Nontoxic Formulations of Scintillation Nanocrystals for Use as X-ray

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Non-Toxic Formulations of Scintillation Nanocrystals for Use as X-Ray Computed Tomography (CT) Contrast Agents Jaewon Lee, Seulgi Choi, Ki Hyun Kim, Hock Gan Heng, Sandra E. Torregrosa-Allen, Benjamin S. Ramsey, Bennett D. Elzey, and You-Yeon Won Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00451 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on October 30, 2016

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Non-Toxic Formulations of Scintillation Nanocrystals for Use as X-Ray Computed Tomography (CT) Contrast Agents Jaewon Lee,1 Seulgi Choi,1 Ki Hyun Kim,1 Hock Gan Heng,2 Sandra E. Torregrosa-Allen,3,4 Benjamin S. Ramsey,3,4 Bennett D. Elzey,3,4 You-Yeon Won1,4,* 1

School of Chemical Engineering, Purdue University, West Lafayette, Indiana 47907, United States, 2

Department of Veterinary Clinical Sciences, Purdue University, West Lafayette, Indiana 47907, United States

3

Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana 47907, United States

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Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, United States

Abstract X-ray computed tomography (CT) is currently one of the most powerful, non-invasive, clinical in vivo imaging techniques, which has resulted from advances in both X-ray device and contrast enhancement technologies. The present study demonstrates, for the first time, that metal tungstates (such as CaWO4) are promising contrast agents for X-ray, radiation and CT imaging, because of the high X-ray mass attenuation of tungsten (W). We have developed a method of formulation, in which CaWO4 (CWO) nanoparticles (NPs) are encapsulated

*

To whom correspondence should be addressed. E-mail: [email protected] 1


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within a bio-compatible poly(ethylene glycol-b-D,L-lactic acid) (PEG-PLA) block copolymer (BCP) capsule. We show that these PEG-PLA-encapsulated CWO NPs (170 ± 10 nm hydrodynamic diameter) produce a higher CT contrast (by a factor of about 2) than commercial iodine-based radio-contrast agents (e.g., Iohexol) at identical molar concentrations of W or I atoms. PEG-PLA-coated CWO NPs are chemically stable and completely non-toxic. It was confirmed that the maximum tolerated dose (MTD) of this material in mice is significantly higher (250 ± 50 mg per kg body weight following a single intravenous (IV) administration) than, for instance, commercially available dextran-coated iron oxide nanoparticles that are currently used clinically as MRI contrast agents (MTD in mice ≈ 168 mg/kg per dose IV). IV-injected PEG-PLA/CWO NPs caused no histopathologic damage in major excretory organs (heart, liver, lungs, spleen, and kidney). When an IV dose of 100 mg/kg was given to mice, the blood circulation half-life was measured to be about 4 hours, and more than 90 percent of the NPs were cleared from the mice within 24 hours via the renal and hepatobiliary systems. When intratumorally administered, PEG-PLA-coated CWO NPs showed complete retention in a tumor-bearing mouse model (measurements were made up to one week). These results suggest that PEG-PLA-coated CWO NPs are promising materials for use in CT contrast.

Introduction X-ray computed tomography (CT) is an efficient, non-invasive method of obtaining real-time images of internal organs. The short imaging time makes this technique especially suitable for routine diagnosis.1 Unfortunately, however, the lack of natural contrast between different tissues2 often necessitates the use of contrast agents to enhance the visibility of 2



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specific structures in the body under X-ray. Current commercial radio-contrast agents are based on two chemistries: iodine and barium sulfate. In iodine-based contrast media, iodine is chemically conjugated to organic substrate compounds. Due to their water solubility, these iodine-containing organic agents are molecularly dissolved in water. These aqueous iodine formulations are administered, for instance, intravascularly for the radiographic visualization of blood vessels, intrathecally in discography of the spine, and intraabdominally for investigation of any body cavity or potential space. Iodine-based substances are popular as CT contrast media, not necessarily because they are best in terms of their X-ray attenuation properties, but simply because iodine more or less strikes a balance between toxicity and cost.2

The use of iodine compounds for microvascular imaging is limited mainly by two

factors: first, the non-specific biodistribution, and second, the rapid clearance of these compounds from the body.3, 4 Also, the accumulation of iodine in the kidney often causes renal toxicity.2 The second type of contrast agent, barium sulfate, is typically formulated in the form of aqueous slurry (because it exists as a water-insoluble powder) and administered directly into the stomach or gastrointestinal tract for imaging of the digestive system. X-ray absorbing nanoparticles (NPs) can fill the gap between these two types of radio-contrast agents. For instance, appropriately sized nanoparticles (between 10 and 1000 nm) can be used for X-ray examinations of tumors, for which iodine-based compounds (< a few nanometers) and barium sulfate powder (> a few micrometers) are not suitable. Gold nanoparticles and bismuth sulfide nanoparticles are two examples of materials that have been extensively investigated for CT contrast applications.1,

4

Unfortunately, most of these

previously studied nanoparticles have been found to be toxic at the cellular or genetic level because they can bind to the cell membrane causing lysis and apoptosis,5-7 and they can also 3


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liberate ions of heavy metals that can even damage the host cell.5, 6 The release of metal ions can be facilitated in a corrosive (e.g., acidic) physiological environment.5,

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It has been

reported that bismuth sulfide (Bi2S3), for instance, dissolves into bismuth (Bi3+) and sulfide (S2-) ions in physiological media to a certain degree, and as a result, cause significant toxicity in cells.9, 10 Toxicity of gold nanoparticles is still a debated issue at present. Although bulk gold is known to be chemically inert, there is evidence that nanoparticulate gold is toxic; the level of gold’s toxicity is influenced by such parameters as cell type, nanoparticle size, surface functionality, concentration and expose time.11 Chemical stability becomes an especially important issue, when certain amounts of nanoparticles stay in the body long-term; for instance, poly(ethylene glycol)-coated (“PEGylated”) quantum dot nanoparticles have been found to remain in the body for more than two years following intratumoral injection in mice.12 Therefore, there is currently a need for exploring safer materials in clinical applications. Scintillation (i.e., radio-luminescent) materials, particularly those belonging to the category of inorganic semiconductors (such as ZnO, CaWO4, etc.), have the potential for use as CT contrast agents, because they are often strong absorbers of X-rays; the X-ray absorption occurs by multiple mechanisms including the photoelectric, Compton scattering and Auger effects.13-17 Scintillating materials have tunable luminescence properties.18-26 The optically excited luminescence of scintillation materials have been investigated for potential applications in in vitro/in vivo protein,22 DNA25 and virus26 detection. However, to our knowledge, little work has been done to test whether scintillation nanocrystals can indeed be used for X-ray contrast enhancement. We believe that, particularly, CaWO4 nanoparticles have the tremendous potential to be adopted as standard radio-contrast agents by clinical 4



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radiologists for the reasons to be discussed below. Unlike many metal (such as gold) and other semiconductor (e.g., ZnO) nanoparticles, the CaWO4 (CWO) material is chemically very stable, does not liberate ions and is completely insoluble in aqueous solution; CWO is a naturally abundant mineral. Compared with the conventional radio-contrast agents (iodine and barium), CWO has a significantly higher X-ray absorbing capability because X-ray mass attenuation increases as approximately Z3 (Z is the atomic number, Z = 30 (Zn), 53 (I), 56 (Ba), 74 (W) and 79 (Au));27 in the range of X-ray energy used for most diagnostic studies (i.e., 10 – 100 keV) the mass attenuation coefficient of W is three to four-fold higher than that of I or Ba.28 Monodisperse CWO nanoparticles in the size range between about 2 and 100 nm can readily be prepared by the solvothermal reaction of sodium tungstate dihydrate with calcium salt.29 We have also demonstrated that fully PEGylated CWO NPs that are stable against fouling under physiological conditions can be produced by encapsulating these NPs with amphiphilic block copolymers (BCPs).29 In the present study, we demonstrate that BCPencapsulated (PEGylated) CWO NPs are non-toxic to live animals (mice); the maximum tolerated dose (MTD) in mice has been determined to be about 250 ± 50 mg per body weight following a single intravenous (i.v.) injection. In this paper, we also report the biodistribution and pharmacokinetic properties of systemically administered PEGylated CWO NPs in mice; CWO NPs are almost completely cleared from the body (mainly by the liver) within 24 hours following i.v. administration. We also show that, on the other hand, when injected directly into mouse xenografts of human cancer, PEGylated CWO NPs remain at the tumor sites for long periods of time (at least for one week). The X-ray attenuation properties of BCP-coated CWO NPs were measured in comparison with a conventional iodine-based contrast agent 5


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(Iohexol). Data confirm that at identical molar concentrations of I vs. W atoms, PEGylated CWO NPs produce a higher contrast and lower transmission than Iohexol. These results overall support that PEG-coated CWO NPs indeed have the great potential to be used as Xray contrast agents.

Results and Discussion The CWO NP sample used in this study was synthesized by a micro-emulsion method under high temperature (160 °C) and pressure

conditions (we cannot measure

presseure, high pressure was resulted from vapor pressure) . The crystalline lattice structure of the CWO NPs was characterized by X-ray diffraction (XRD) (SmartLab, Rigaku). As shown in Figure 1a, the XRD analysis confirmed the scheelite CWO phase. The full width at half maximum (FWHM) at 2θ = 28.775° was 0.49°, suggesting a high crystallinity with an estimated grain size of about 18.5 nm (estimated using the Scherrer equation30). The morphology of the CWO NPs was evaluated using a transmission electron microscope (TEM) (Tecnai 20, FEI). As shown in Figure 1b, the NPs were monodisperse in size and had a mean diameter of about 15 ± 2 nm.

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Figure 2. (a) 1H NMR spectra of the PEG-PLA BCP used in this study. (b) GPC traces for the PEG-PLA (Mw/Mn=1.17) and polystyrene (PS) standards with molecular weights of 357400, 112300, 32300, 12700 and 200 Da. The BCP used in this study for CWO NP encapsulation was poly(ethylene glycol)poly(lactic acid) (PEG-PLA). A PEG-PLA sample, PEG113-PLA45, was prepared by ring opening polymerization of (racemic) lactide under the catalytic influence of 1,8diazabicyclo[5.4.0]undec-7-ene (DBU);31 the subscripts in the BCP notation denote the 7


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number-average degrees of polymerization of the individual blocks determined by 1H NMR spectroscopy (Figure 2a). The polydispersity index of the PEG113-PLA45 material was determined by gel permeation chromatography (GPC) (Waters Breeze HPLC System) to be 1.17 (Figure 2b). PEG-PLA-encapsulated CWO NP samples were prepared using a solvent exchange method.29 The mean hydrodynamic diameter (DH) of BCP-encapsulated CWO NPs in MilliQ-purified water was determined by dynamic light scattering (DLS) (Brookhaven Instruments) measurements; the DH value was 173 nm (Figure 3a). The morphologies of BCP-encapsulated CWO NPs were examined using TEM. Representative images are presented in Figure 3b. As shown in the TEM micrograph, CWO NPs formed clusters during the encapsulation process, and as a result, multiple NPs were incorporated within each BCP assembly. This result is consistent with the size data obtained by DLS testing. The X-ray absorption properties of the BCP-coated CWO NPs were measured in

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Figure 3. (a) Mean hydrodynamic diameters of PEG-PLA-coated CWO NPs in Milli-Q water (red) and 150 mM NaCl (blue). Measurements were performed at a CWO concentration of 0.05 mg/ml. (b) A representative TEM image of PEG-PLA-coated CWO NPs. The sample was negatively stained with 2% uranyl acetate. 8



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comparison with a commercial iodine-based contrast agent, Iohexol. Aqueous solutions containing BCP-encapsulated CWO NPs (or Iohexol) at various different concentrations were prepared in 1.5 ml Eppendorf tubes, and were imaged using a clinical CT scanner instrument (80 kV, 200 mA) (Figure 4a). For each sample, the CT value was calculated as an average for the whole solution in Hounsfield units (HU); the HU scale is a linear transformation of the original linear attenuation coefficient measurement into one in which the radio density of distilled water at standard pressure and temperature (STP) is defined as 0 HU, while the radio density of air at STP is defined as -1000 HU.32 As shown in Figure 4b, the CT values increased linearly with contrast agent concentration. Notably, the CT curve for PEG-PLAcoated CWO NPs showed a higher slope (≈ 7.94 HU/mM) compared with Iohexol (≈ 4.51 HU/mM); at identical molar concentrations of I vs. W atoms, the PEGylated CWO NPs produced a higher contrast and lower transmission (by a factor of about 2) than Iohexol, which was also clearly visible in the cross-sectional CT images shown in Figure 4a (in Figure 4(a), if we compare intensities in each pair of vertically aligned images, it becomes clear that CWO NPs produce a higher CT contrast than Iohexol at low molar concentrations). This result confirms that CWO is a stronger X-ray absorber than Iohexol, as anticipated from the fact that the mass attenuation coefficient scales approximately with the cube of the atomic number, Z (Z = 53 (I), and 74 (W));27 the actual reported values of the X-ray mass attenuation coefficients are 4.438 cm2/kg for tungsten and 1.94 cm2/kg for iodine under, for instance, 100 keV X-ray irradiation.33

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We would like note that reducing the size of BCP-encapsulated CWO NPs (while keeping the total NP mass constant) will not likely increase the overall X-ray attenuation,

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Figure 4. (a) CT images of PEG-PLA-coated CWO NPs (173 nm hydrodynamic diameter) and Iohexol (control) as functions of molar concentration of W or I (data obtained using the GE Light Speed Clinical CT Scanner). (b) CT values in Hounsfield Units (HU) estimated from the images shown in (a). because the X-ray attenuation length of CaWO4 is significantly greater than the overall size of the BCP-coated CWO NPs (DH ≈ 173 nm); the X-ray attenuation length is defined as the depth into the material at which the intensity of the radiation decays to a level of 1/e (approximately 0.37) of its value at the surface, and for CaWO4 (ρ ≈ 6.06 g/cm3) the attenuation length is estimated to be about 119 µm at an X-ray energy of 30 keV.34 That is, in this small particle limit the NPs will be completely transparent to X-rays, and as a result, the size dependence of the nanoparticle’s attenuation strength would disappear. This is, in fact, what has been observed with small-sized (< 60 nm diameter) gold nanoparticles;35 the X-ray attenuation length of Au (ρ ≈ 19.3 g/cm3) is about 20 µm at an X-ray energy of 30 keV.34 Our recent simulation results also support this behavior that the energy absorption per particle mass is independent of nanoparticle size.36 One might also question whether decreasing NP size would be beneficial in terms of preventing NP sedimentation during storage and experimentation. However, sedimentation is 10



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not an important factor, because the sedimentation velocity of 173-nm DH CWO particles in water is estimated to be about 82.5 nm/s; this means that it will take about 61 hours (2.5 days) for these particles to sediment a distance of about 1.8 cm, for instance, in a vertically situated Eppendorf tube. It is known in the literature that NPs with sizes between 100 and 200 nm exhibit prolonged blood circulation time following systemic (e.g., intravenous) administration because they are sufficiently large to avoid renal clearance but at the same time sufficiently small not to be captured by the reticuloendothelial system (RES);37 this is why we chose the current NP size (DH ≈ 173 nm) in this study. However, it should also be noted that where it is desirable depending on specific applications, the NP size may need to be further adjusted; for instance, for achieving a uniform spreading of NPs within tumor tissue following direct intratumoral administration, the particle diameter should be less than about 60 nm.38 Similar sizes are also desirable if one wants to systemically deliver NPs to solid tumors via the utilization of the enhanced permeability and retention (EPR) phenomenon.39,

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recently demonstrated that CWO NPs can also be formulated into sizes less than about 60 nm. The CT contrast and in vivo pharmacological properties of these smaller sized formulations will be investigated in a future study. The time-dependent aggregation behaviors of various uncoated and coated CWO NPs in Milli-Q water or with added 150 mM NaCl have previously been investigated by dynamic light scattering (DLS) (Brookhaven Instruments).41 We repeated these same measurements on newly prepared NP samples. The same general results were confirmed. Uncoated CWO NPs are typically very unstable because of the hydrophobicity of the CWO material. CTAB-coated CWO NPs also undergo rapid aggregation upon the addition of 150 mM NaCl, because the salt screens the electrostatic repulsion between CTAB-coated particles. 11


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The adsorption of PEG-PLA BCPs onto CWO NPs produces PEG brushes of high grafting density on the surfaces of the NPs; the PEG brushes confer long-term colloidal stability to the CWO NPs even under physiological electrolyte conditions. As shown in Figure 5, the mean hydrodynamic diameter of PEG-PLA-coated CWO NPs in deionized water was approximately 163 nm, and this value did not change over 30 days (and likely beyond that as well). The figure also demonstrates that the size and stability characteristics of the PEG-PLAcoated CWO NPs remain unchanged upon addition of 150 mM NaCl. The DLS correlation functions and intensity-based size distributions for PEG-PLA-coated CWO NPs (not shown) confirmed the size monodispersity of these NPs both in deionized water and in the NaCl solution; the polydispersity indices were 0.167 and 0.182, respectively, for the two different conditions (Figure S1). To investigate acute toxicities associated with PEG-PLA-encapsulated CWO NPs (173 nm hydrodynamic diamter), we performed maximum tolerated dose (MTD) studies, along with histopathologic examination of major organs and serology, following a single intravenous (IV) administration of an aqueous NP formulation (200 µl) in normal BALB/C mice (female, 10 weeks old, 20 g body weight). We used the MTD protocol employed by the National Cancer Institute which can be found at 42; According to the NCI guidelines, the MTD is defined as the dose level at which the mouse loses more than 20% of its body weight or there are other signs of significant toxicity. Mice injected with various doses of PEG-PLAcoated CWO NPs were tested for body weight and symptoms of toxicity, in terms of their responses to bright light, reversed position of the body (recovery speed) and sound (of beating the cage), for a period of two weeks. Three series of MTD studies were performed. In the 1st MTD study, the effects of three (relatively low) dose levels (20, 50 and 100 mg of 12



CWO NPs per kg of body weight) were studies (N = 1). The results of this study are summarized in Table S1 (visual observation) and Figure S3 (body weights). At all dose levels, no symptom of toxicity (in terms of the mice’s body weights and responses to light, sound and motion) was observed for two weeks. In the 2nd MTD study, the next 3 higher dose levels (100, 200 and 300 mgCWO/kgbody weight)

were tested in a similar manner (N = 1). The results of this 2nd MTD study are

summarized in Table S2 (visual observation), and Figures S4 (body weights), S5 (histological sections for the 100 mg/kg dose), S6 (histological sections for the 200 mg/kg dose) and S7 (histological sections at the 300 mg/kg dose). The mice that received 100 and 200 mg/kg doses showed mildly squinting eyes, irregular respiration, and weak grabs. However, they completely recuperated from these mild symptoms within a few hours (Table S2). The mice injected with 100 and 200 mg/kg doses also showed slight weight loss during the first two days (Figure S4); the mouse given 200 mg/kg CWO NPs lost about 10% of its body weight by the end of day 2. However, the starting weights were recovered within the next few days.

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At the end of the two-week period, mice were euthanized, major organs (heart, liver, lungs, spleen and kidney) were harvested for histopathologic examination. As shown in Figures S5 and S6, no local abnormalities or necrosis was detected in these organs. At the 300 mg/kg dose level, the mouse immediately died upon injection of the NPs. Histological sections show no evidence of organ damage (Figure S7). Therefore, the death was likely caused by blockage of blood vessels.

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Table 1. Visual observation of mice injected with PEGylayed CWO NPs (173 nm hydrodynamic diameter) from the 3rd MTD study (N = 3). Mice were tested for their responses to bright light, reversed position of the body (recovery speed) and sound (of beating the cage) and their respiration pattern. All mice had strong grabs at all times. A circle (“O”) means normal.

In the 3rd MTD study, we repeated these tests at the higher dose range (i.e., 200 and 300 mg/kg, plus PBS buffer as control) in triplicates (N = 3) to verify the N = 1 results at 200 and 300 mg/kg and thus to conclusively determine the upper dose limit. Interestingly, even at the highest NP dose, 300 mg/kg, all mice survive. At the 200 and 300 mg/kg dose levels, the reproducibility of the (mild) negative effects (in terms of body weight loss in mice, and their responses to light, sound and motion) was confirmed (Table 1, and Figure 5); the mice given 300 mg/kg lost maximum about 10.46% body weight on day 1. This time again, major excretory organs (heart, liver, lungs, spleen and kidney) were harvested at the end of the 2week period for histopathologic detection of gross organ damage. In both the 200 and 300 mg/kg-treated groups, no histopathological changes were detected in major organs relative to those of the control(PBS)-treated mice (Figures 6a through 6e). Taken all together, our conclusion is that the MTD of PEG-PLA-coated CWO NPs is, at least, greater than about 250

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Figure 6. H&E-stained histological sections of mouse (a) heart, (b) kidney, (c) liver, (d) lung and (e) spleen taken at 2 weeks after IV injection of 300 mg PEG-PLA-coated CWO NPs per kg body weight in mice in the 3rd MTD study (N = 3). Images are at 200× magnification. ± 50 mg per kg body weight following a single IV administration; this material is thus much safer than, for instance, commercially available dextran-coated iron oxide nanoparticles that are currently used clinically as MRI contrast agents (MTD in mice ≈ 168 mg/kg per dose IV).43, 44

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Figure 7. (a) Whole body and (b) abdominal in vivo CT tomographs of a mouse that received a single dose of 100 mg CWO/kgbody weight PEG-PLA-coated CWO NPs (173 nm hydrodynamic diameter) through IV route (200 μl total injection volume). Pink and blue arrows, respectively, indicate the heart and liver regions where significant contrast enhancement was observed. The feasibility of using intravenously administered PEG-PLA-coated CWO NPs (173 nm hydrodynamic diameter) for the radiographic visualization of blood vessels/organs

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(angiography) was evaluated in mice. Whole body CT scans were performed on mice (normal BALB/C, female, N = 3) before and at various times (2, 30, 60 and 120 minutes) following a single injection of 100 mg/kg CWO NPs (200 µl injection volume) (Figure 7a). All CT measurements were taken while the mice were under anesthesia; each time the mice were anesthetized for 5 minutes with 2% isoflurane in oxygen. From the resulting X-ray tomographs, the average CT values were calculated in the Hounsfield units for liver, heart, bladder, kidney, lung and brain tissues. The results are plotted in Figure 8. As shown in the figure, within 2 minutes after the IV injection of the NPs, significant amounts of CWO NPs were detected in the heart region (also see Figure 7a (red arrows)) and the liver region (Figure 7a, blue arrows). The initial high CT contrast in the heart region was because the heart region

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Time (min) Figure 8 Mean CT values for different organs in mice (N = 3) at different times after 100 mg/kg IV injection of PEG-PLA-coated CWO NPs (173 nm hydrodynamic diameter). Error bars represent standard deviations.

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contains a relatively large amount of blood tissue. However, the CT signals from the heart region rapidly decayed and nearly disappeared within approximately 60 minutes, because the CWO NPs were increasingly captured by the liver (Figures 7b and 8). It is well known that during blood circulation, NPs typically exhibit uptake in the reticuloendothelial system (RES) (liver and spleen are two major organs of the RES).1 We would like to note that the measured CT value can vary from measurement to measurement, influenced by such parameters as water content, blood circulation rate, and uptake of contrast agents in tissues.45 In our measurements demonstrated in Figure 8, the standard deviations were in the range of ±2 to 10 HU, which is, in fact, significantly less than typical errors associated with in vivo CT imaging. For instance, a typical range of CT value variation in gold nanoparticle-contrast-enhanced CT

Brain Heart Lung Kidney Spleen Liver Blood Total

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8

Total Mass of CaWO4 (mg)

(a)

Organ Distribution (%)

images of mouse tumor xenografts is about ±12 HU at the smallest.46

Total Mass of CaWO4 (mg)

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Blood Liver Spleen Kidney Lung Heart Brain

24

Time (h)

Time (h)

Figure 9. (a) Biodistribution and pharmacokinetics of PEG-PLA-coated CWO NPs (173 nm hydrodynamic diameter) over initial 24 hours following IV injection in mice (N = 3) determined by AAS. (b) Relative distributions (bar graphs) of the CWO NPs among different organs at various times. Error bars represent standard deviations. The biodistribution and pharmacokinetics of IV-administered PEG-PLA-coated CWO NPs (173 nm hydrodynamic diameter) in mice (N = 3) were determined by analysis of tissues by atomic adsorption spectroscopy (AAS). The concentrations of CWO NPs in 19


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various organs were determined by AAS as functions of time over a 24-hour period (Figure 9a). It is notable that at the shortest time measured (20 minutes post-injection), no significant amount of NPs was detected in the heart tissue, while a significantly larger amount of NPs were found in blood, which indicates that PEG-PLA-coated CWO NPs remain dispersed in the blood, and the observed enhancement of CT contrast in the heart region was not due to the uptake of PEG-PLA-coated CWO NPs by heart cells. As shown in Figure 9b, the majority of the injected PEG-PLA-coated CWO NPs were captured by the RES (mainly the liver);47-49 this result is consistent with previous reports that bare PEG-PLA micelles (having hydrodynamic diameters in the range of 140 – 500 nm regardless of whether the distal ends of the PEG chains are capped with methoxy or carboxylic acid groups) well accumulate in the liver.47-49 The amount in the liver decreased over time. The total amount of PEG-PLAcoated CWO NPs similarly decreased with time. Another important trend was that the CWO NP concentration showed a significant increase with time in the kidneys and also in the spleen. These results suggest that PEG-PLA-coated CWO NPs initially taken up by the Kupffer cells in the RES of the liver were eventually cleared from the body through the spleen and the kidneys.1, 4 PEG-PLA-coated CWO NPs have a long circulation time. From the plot shown in Figure 9b, the circulation half-life of PEG-PLA-coated CWO NPs was estimated to be about 4 hours, which is far longer than that of the commercial CT contrast medium, Iohexol (12.3 ± 0.5 minutes). The longer circulation time is advantageous for assessing vascular disease because it enables the imaging of the vasculature during the steady-state phase.1 At 24 hours, about 91 percent of the injected CWO NPs were cleared from the body through the kidney and liver (Figure 9). The accumulation of nanoparticles in organs can cause local toxicity.4 PEG-PLA-coated CWO NPs will not have this type of problem. 20



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Figure 10. (a) 3D whole body in vivo CT images of a tumor-bearing mouse in which PEGPLA-coated CWO NPs (173 nm hydrodynamic diameter) were intratumorally administered (10 mg per cc of tumor). (b) Average CT values of the tumor relative to the average background CT signal inside the tumor (dotted line) (N = 5). (c) Biodistribution of PEGPLA-coated CWO NPs at 7 days after intratumoral injection (N = 5) determined by AAS. Error bars represent standard deviations. For potential tumorography applications, we evaluated the tumor retention characteristics of intratumorally infused PEG-PLA-coated CWO NPs (173 nm hydrodynamic diameter). About 10 mg of PEG-PLA-coated CWO NPs per cc of tumor were injected into human head and neck cancer (HN31) xenografts grown in BALB/C nude mice (male, N = 5). CT scans of the injected area (and the whole body as well) were performed at multiple times over a period of several hours immediately following the NP injection and then regularly over the next few days afterwards. The results are summarized in Figure 10. The average background CT value inside the tumor (about 300 mm3) was measured to be about 26 HU 21


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(i.e., prior to the CWO NP injection), which was comparable to the background level from soft-tissue (20 – 30 HU). The PEG-PLA-coated CWO NPs produced significant contrast for the tumor (the CT value inside the NP infused region was about 290 HU) (Figure 10b). As shown in Figure 10b, the 173-nm PEG-PLA-coated CWO NPs exhibited a tumor retention

a

d

b

c

e

f

g

Figure 11. H&E-stained histological sections of mouse (a) brain, (b) heart, (c) kidney, (d) liver, (e) lung, (f) spleen and (g) tumor taken at 7 days after intratumoral injection of 10 mg PEG-PLA-coated CWO NPs per cc of tumor in mouse HN31 xenografts (N = 5). Images are at 200× magnification. time at least greater than one week following direct intratumoral injection; the intratumoral CT value did not decrease over the 1-week period (further measurements were not possible because the tumors grew too large > 2000 mm3). TEM analysis was performed on tumor biopsies collected at day 7. Results of this TEM examination (Figure S13) confirmed the cellular uptake of the PEG-PLA-coated CWO NPs. A study is currently underway to investigate how particle size affects the intratumoral retention and distribution, and the cellular internalization of PEG-PLA-coated CWO NPs.

Conclusion

22



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CaWO4 (CWO) nanocrystal particles (NPs) have the potential for use as contrast agents in X-ray CT imaging. We developed CWO NP formulations that are suitable for in vivo applications; the formulated nanoparticles are sufficiently small, chemically and biologically inert, and stable against aggregation under physiological electrolyte conditions. By way of demonstration, mono disperse 10-nm diameter CWO NPs were encapsulated in an enclosure formed by FDA-approved poly(ethylene glycol-block-D,L-lactic acid) (PEG-PLA) block copolymer (BCP) materials using a solvent exchange method. PEG-PLA BCPs were able to reproducibly produce monodisperse, fully PEGylated CWO NPs having a mean hydrodynamic diameter of about 170 ± 10 nm that are stable against aggregation under physiological salt conditions for long periods of time (> 1 month). The X-ray attenuation properties of PEG-PLA-coated CWO NPs were measured in comparison with a commercial iodine-based radio-contrast agent (Iohexol). The data confirm that at identical molar concentrations of iodide (I) versus tungstate (W) atoms, PEG-PLA-coated CWO NPs produce a higher contrast and lower transmission than Iohexol. PEG-PLA-coated CWO NPs are nontoxic to mice. A study was performed to determine the maximum tolerated dose (MTD) of PEG-PLA-encapsulated CWO NPs in mice. The MTD of this material was estimated to be about 250 ± 50 mg per body weight following a single intravenous (IV) administration; this material is, therefore, safer than, for instance, commercially available iron oxide MRI contrast agents. The biodistribution and pharmacokinetics of PEG-PLA-coated CWO NPs were determined following IV administration in mice. The blood circulation half-life for PEGylated CWO NPs was estimated to be significantly longer (12 hours) than that of a commercial X-ray contrast agent, Iohexol (12 minutes). The IV-injected CWO NPs are nearly completely cleared from the body of a mouse, mainly through the liver and the kidney, without causing histologic toxicity in these and other excretory organs. PEG-PLA-coated 23


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CWO NPs also exhibited a tumor retention time at least greater than 7 days following direct intratumoral injection. We would like to note that tungsten oxide (WO3) nanoparticles have previously been studied as potential CT contrast agents. In terms of their X-ray mass attenuation capabilities, CaWO4 versus WO3 nanoparticles are quite comparable to each other, because the X-ray attenuation coefficient depends primarily on the atomic number and physical density of the material (rather than the crystal structure of the material).27 Unlike other metal oxides, WO3 nanoparticles have been shown to be non-toxic.50,

51

We have also confirmed that both

uncoated and PEGylated (i.e., PEG-PLA-coated) CaWO4 nanoparticles are non-toxic to cultured human cells (e.g., HN31 cells) (unpublished results). In the present paper, we report that the MTD of PEGylated CaWO4 nanoparticles in mice is significantly higher than that of a commercial MRI contrast agent. Overall, CaWO4 and WO3 nanoparticles are equally promising as CT contrast agents; see Ref.

43

for demonstration of the use of WO3

nanoparticles for CT imaging. However, the crystal structures and optical properties of CaWO4 and WO3 are very different from each other. In WO3 crystals, W atoms are located at the octahedral centers of the oxygen ions.52 In CaWO4 crystals, W atoms are located at the tetragonal centers of the oxygen ions, and Ca atoms are located at the deltahedral centers of the oxygen ions,53 which makes this material UV luminescent under X-ray/γ ray irradiation.14 As we pointed out in a previous publication,29 because of this radio-luminescence property, CaWO4 nanoparticles are also very promising for use in biomedical theranostics. In this regard, CaWO4 is a more attractive material to work with than WO3.

Experimental Procedures 24



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Synthesis of CaWO4 Nanoparticles (CWO NPs). CWO NPs were synthesized using a micro-emulsion method under high pressure and high temperature.29, 54 First, 2 ml of 1hexanol and 2 mmol of cetyltrimethylammonium bromide (CTAB, ≥99%, Sigma) were added to 20 ml of cyclohexane (99%, BDH), and then the mixture was heated to 70℃ (Solution 1). Meanwhile, 0.4 mmol of Na2WO4 (99%, Acros Organics) was dissolved in 0.6 ml of Milli-Q water (Solution 2). Next, 0.4 mmol of CaCl2 (99%, Acros Organics) was dissolved in a mixture of 0.564 ml Milli-Q water and 0.036 ml 0.1 m HCl solution (Solution 3). Solutions 2 and 3 were immediately injected to Solution 1 under vigorous stirring. After approximately one minute, the mixture was transferred into a Teflon-lined stainless steel autoclave reactor. The autoclave was heated to 160 ℃ and maintained at that temperature for 24 hours. Afterwards, the autoclave was gradually cooled to room temperature. The product was purified by centrifugation with ethanol (twice) to remove excess CTAB and with chloroform (five times) to remove 1-hexanol. Synthesis of PEG-PLA Block Copolymers (BCPs). The PEG-PLA BCP used in this study was synthesized by 1,8-diazabicyclo [5.4.0] undec-7-ene(DBU, 98%, Aldrich)-catalyzed ring-opening polymerization of lactide (LA).55,

56

First, 2.235 g of monomethoxy,

monohydroxy-terminated PEG (PEG-ME, Mn = 5,000 g/mol, Aldrich) and 1.565 g of LA were dried under vacuum, and dissolved in 22 ml of dichloromethane (DCM, ≥99.8%, Sigma-Aldrich). Then, the polymerization was initiated by adding 2 ml of a DBU solution (3.35 mmol of DBU dissolved in 30 ml of DCM) to the LA/PEG-ME mixture at room temperature. After one hour of reaction time at room temperature, the reaction was terminated by adding 50 mg of benzoic acid (≥99.5%, Sigma-Aldrich). The polymerization mixture was added drop-wise to 1000 ml of petroleum ether for purification. After the PEG-PLA product 25


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precipitated to the bottom, the supernatant was decanted. The polymer was dried under vacuum at room temperature. Encapsulation of CWO NPs with PEG-PLA BCPs. PEG-PLA-encapsulated CWO NPs were prepared using a solvent exchange method.41 Typically, 5.0 mg of CWO NPs and 0.5 g of the PEG-PLA BCP were first co-dissolved in 3.9 g of dimethylformamide (DMF, ≥99.9%, Sigma-Aldrich). The mixture was then stirred using a high-speed overhead mechanical stirrer (at 15000 rpm) under sonication. Next, 2.1 ml of Milli-Q water was injected into the DMF solution under the stirring/sonication. The emulsification process was allowed to proceed for 30 minutes at room temperature. Finally, the product was transferred to a dialysis bag (molecular weight cutoff 50 kDa) and dialyzed for three days against a total of 1.0 liter of Milli-Q water (regularly replaced with fresh Milli-Q water every 3 – 6 hours) to remove the DMF. X-ray absorption properties. Aqueous solutions containing BCP-coated CWO NPs and Iohexol at various different concentrations were prepared in 1.5 ml Eppendorf tubes, and the tubes were placed in a micro-centrifuge rack; the whole rack was placed on the stage inside a GE Light Speed clinical CT scanner. CT images of the samples were obtained using a GE Light Speed VCT/64 Slice Detector. The imaging parameters were set as follows: slice thickness = 0.625 mm; pitch = 1; voltage = 80 kV; current = 200 mA; field of view = 23 cm, gantry rotation time = 1 s. Mice. Female BALB/C mice (seven weeks old) were purchased from the Jackson Laboratory, and Male nude BALB/C mice (13 weeks old) were purchased from Harlan Laboratories (Indianapolis, IN). The mice were housed in a specific pathogen-free environment that includes a group of five in standard cages with free access to food and water, and an 26



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automatically controlled 12 hour light/dark cycle. The mice were cared for in accordance with the guidelines set forth by the American Association for Accreditation of Laboratory Animal Care (AAALAC). All mice were acclimated to the animal facility for one week prior to tests. Maximum Tolerated Dose (MTD) Studies. PEG-PLA-coated CWO NPs were dispersed in a sterile 1× phosphate buffered saline (PBS), and injected intravenously to mice in the amount of 200 µl per mouse under isoflurane anesthesia. The starting dose was 50 mg CWO NPs per kg of mouse body weight. The symptoms of toxicity that were looked at were: immediate death, abnormal responses to light, sound and motion, breathing irregularity, and > 10% weight loss over continuous days, and histologic organ damage at 14 days after injection. If any evidence of toxicity occurred over a 14-day observation period, mice were scarified at the end of that period, and blood, urine and tissue samples were collected for further analysis. In the next studies, the dose level was increased to 100, 200, and then 300 mg/kg. If major toxic response was observed at any time within the 14-day window, the mouse was euthanized, and blood, urine and tissue samples were collected for further analysis. The MTD was determined as the maximum dose that was well tolerated by all mice. Histologic Analysis of Major Excretory Organs. Major organs (including liver, kidney, spleen, heart, and lung) were harvested immediately after euthanasia/sacrifice. The organs were fixed with formalin-free zinc fixative (BD Bioscience) in PBS, and stored at 4 ℃. The tissues were mounted on paraffin blocks, and then sectioned into 5 µm thick slides. These tissue slices were placed on glass slides and stained with hematoxylin and eosin (H&E). Images were recorded using a confocal microscope. Biodistribution and Pharmacokinetics. Six treatment groups, each group comprising three 27


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mice (female, BALB/C, 11 weeks old), were prepared. Under isoflurane anesthesia, 200 µl of a sterile solution containing 10 mg/ml PEG-PLA-coated CWO NPs in PBS was injected into mice through the tail veins at the dose level of 100 mg CWO NPs per kg of mouse body; PBS only was used as control. Blood samples were taken from the heart at designated times (immediately after PBS injection, and at 0.5, 2, 4, 8 and 24 hours after PEG-PLA/CWO NP injection), and placed in heparin-coated centrifuge tubes. These blood samples were centrifuged at 1000 rpm for one minute to obtain the plasma for blood chemistry evaluations. After collecting urine and blood, the mice were immediately euthanized, and then the liver, spleen, kidneys, lungs, heart and brain were collected and stored in glass vials. All organs were dried in an oven at 90 ℃ for two days, and the dry weights were measured. For atomic absorption spectrometry (AAS) analysis, the dried organ samples were digested using the following procedures:57 (1) the organ sample was placed in a flask containing 10 ml of 18 M H2SO4 (trace metal grade, Fisher); (2) the flask was heated at 400 ℃ using a temperaturecontrolled heating mantle for one hour; (3) the sample was then cooled down to room temperature; (4) 5 ml of 10 M H2O2 (trace metal grade, Fisher) was added to the sample, and the sample was kept in a dark room until the solution became transparent; (5) the temperature was increased to 100 ℃ to let the solvent evaporate completely; (6) 1 ml of 12 M HCl (trace metal grade, Fisher) was added at room temperature, and the solution was ultra-sonicated for 10 minutes; (7) the solution was diluted to 25 ml by adding deionized (DI) Milli-Q-purified water, and filtered with a 0.2-µm PTFE filter (Fisher Scientific). Cell Culture. A human head and neck cancer cell line, HN31 (p53 mutant), was generously provided by Dr, Jeffrey N. Myers at MD Anderson Cancer Center. HN31 cells were cultured in DMEM medium (Corning Cellgro) supplemented with 10% fetal bovine serum plus 1% 28



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antibiotics/antimycotics. The cells were incubated in humidified atmosphere containing 5% CO2 at 37 ℃, and passaged twice a week typically at a split ratio of 1:3.

Mouse HN31 Xenografts. Ectopic tumor xenografts were established by subcutaneous inoculation of 3 × 106 HN31 cells (in a serum-free medium containing 50% Matrigel (BD Bioscience)) at a total injection volume of 0.1 ml into the right thighs of immunodeficient mice (male, BALB/C nude, 13 weeks old) under isoflurane anesthesia. The tumors became visible and palpable at about 7 days, and grew to appreciable sizes at 10 days after inoculation. Tumor volumes (V) were determined by measuring the largest (L) and smallest (W) diameters and the height (H) (V = (π/6)×L×W×H) using digital calipers. Intratumoral NP injection studies were performed when the tumor volume reached a value of about 300 mm3. In Vivo CT Scans. CT examinations of mice were performed using a GE Light Speed VCT/64 Slice Detector. The imaging parameters were set as follows: slice thickness = 0.625 mm; pitch = 1; voltage = 80 kV; current = 200 mA; field of view = 23 cm, gantry rotation time = 1 s. CT scans were obtained while mice were under isoflurane anesthesia.

Acknowledgement This work was supported by the “NIH New R01 Program” of the Purdue University Office of Executive Vice President for Research and Partnerships, the Indiana Clinical and Translational Sciences Institute (CTSI) Collaboration in Translational Research (CTR) Pilot Grant Program, the Purdue University Center for Cancer Research (PCCR) Shared Resource 29


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Biological Evaluation Project Program, and the US National Science Foundation (CBET1264336). The authors gratefully acknowledge the support from the Purdue University Center for Cancer Research (NIH Grant P30 CA023168). We thank Dr. Yava Jones-Hall in the Histology Research Laboratory of the College of Veterinary Medicine at Purdue University for assistance with preparation of histology sections.

Supporting Information Available: DLS intensity correlations for NPs (Figure S1); photographs of mice treated with NPs (Figure S2); mouse body weights during the 1st and 2nd MTD studies (Figures S3 and S4); H&E sections of various organs from mice IV treated with NPs (Figures S5, S6, S7, S8 and S9); abdominal CT tomographs of a mouse IV treated with NPs (Figure S10); H&E sections of various organs from mice intratumorally treated with NPs (Figure S11); abdominal CT tomographs of a mouse intratumorally treated with NPs (Figure S12); TEM images of HN31 tumor tissues intratumorally treated with NPs (Figure S13); HN31 xenograft volumes in mice (Figure S14); Summaries of the 1st and 2nd MTD study results (Tables S1 and S2).

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