlgNC was investigated by observing bubble generation and measuring the CO2 amount from the particle in PBS (pH 7.4 and 6.5) (Figure 3). From the observation
Figure 2. Characterization of CaCO3AlgNC compared with different samples by (A) FTIR and (B) XRD analysis. CaCO3 is CaCO3 prepared by direct reaction between Ca2+ and CO32− without NC for 15 h. CaCO3 (15 min) and CaCO3 (3 sec) indicate CaCO3 prepared similarly by reaction for 15 min and 3 s, respectively.
affected by alginate loading. However, in the case of Ca2+-added AlgNC, additional XRD patterns occurred at 30−32.5° and 45−47.5°, which indicated that the rearrangement of alginate inside the AlgNC occurred by the interaction with Ca2+. In addition, after the formation of CaCO3 in AlgNC had been induced, the XRD spectrum of CaCO3AlgNC showed the peaks from Ca2+-added AlgNC as well as the peaks from CaCO3 prepared at various reaction times. CaCO3 samples showed different crystalline states depending on incubation time;47,48 CaCO3, which was reacted for 15 h, can be considered as calcite that is most stable anhydrous state of CaCO3 with XRD peaks at 27−30°.47,49 On the other hand, CaCO3 (3 sec) and CaCO3 (15 min) with three peaks between 25°, 27°, and 32° can be considered as vaterite, a significantly unstable anhydrous state of CaCO3.47,49,50 In addition to the XRD results, because CaCO3AlgNC was an assembly of CaCO3 nanoparticles in the nanocarrier by its morphology (TEM image in Figure 1A), CaCO3AlgNC could be considered as the mixture of CaCO3 nanoparticles with calcite and vaterite states. Furthermore, the presence of both calcite and vaterite states in CaCO3AlgNC suggested that CaCO3 formed in the nanocarrier was not very stable state; thus, it could generate gas in the aqueous environment including physiological condition or by ultrasound pulse. The composition of CaCO3AlgNC was analyzed by TGA (Figure S2). Because CaCO3 showed a good thermal stability up to 800 °C in TGA,51 thermal degradation of CaCO3AlgNC below 800 °C could be considered as the degradation of the organic part. From TGA measurement, the composition of CaCO3 and that of organic part were 41 ± 5 and 59 ± 5 wt %, respectively. Furthermore, the amount of alginate in CaCO3AlgNC was analyzed to be 12 ± 2 wt % by DMMB assay. By combining these data, the composition of
Figure 3. CO2 generation from CaCO3AlgNC at different pH values: (A) images of bubble generation; (B) amounts of CO2 generation from CaCO3AlgNC in PBS at pH 6.5 and 7.4. n = 3; cc-atm, gas volume (cc) in 1 atm pressure; ∗, p < 0.05 from t test by comparison between CaCO3AlgNC (pH 7.4) and CaCO3AlgNC (pH 6.5).
(Figure 3A), initially, CaCO3AlgNC showed some bubbling at pH 7.4, but much more bubble generation was observed at pH 6.5. The degree of bubble generation decreased over time, but at all time points, bubble generation at pH 6.5 was higher than that at pH 7.4. The CO2 generation from CaCO3AlgNC was quantitatively analyzed by an air quality meter (Figure 3B), and this result coincided well with the visual observation; the CO2 generation from CaCO3AlgNC at pH 6.5 was significantly higher than that at pH 7.4, particularly at early time points. With time, the amount of CO 2 gas generation from CaCO3AlgNC was decreased. Furthermore, AlgNC without CaCO3 did not show any CO2 generation with PBS at both pH 7.4 and 6.5. Thus, it could be concluded that CaCO3 in the nanocarrier generated CO2 gas. This result indicates that the CaCO3AlgNC can improve US signal in the aqueous environment by gas generation. This gas generation is triggered by lowering the pH, because CaCO3 forms more CO2 gas in an acidic environment52 by enhanced dissolution, which would be beneficial for imaging the tumor site due to a little acidic microenvironment of the tumor.24,53,54 After that, the ultrasound enhanced-contrast ability of CaCO3AlgNC was confirmed and compared with control groups including PBS alone, bare NC, and AlgNC by using an ultrasound imaging medical unit after the injection of samples into agarose gel with different pH values (Figure 4). In all pH environments from pH 7.4 to 5.0, CaCO3AlgNC only showed US signal improvement, proving that it could be detected by 8413
DOI: 10.1021/acsami.6b02115 ACS Appl. Mater. Interfaces 2016, 8, 8409−8418
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Figure 4. Ultrasound images of CaCO3AlgNC compared to different samples injected into 1 wt % agarose gel at different pH values. Scale bar = 5 mm; red circle, agarose gel.
imaging. On the other hand, all other control samples did not show any sign of US signal in all pH environments from pH 7.4 to 5.0. Therefore, the presence of CaCO3 in the nanocarrier was essential for the detection and imaging by US signal resulting exclusively from gas generation, and other organic parts in the nanocarrier did not contribute at all to the US signaling.24 To further characterize the ultrasound enhanced-contrast enhancement ability of CaCO3AlgNC, US signal from the particle injected agarose gel was observed over time in different pH environments (pH 7.4, 6.5, and 5) (Figure 5 and Figure S3). At pH 7.4, CaCO3AlgNC showed ultrasound enhancedcontrast image at the injection spot at early time points until 1 h. However, 3 h after injection, no more US signal at the injection spot of CaCO3AlgNC was observed at pH 7.4. In contrast, the injection spot of CaCO3AlgNC was observable over 12 h at pH 6.5, and it was observable over 24 h at pH 5.0 (Figure 5 and Figure S3). These results clearly indicate that CaCO3AlgNC could generate enough CO2 for imaging by US, and a lower pH further facilitated the gas generation by accelerating the dissolution of mineralized CaCO3 in the nanocarrier for a long time,24 which corresponded well to the prior results of bubbling (Figure 3) in this section. Also, this in vitro result supports that the presence of a rather unstable state of CaCO3 formed in CaCO3AlgNC can generate gas even at pH 7.4, but the gas generation from CaCO3AlgNC at pH 7.4 did not last long enough to be used for US imaging, whereas the US enhanced contrast by CaCO3AlgNC at slightly acidic condition, like a tumor, lasted long enough for US imaging. 3.3. In Vivo Tumor Imaging by Ultrasound Enhanced Contrast of CaCO3AlgNC. Before in vivo application of CaCO3AlgNC, the toxicity of CaCO3AlgNC was analyzed in vitro (Figure S4). No cytotoxicity was observed on SCC7 cells up to 1.25 mg/mL concentration of CaCO3AlgNC as well as bare NC and AlgNC. The nontoxicity of pluronic nanocarriers with different kinds of loading was reported previously.31,41 Also, CaCO3 itself is reported to be biocompatible in the range used in this experiment.55,56 Furthermore, CaCO3AlgNC showed cellular uptake to cancer cells (Figure S5). Both nontoxic and cell uptake characteristics of CaCO3AlgNC made this nanosystem suitable for in vivo imaging application. After iv injection of CaCO3AlgNC to tumor-bearing mice, the tumor site was monitored by US imaging (Figure 6). Enhanced US signal was observed 30 min after CaCO3AlgNC
Figure 5. (A) Ultrasound images and (B) ultrasound contrast enhanced ratio (I/I0, I0, US signal intensity with PBS injection in ROI; I, US signal intensity with CaCO3AlgNC injection in ROI) of CaCO3AlgNC injected into 1 wt % agarose gel at different pH values. Scale bar = 5 mm; red dash line circle, region of interest (ROI), n = 3.
injection, compared to before injection state. This US signal at the tumor site slightly increased further at 1 h postinjection. Therefore, it was possible to detect the tumor site by US imaging upon iv injection of CaCO3AlgNC. In contrast, AlgNC showed no difference in US signal before and after the sample injection. Thus, the enhanced US contrast resulted from the gas generated by CaCO3 in the nanocarrier. On the other hand, when the liver was monitored by US imaging after iv injection of CaCO3AlgNC injection via tail vein, no significant change in ultrasound signal was detected (Figure S6). Therefore, CaCO3AlgNC could result in selective US signal enhancement of the tumor site upon iv injection. This selective ultrasound enhanced contrast of CaCO3AlgNC might result from the selective targeting of the nanocarrier at the tumor site due to the nanosized UECA by the EPR effect as well as the accelerated gas generation at more acidic tumor site than the liver.24,27 In addition, these results indicate that the local concentration of CaCO3AlgNC in the tumor was high enough to reach the threshold of CaCO3 concentration to be detected by US signal enhancement in contrast to the low CaCO3AlgNC concentration in the liver resulting from low accumulation of UECA to be detected by US signal enhancement.27,29 As a result, the CaCO3AlgNC could be applied for US imaging to distinguish tumor from the normal tissues such as the liver after iv injection. 3.4. In Vivo Biodistribution and Clearance of CaCO3AlgNC by Optical Imaging. To characterize the 8414
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Figure 6. In vivo ultrasound imaging of tumor before and after iv injection of (A) AlgNC and (B) CaCO3AlgNC. (C) Contrast enhanced ratio (Ipostinjection/Iinitial in ROI) of CaCO3AlgNC and AlgNC. Scale bar = 5 mm, n = 3; red dash line circle, region of interest (ROI); ∗, p < 0.05, and #, p > 0.05, from t test by comparison between samples.
nonspecific liver accumulation of the nanocarrier.57 Signals were also observed from the lung and liver, so some accumulation of the nanoparticle, probably slightly larger particles considering the particles are not monodispersed, in the lung, occurred. Although the signals at the lung and liver were lower than that in the tumor and also decreased over time, this might cause a problem later.58 The relatively fast clearance of CaCO3AlgNC from the body as well as the tumor site must result from no surface modification of the pluronic-based nanocarrier, as reported by us.31 If a nanosystem is not aimed for drug delivery, but only for detection and imaging, the characteristic of CaCO3AlgNC showing tumor targeting within 1 h upon iv injection, followed by a rapid clearance, would be beneficial because it is desired that the contrast agent needs to detected in a short time after injection and be cleared out after images have been taken for the patient’s convenience and safety. Resultantly, this CaCO3AlgNC might be suitable as a nano-UECA for tumor imaging upon iv injection. The actual presence of CaCO3 at the tumor site at 1 h postinjection via tail vein was further confirmed by staining the tumor section with Alizarin Red S (Figure S8). Red color in the tumor at 1 h postinjection only appeared from CaCO3AlgNC, whereas no staining was observed from PBS or AlgNC. In addition, the tumor at 6 h postinjection of CaCO3AlgNC showed diminished red color, corresponding well to the fast
biodistribution and clearance of CaCO3AlgNC upon iv injection, Cy5.5-attached CaCO3AlgNC was injected and optical fluorescence was monitored by IVIS (Figure 7 and Figure S7). At the tumor site, the fluorescence signal from CaCO3AlgNC increased until 1 h postinjection, followed by a fast decrease showing almost no detectable fluorescence signal at 24 h, similar to bare NC, as previously reported.31 To analyze more correctly, mice were sacrificed at 1 and 24 h, and the fluorescence signals from the ex vivo tumor and major organs were analyzed and compared with the control, free Cy5.5. At 1 h, the strongest signal was observed in the tumor compared to other major organs including the liver, which clearly indicates that selective tumor targeting was achieved by nanosized CaCO3AlgNC upon iv injection. On the other hand, at 24 h, the signal from the tumor as well as those from other organs including the liver decreased significantly, suggesting a rapid clearance of CaCO3AlgNC from the body. In contrast, free Cy5.5 showed a minimum fluorescence compared to CaCO3AlgNC without tumor accumulation, indicating the fluorescence signal of the nanoparticle was not from the degraded free dye but the particle itself. The initially selective tumor targeting of CaCO3AlgNC might be associated with the EPR effect of the present nanosystem,25,26,31 and the flexible state of the nanocarrier containing lots of small CaCO3 particles instead of a single, large CaCO3 might also help to reduce the 8415
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Figure 7. Representative NIR fluorescence images of tumor-bearing mouse (A) and explanted ex vivo tumor and major organs (B) after iv injection of CaCO3AlgNC. Quantitative comparison of corresponding fluorescence intensities (C, D). n = 3, iv injection of free Cy5.5 was used as control; black circle, tumor area; ∗, p < 0.05, from t test by comparison between 1 h postinjection and 24 h postinjection of CaCO3AlgNC.
nanosized UECA showed high colloidal stability in serumcontaining media. CaCO3 nanoparticles contained inside the nanocarrier were not in a perfect crystalline state, but a mixture with a relatively unstable state. In aqueous environment, this UECA generated bubbling, more vigorously and longer at a slightly acidic environment. This UECA showed the enhanced US contrast in vitro, more strongly at an acidic condition. More importantly, the selective tumor detection by US imaging with a commercial US medical unit was possible upon intravenous injection of this nano-UECA into tumor-bearing mice in contrast to no change of US imaging in the liver. Upon injection, without targeting ligand, CaCO3AlgNC was preferentially targeted to the tumor site in 1 h, followed by a rapid clearance from the body. Therefore, the present nanosized UECA, CaCO3AlgNC, could be applied as an in vivo US diagnostic agent by intravenous injection for the patient’s convenience and safety for tumor imaging.
clearance of the pluronic carrier in Figure 7 and indicating that CaCO3 in CaCO3AlgNC moved together with pluronic nanocarrier without the destruction of the particle or the escape from the carrier. Therefore, all of these results support that the nanosized CaCO3 was preferentially delivered to the tumor site upon iv injection, which enabled the selective tumor detection by US imaging. Compared to the previous reports of nanoparticle systems for US enhanced tumor imaging, CaCO3AlgNC with a good colloidal stability showed the increase in CO2 generation and US signal with decrease in pH (Figures 3 and 5), similar to the refs 24 and 28. However, without targeting ligand, CaCO3AlgNC showed a relatively fast selective tumor targeting in 1 h upon intravenous injection, not intratumoral injection. Also, it showed a rapid clearance after that, thus minimizing toxicity issues. Furthermore, upon intravenous injection of CaCO3AlgNC, generated CO2 from CaCO3AlgNC in the tumor site was large enough to show the US signal significantly different from the liver (Figure 6 and Figure S6). In addition, this in vivo ultrasound enhancement ratio by CaCO3AlgNC increased up to ∼1.8 (Figure 6C), which is higher than in vivo results of intratumoral injection of ref 24. In view of these facts, this CaCO3AlgNC can be useful for tumor imaging by US because of its intravenous injectability, fast tumor targeting ability without targeting ligand, and rapid clearance after that.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b02115. Stability of CaCO3 AlgNC, TGA analysis of CaCO3AlgNC, long-term US images of CaCO3AlgNC, cytotoxicity of CaCO 3 AlgNC, cellular uptake of CaCO3AlgNC, comparison of US imaging of liver between pre- and postinjection of CaCO3AlgNC, NIR fluorescence images in vivo and ex vivo after injection of CaCO3AlgNC, Alizarin Red S staining of tumor after CaCO3AlgNC injection (PDF)
4. CONCLUSION Nanosized CaCO3-loaded nanocarrier (CaCO3AlgNC) was successfully prepared as an ultrasound enhanced-contrast agent (UECA) for ultrasound imaging of tumor upon intravenous injection. With ∼160 nm in size and neutral surface charge, the 8416
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(17) Stride, E.; Saffari, N. Microbubble Ultrasound Contrast Agents: A Review. Proc. Inst. Mech. Eng., Part H 2003, 217 (6), 429−447. (18) Zhou, Q.-L.; Chen, Z.-Y.; Wang, Y.-X.; Yang, F.; Lin, Y.; Liao, Y.-Y. Ultrasound-Mediated Local Drug and Gene Delivery Using Nanocarriers. BioMed Res. Int. 2014, 2014, 1−13. (19) Liberman, A.; Martinez, H. P.; Ta, C. N.; Barback, C. V.; Mattrey, R. F.; Kono, Y.; Blair, S. L.; Trogler, W. C.; Kummel, A. C.; Wu, Z. Hollow Silica and Silica-Boron Nano/microparticles for Contrast-Enhanced Ultrasound to Detect Small Tumors. Biomaterials 2012, 33 (20), 5124−5129. (20) Ta, C. N.; Liberman, A.; Martinez, H. P.; Barback, C. V.; Mattrey, R. F.; Blair, S. L.; Trogler, W. C.; Kummel, A. C.; Wu, Z. Integrated Processing of Contrast Pulse Sequencing Ultrasound Imaging for Enhanced Active Contrast of Hollow Gas Filled Silica Nanoshells and Microshells. J. Vac. Sci. Technol. B 2012, 30 (2), 02C104. (21) Ferrara, K.; Pollard, R.; Borden, M. Ultrasound Microbubble Contrast Agents: Fundamentals and Application to Gene and Drug Delivery. Annu. Rev. Biomed. Eng. 2007, 9, 415−447. (22) Chung, M.-F.; Chen, K.-J.; Liang, H.-F.; Liao, Z.-X.; Chia, W.T.; Xia, Y.; Sung, H.-W. A Liposomal System Capable of Generating CO 2 Bubbles to Induce Transient Cavitation, Lysosomal Rupturing, and Cell Necrosis. Angew. Chem., Int. Ed. 2012, 51 (40), 10089− 10093. (23) Kang, E.; Min, H. S.; Lee, J.; Han, M. H.; Ahn, H. J.; Yoon, I.-C.; Choi, K.; Kim, K.; Park, K.; Kwon, I. C. Nanobubbles from GasGenerating Polymeric Nanoparticles: Ultrasound Imaging of Living Subjects. Angew. Chem., Int. Ed. 2010, 49 (3), 524−528. (24) Min, K. H.; Min, H. S.; Lee, H. J.; Park, D. J.; Yhee, J. Y.; Kim, K.; Kwon, I. C.; Jeong, S. Y.; Silvestre, O. F.; Chen, X.; Hwang, Y.-S.; Kim, E.-C.; Lee, S. C. pH-Controlled Gas-Generating Mineralized Nanoparticles: A Theranostic Agent for Ultrasound Imaging and Therapy of Cancers. ACS Nano 2015, 9 (1), 134−145. (25) Figueiredo, M.; Esenaliev, R. PLGA Nanoparticles for Ultrasound-Mediated Gene Delivery to Solid Tumors. J. Drug Delivery 2012, 2012, 1−20. (26) Zheng, R.; Yin, T.; Wang, P.; Zheng, R.; Zheng, B.; Cheng, D.; Zhang, X.; Shuai, X.-T. Nanobubbles for Enhanced Ultrasound Imaging of Tumors. Int. J. Nanomed. 2012, 895. (27) Díaz-López, R.; Tsapis, N.; Santin, M.; Bridal, S. L.; Nicolas, V.; Jaillard, D.; Libong, D.; Chaminade, P.; Marsaud, V.; Vauthier, C.; Fattal, E. The Performance of PEGylated Nanocapsules of Perfluorooctyl Bromide as an Ultrasound Contrast Agent. Biomaterials 2010, 31 (7), 1723−1731. (28) Lee, J.; Min, H.-S.; You, D. G.; Kim, K.; Kwon, I. C.; Rhim, T.; Lee, K. Y. Theranostic Gas-Generating Nanoparticles for Targeted Ultrasound Imaging and Treatment of Neuroblastoma. J. Controlled Release 2016, 223, 197−206. (29) Yang, P.; Li, D.; Jin, S.; Ding, J.; Guo, J.; Shi, W.; Wang, C. Stimuli-Responsive Biodegradable Poly(methacrylic Acid) Based Nanocapsules for Ultrasound Traced and Triggered Drug Delivery System. Biomaterials 2014, 35 (6), 2079−2088. (30) Choi, W. I.; Tae, G.; Kim, Y. H. One Pot, Single Phase Synthesis of Thermo-Sensitive Nano-Carriers by Photo-Crosslinking of a Diacrylated Pluronic. J. Mater. Chem. 2008, 18 (24), 2769. (31) Kim, J.-Y.; Choi, W. I.; Kim, Y. H.; Tae, G.; Lee, S.-Y.; Kim, K.; Kwon, I. C. In-Vivo Tumor Targeting of Pluronic-Based NanoCarriers. J. Controlled Release 2010, 147 (1), 109−117. (32) Kim, J.-S.; Kim, M.; Won, D.-A.; Tae, G. Preparation of Nanosize Alginate Gel Using Pluronic-Based Nano-Carrier as a Template. Eur. Polym. J. 2015, 72, 632−641. (33) Zhai, P.; Chen, X. B.; Schreyer, D. J. Preparation and Characterization of Alginate Microspheres for Sustained Protein Delivery within Tissue Scaffolds. Biofabrication 2013, 5 (1), 015009. (34) LeRoux, M. A.; Guilak, F.; Setton, L. A. Compressive and Shear Properties of Alginate Gel: Effects of Sodium Ions and Alginate Concentration. J. Biomed. Mater. Res. 1999, 47 (1), 46−53.
AUTHOR INFORMATION
Corresponding Author
*(G.T.) E-mail:
[email protected]. Phone: +82-62-715-2305. Fax: +82-62-715-2304. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
This research was financially supported by the National Research Foundation of Korea (NRF) funded by MSIP of Korea (2013R1A2A2A03068802) and by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare of Korea (Grant HI15C2532).
(1) Yeh, C.-K. Ultrasound Microbubble Contrast Agents for Diagnostic and Therapeutic Applications: Current Status and Future Design. Chang Gung Med. J. 2012, 35 (2), 125−139. (2) Foster, F. S.; Hossack, J.; Adamson, S. L. Micro-Ultrasound for Preclinical Imaging. Interface Focus 2011, 1 (4), 576−601. (3) Taeymans, O.; Penninck, D. G.; Peters, R. M. Comparison between Clinical, Ultrasound, CT, MRI, and Pathology Findings in Dogs Presented for Suspected Thyroid Carcinoma: Imaging Of Thyroid Carcinoma In Dogs. Vet. Radiol. Ultrasound 2013, 54 (1), 61− 70. (4) Eskandar, O.; Eckford, S.; Watkinson, T. Safety of Diagnostic Imaging in Pregnancy. Part 2: Magnetic Resonance Imaging, Ultrasound Scanning and Doppler Assessment. Obstet. Gynaecol. 2010, 12 (3), 171−177. (5) Kanal, E.; Shellock, F. G.; Talagala, L. Safety Considerations in MR Imaging. Radiology 1990, 176 (3), 593−606. (6) Kanal, E.; Shellock, F. G. Burns Associated with Clinical MR Examinations. Radiology 1990, 175 (2), 585−585. (7) Shellock, F. G.; Crues, J. V. Corneal Temperature Changes Induced by High-Field-Strength MR Imaging with a Head Coil. Radiology 1988, 167 (3), 809−811. (8) Tan, Y. K.; Ostergaard, M.; Conaghan, P. G. Imaging Tools in Rheumatoid Arthritis: Ultrasound vs Magnetic Resonance Imaging. Rheumatology 2012, 51 (Suppl. 7), vii36−vii42. (9) Ferreira, A. M.; Mendes, L.; Soares, L.; da Graça Correia, M.; Gil, V. Cardiac Magnetic Resonance in a Patient with MRI-Conditional Pacemaker. Rev. Port. Cardiol. 2013, 32 (2), 159−162. (10) Martin, E. T.; Coman, J. A.; Shellock, F. G.; Pulling, C. C.; Fair, R.; Jenkins, K. Magnetic Resonance Imaging and Cardiac Pacemaker Safety at 1.5-T. J. Am. Coll. Cardiol. 2004, 43 (7), 1315−1324. (11) McCollough, C. H.; Guimarães, L.; Fletcher, J. G. In Defense of Body CT. AJR, Am. J. Roentgenol. 2009, 193 (1), 28−39. (12) Rogers, L. F. Taking Care of Children: Check out the Parameters Used for Helical CT. AJR, Am. J. Roentgenol. 2001, 176 (2), 287−287. (13) Paterson, A.; Frush, D. P.; Donnelly, L. F. Helical CT of the Body: Are Settings Adjusted for Pediatric Patients? AJR, Am. J. Roentgenol. 2001, 176 (2), 297−301. (14) Brenner, D. J.; Elliston, C. D.; Hall, E. J.; Berdon, W. E. Estimated Risks of Radiation-Induced Fatal Cancer from Pediatric CT. AJR, Am. J. Roentgenol. 2001, 176 (2), 289−296. (15) Goldberg, B. B.; Liu, J.-B.; Forsberg, F. Ultrasound Contrast Agents: A Review. Ultrasound Med. Biol. 1994, 20 (4), 319−333. (16) Hassani, S. Principles of Ultrasonography. J. Natl. Med. Assoc. 1974, 66 (3), 205−207. 8417
DOI: 10.1021/acsami.6b02115 ACS Appl. Mater. Interfaces 2016, 8, 8409−8418
Research Article
ACS Applied Materials & Interfaces
Microparticles on HeLa Cells. World J. Nano Sci. Eng. 2012, 02 (01), 25−31. (56) Kamba, A.; Ismail, M.; Ibrahim, T.; Zakaria, Z. Biocompatibility of Bio Based Calcium Carbonate Nanocrystals Aragonite Polymorph on NIH 3T3 Fibroblast Cell Line. Afr. J. Tradit., Complementary Altern. Med. 2014, 11 (4), 31. (57) Choi, W. I.; Kim, J.-Y.; Heo, S. U.; Jeong, Y. Y.; Kim, Y. H.; Tae, G. The Effect of Mechanical Properties of Iron Oxide NanoparticleLoaded Functional Nano-Carrier on Tumor Targeting and Imaging. J. Controlled Release 2012, 162 (2), 267−275. (58) Yildirimer, L.; Thanh, N. T. K.; Loizidou, M.; Seifalian, A. M. Toxicology and Clinical Potential of Nanoparticles. Nano Today 2011, 6 (6), 585−607.
(35) Choi, W. I.; Kim, Y. H.; Tae, G. Controlled Release of Proteins from Pluronic-Based Nano-Carrier. Macromol. Res. 2011, 19 (6), 639− 642. (36) Han, S. B.; Shin, Y. J.; Hyon, J. Y.; Wee, W. R. Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells▽. Antimicrob. Agents Chemother. 2011, 55 (10), 4519−4523. (37) Ishiyama, M.; Tominaga, H.; Shiga, M.; Sasamoto, K.; Ohkura, Y.; Ueno, K. A Combined Assay of Cell Vability and in Vitro Cytotoxicity with a Highly Water-Soluble Tetrazolium Salt, Neutral Red and Crystal Violet. Biol. Pharm. Bull. 1996, 19 (11), 1518−1520. (38) Kim, B. J.; Min, K. H.; Hwang, G. H.; Lee, H. J.; Jeong, S. Y.; Kim, E.-C.; Lee, S. C. Calcium Carbonate-Mineralized Polymer Nanoparticles for pH-Responsive Robust Nanocarriers of Docetaxel. Macromol. Res. 2015, 23 (1), 111−117. (39) Grant, G. T.; Morris, E. R.; Rees, D. A.; Smith, P. J.; Thom, D. Biological Interactions between Polysaccharides and Divalent Cations: The Egg-Box Model. FEBS Lett. 1973, 32 (1), 195−198. (40) Lee, K. Y.; Mooney, D. J. Alginate: Properties and Biomedical Applications. Prog. Polym. Sci. 2012, 37 (1), 106−126. (41) Choi, W. I.; Kim, J.-Y.; Kang, C.; Byeon, C. C.; Kim, Y. H.; Tae, G. Tumor Regression In Vivo by Photothermal Therapy Based on Gold-Nanorod-Loaded, Functional Nanocarriers. ACS Nano 2011, 5 (3), 1995−2003. (42) Alexis, F.; Pridgen, E.; Molnar, L. K.; Farokhzad, O. C. Factors Affecting the Clearance and Biodistribution of Polymeric Nanoparticles. Mol. Pharmaceutics 2008, 5 (4), 505−515. (43) Sonavane, G.; Tomoda, K.; Makino, K. Biodistribution of Colloidal Gold Nanoparticles after Intravenous Administration: Effect of Particle Size. Colloids Surf., B 2008, 66 (2), 274−280. (44) Otsuka, H.; Nagasaki, Y.; Kataoka, K. PEGylated Nanoparticles for Biological and Pharmaceutical Applications. Adv. Drug Delivery Rev. 2003, 55 (3), 403−419. (45) Talelli, M.; Rijcken, C. J. F.; van Nostrum, C. F.; Storm, G.; Hennink, W. E. Micelles Based on HPMA Copolymers. Adv. Drug Delivery Rev. 2010, 62 (2), 231−239. (46) Ma, X.; Zhou, B.; Deng, Y.; Sheng, Y.; Wang, C.; Pan, Y.; Wang, Z. Study on CaCO3/PMMA Nanocomposite Microspheres by Soapless Emulsion Polymerization. Colloids Surf., A 2008, 312 (2− 3), 190−194. (47) Kontoyannis, C. G.; Vagenas, N. V. Calcium Carbonate Phase Analysis Using XRD and FT-Raman Spectroscopy. Analyst 2000, 125 (2), 251−255. (48) Demichelis, R.; Raiteri, P.; Gale, J. D.; Dovesi, R. The Multiple Structures of Vaterite. Cryst. Growth Des. 2013, 13 (6), 2247−2251. (49) Gopi, S.; Subramanian, V. K.; Palanisamy, K. Aragonite− Calcite−Vaterite: A Temperature Influenced Sequential Polymorphic Transformation of CaCO3 in the Presence of DTPA. Mater. Res. Bull. 2013, 48 (5), 1906−1912. (50) Schüler, T.; Tremel, W. Versatile Wet-Chemical Synthesis of Non-Agglomerated CaCO3 Vaterite Nanoparticles. Chem. Commun. 2011, 47 (18), 5208. (51) Abdolmohammadi, S.; Siyamak, S.; Ibrahim, N. A.; Yunus, W. M. Z. W.; Rahman, M. Z. A.; Azizi, S.; Fatehi, A. Enhancement of Mechanical and Thermal Properties of Polycaprolactone/Chitosan Blend by Calcium Carbonate Nanoparticles. Int. J. Mol. Sci. 2012, 13 (12), 4508−4522. (52) Choi, B. Y.; Park, H. J.; Hwang, S. J.; Park, J. B. Preparation of Alginate Beads for Floating Drug Delivery System: Effects of CO2 GasForming Agents. Int. J. Pharm. 2002, 239 (1), 81−91. (53) Wang, Y.; Zhou, K.; Huang, G.; Hensley, C.; Huang, X.; Ma, X.; Zhao, T.; Sumer, B. D.; DeBerardinis, R. J.; Gao, J. A NanoparticleBased Strategy for the Imaging of a Broad Range of Tumours by Nonlinear Amplification of Microenvironment Signals. Nat. Mater. 2014, 13 (2), 204−212. (54) Manchun, S.; Dass, C. R.; Sriamornsak, P. Targeted Therapy for Cancer Using pH-Responsive Nanocarrier Systems. Life Sci. 2012, 90 (11−12), 381−387. (55) Zhang, Y.; Ma, P.; Wang, Y.; Du, J.; Zhou, Q.; Zhu, Z.; Yang, X.; Yuan, J. Biocompatibility of Porous Spherical Calcium Carbonate 8418
DOI: 10.1021/acsami.6b02115 ACS Appl. Mater. Interfaces 2016, 8, 8409−8418
