Biobehavior in Normal and Tumor-Bearing Mice of Tobacco Mosaic

Oct 4, 2013 - Viruses, Artificial Viruses and Virus-Based Structures for Biomedical Applications. Patrick van Rijn , Romana Schirhagl. Advanced Health...
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Biobehavior in Normal and Tumor-Bearing Mice of Tobacco Mosaic Virus Man Wu,†,‡ Jiyun Shi,†,§,∥ Di Fan,§ Quan Zhou,‡ Fan Wang,*,§ Zhongwei Niu,*,‡ and Yong Huang‡ ‡

National Engineering Research Center of Engineering Plastics, Technical Institute of Physics and Chemistry, CAS, Beijing 100190, China § Medical Isotopes Research Center, Department of Radiation Medicine, School of Basic Medical Sciences, and ∥Medical and Healthy Analytical Center, Peking University, Beijing 100191, China S Supporting Information *

ABSTRACT: Viral nanoparticles (VNPs) have shown great potential as platforms for biomedical applications. Before using VNPs for further biomedical applications, it is important to clarify their biological behavior in vivo, which is rare for rodlike VNPs. In this paper, a study of tobacco mosaic virus (TMV), a typical rod-like VNP, is performed on blood clearance kinetics, biodistributions in both normal and tumorbearing mice, histopathology and cytotoxicity. TMV was radiolabeled with 125I using Iodogen method for in vivo quantitative analysis and imaging purpose. In the normal mice, the accumulation of TMV in the immune system led to a rapid blood clearance. The uptake of TMVs in the liver was less than that in the spleen, which is opposite to the results observed in the case of spherical VNPs. No signs of overt toxicity were observed in examined tissues according to the results of histological analysis. In addition, similar biodistribution patterns were observed in U87MG tumor-bearing mice.

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INTRODUCTION Over the past decade, viral nanoparticles (VNPs) issued from plant viruses and bacteriophages have shown great potential as platforms for biomedical applications.1−4 VNPs consist of repeating protein units, which self-assemble to form a hollow capsid surrounding the viral genome. VNPs offer great advantages over synthetic nanoparticles,2,4,5 for example, (i) good biocompatibility and biodegradability, (ii) well-defined structure and monodispersity in shape and size, and (iii) diverse modifications using chemical and biological methods. Moreover, they are considered noninfectious and nonhazardous in human and mammalian because of their specific host selectivity. VNPs being developed for biomedical applications mainly include cowpea mosaic virus (CPMV), cowpea chlorotic mottle virus (CCMV), and bacteriophages MS2 and M13.1,5 Although VNP-based nanomaterials have exhibited fantastic performance as nanocarriers in drug delivery,6,7 targeting,8−10 bioimaging,10−12 and biosensing,4 much attention is still focused on the design of new nanoplatform to promote the pharmacokinetic properties. Recent findings illustrate that the shape of nanoparticles plays an important role in both clearance and in vivo distribution.13−16 Polymer-based long “filomicelles”,14 for example, represented a longer blood circulation than spherical particles of the same polymer composition, because they can align with blood flows, and evade the uptake by phagocyte cells. Moreover, the long filomicelles delivered more drugs to tumor © 2013 American Chemical Society

than other organs. Likewise, shape effect on in vivo behavior has been investigated with mesoporous silica nanoparticles (MSNs) modified with poly(ethylene glycol) (PEG).16 The rod-shaped MSNs with higher aspect ratio circulated for longer periods in the blood than those with lower aspect ratio. In addition, the effect of particle shape led to an apparent difference in biodistribution. Tobacco mosaic virus (TMV) is a typical rod-like VNP with a length of 300 nm, diameter of 18 nm, and a hollow core with 4 nm in diameter (Scheme 1a). Its capsid is consisted of 2130 identical protein subunits, which arrange helically around a single stranded RNA. TMV is stable in a wide range of pH values (3.5−9) and temperatures (up to 90 °C).1 Further, TMV can be chemically modified on both the exterior and interior of the capsid using bioconjugation protocol.17,18 It endows TMV with more possibilities of multifunctional properties by covalent attachment of sensor,4,19 targeting,20,21 drug22 molecules, and PEGs.23 Furthermore, TMV coat protein is capable to self-reassemble in disk-shaped or rod-shaped particles.17,24−26 The aspect ratio of assemblies can be controlled by pH, ionic strength, and protein concentration. This character offers more flexibility to design various particles with different aspect ratio and, further, to optimize the Received: July 31, 2013 Revised: September 23, 2013 Published: October 4, 2013 4032

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mM pH 7.4 phosphate buffer (0.1 mL) were added to a vial precoated with 50 μg Iodogen. After reacting at room temperature for 10 min, the reaction mixture was purified by a MidiTrap G-25 column using phosphate buffer as eluent. The radioactive fractions containing 125ITMV were collected and passed through a 0.2 μm syringe filter for further in vivo experiments. The quality control was carried out with instant thin layer chromatography (ITLC) method using Gelman Sciences silica gel paper strips and saline as the developing solution. Visualization of TMV in Organs. A total of 2 mg (equal to 100 mg/kg) native TMV in 0.1 mL of saline was administered intravenously to BALB/c normal mouse. Blood, liver, and spleen were harvested at 24 h postinjection (p.i.). The blood was directly used without further treatment. The liver and spleen were milled and centrifuged at 9500 rpm at 4 °C for 15 min, and then the supernatants were collected for analysis. All specimens were diluted and stained with uranyl acetate before characterization. The visualization of TMV was performed with a transmission electron microscopy (TEM, JEM2100F JEOL, Japan). Dosage Preparation for Animal Studies. 125I-TMV was purified using MidiTrap G-25 column before animal studies. MidiTrap G-25 column was washed with 6 mL phosphate buffer, and was saturated with 4 mL of 1% BSA before purification. After that, the MidiTrap G25 column was loaded with radiotracer (∼100 μL), and was then washed with 3 mL phosphate buffer, the 0.5 mL fraction between 0.75 and 1.25 mL of eluent was collected. Doses for animal study were prepared by dissolving the purified TMV radiotracer in saline to reach a concentration of 100 μCi/mL (∼10 μCi/μg for TMV) for biodistribution and pharmacokinetic study and 5.0 mCi/mL (∼10 μCi/μg for TMV) for imaging. Each mouse was injected with 0.1 mL of radiotracer solution. Blood Clearance Experiment. Five BALB/c normal mice were regarded as one group for the blood clearance experiment of the radiotracer. Ten μCi of 125I-TMV (∼1 μg TMV in 0.1 mL saline) was administered intravenously to each mouse. Blood was harvested from orbital sinus at 1, 5, 7, 10, 15, 20, 30, 60, 90, 120, 240, and 360 min, weighed and measured for radioactivity in a γ-counter (Wallac 1470− 002, Perkin-Elmer, Finland). The radioactivity in blood was calculated as a percentage of the injected dose per gram (%ID/g). In Vivo Biodistribution in Mice. Biodistribution study was performed according to the literatures.31,32 All animal experiments were performed in accordance with guidelines of Peking University Health Science Center Animal Care and Use Committee. A total of 20 BALB/c normal mice were randomly divided into five groups, each of them contained four individuals. A total of 10 μCi of 125I-TMV (∼1 μg TMV in 0.1 mL saline) was administered intravenously to each mouse. Animals were anesthetized with intraperitoneal injection of sodium pentobarbital at a dose of 45.0 mg/kg. Time-dependent biodistribution studies were carried out by sacrificing mice at 1, 4, 24, 48, and 72 h p.i. Blood, heart, liver, spleen, kidney, stomach, intestine, muscle, and bone were harvested, weighed, and measured for radioactivity in a γcounter. The organ uptake was calculated as a percentage of injected doses per gram of wet tissue mass (%ID/g). Additionally, the biodistribution study of 125I-TMV in U87MG tumor-bearing mice was carried out with the same protocol as the above. Tumor and organ tissues were harvested at 4, 24, and 72 h p.i. Scintigraphic Imaging. An imaging study was performed using two BALB/c normal mice. Each animal was administered with 500 μCi of 125I-TMV (∼50 μg TMV) in 0.1 mL of saline. Animals were anesthetized with intraperitoneal injection of sodium pentobarbital at a dose of 45.0 mg/kg, then they were placed supine on a two-headcamera (GE Healthcare, Millennium VG SPECT) equipped with a parallel-hole, low energy, and high-resolution collimator. Anterior images were acquired at 4 and 24 h p.i. and stored digitally in a 128 × 128 matrix. The acquisition count limits were set at 200 k. The γimaging of 125I-TMV in four U87MG human glioma tumor-bearing mice was carried out with the same protocol as the above. Anterior images were acquired at 4, 24, 48, and 72 h p.i. Histological Examination and Flow Cytometry. Six BALB/c normal mice were randomly divided into two groups and intravenously injected with 0.2 mg (equal to 10 mg/kg) of native TMV in 0.1 mL

Scheme 1. Schematic Representation of (a) Native TMV Particle, (b) TMV Coat Protein with Highlight of Phenolic Hydroxyl Group of Tyrosine (Y139) and (c) 125I-Labeling Procedure

performance of nanocarriers. For all the reasons above, TMV would be a promising candidate for biomedical applications. Prior to using VNPs for further biomedical application, the understanding of biological behavior is a fundamental aspect. Up to now, spherical (including CPMV3,27 and CCMV28) and surface-modified filamentous (such as potato virus X29 and M1330) VNPs have been studied in this field, the rod-like VNPs remain poorly understood. Here, a systematical study of TMV is presented on blood clearance kinetics, biodistributions following intravenous administration in both normal and tumor-bearing mice.

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EXPERIMENTAL SECTION

Materials. All reagents were used as received. Poly(ethylene glycol) (PEG 8k) and Iodogen were purchased from Sigma. Na125I was obtained from Beijing Atom High Tech (Beijing, China). Phosphate, dichloromethane, and n-butanol were from Beijing Chemical Works (Beijing, China). β-Mercaptoethanol was from Shenyang Chemical Reagent Factory (Shenyang, China). APC-conjugated rat antimouse CD4, APC-Cy7-conjugated rat antimouse CD8, and PE-conjugated rat antimouse CD19 were from BD Bioscience. Cell counting kit-8 (CCK8) was from Beyotime (Haimen, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Solarbio (Shanghai, China). PD MidiTrap G-25 column was from GE Healthcare. Milli-Q water was used for all experiments. Purification and Characterization of TMV. The general procedure of TMV purification was as follows: infected leaves were pulverized and homogenized in 10 mM pH 7.8 phosphate buffer containing 0.1%v/v β-mercaptoethanol. The resulting mixture was filtered through two layers of cheesecloth. Obtained filtrate was centrifuged and filtered again through four layers of Kleenex. Then, dichloromethane/n-butanol mixture (volume ratio of 1:1) was added into the filtrate under stirring in ice bath. After 30 min, a suspension was obtained and centrifuged. The aqueous phase was carefully collected. After that, sodium chloride (0.2 M) and PEG 8k (8%w/v) were added into this aqueous solution, which resulted in the formation of a white precipitate under stirring in ice bath for 1 h. The product was centrifuged for 30 min and the supernatant was discarded. The pellet was dispersed in 10 mM pH 7.8 phosphate buffer and centrifuged again. The final supernatant was centrifuged at 42000 rpm at 4 °C for 3 h. Obtained pellet was redispersed in water and dialyzed to remove the residual PEG. The concentration of TMV was determined by absorbance at 260 nm using a UV−vis spectrophotometer (UV-3900, Hitachi, Japan), assuming an extinction coefficient of 3.0 for a 1 mg/mL solution of TMV at this wavelength. Preparation and In Vitro Stability of Radiotracer. For 125I radiolabeling of TMV, 100 μg of TMV and 37 MBq of Na125I in 100 4033

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saline. The mice were sacrificed at 24 and 72 h p.i. Blood was harvested, treated with red blood cell lysis buffer and incubated with APC-conjugated rat antimouse CD4, APC-Cy7-conjugated rat antimouse CD8 and PE-conjugated rat antimouse CD19. CD4+, CD8+, and B cells in peripheral blood were detected by fluorescenceactivated cell sorting (FACS) using BD LSRFortessa cell analyzer. In addition, liver, spleen, and kidney were cut out and fixed in 4% buffered paraform, subsequently got trimmed. These tissues were embedded in paraffin and then sectioned into 5 μm sections and mounted on glass slides. After staining with hematoxylin and eosin (H and E), the sections were observed using optical microscopy. Three BALB/c normal mice injected with 0.1 mL pH 7.4 phosphate buffer were used as reference, and all analysis were prepared with the same method as above. In Vitro Cytotoxicity. L929 and HeLa cells were maintained in DMEM, supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL). Cells (2 × 103) were seeded in 96-well plates and incubated overnight to allow the cells to attach to the surface of the wells. The cells were then exposed to TMV solutions with various concentrations and incubated at 37 °C in a humidified atmosphere of 5% CO2 for 24 h. After that, cells were washed with phosphate buffer and stained with CCK8. The absorbance was measured at 450 nm using an iMark microplate reader (Bio-Rad, U.S.A.). The data represented the means of triplicate measurements.

native TMV in 0.1 mL saline was administered intravenously to a BALB/c normal mouse. The blood, liver and spleen were harvested and analyzed at 24 h p.i. TMV particles were observed in all tested organ tissues (Figure S3). Moreover, most of the observed particles remained long rod shape (Figure S3). The blood clearance kinetics of TMV was determined over the first 360 min p.i. with BALB/c normal mice. Five BALB/c normal mice were used as one group, and 10 μCi of 125I-TMV (∼1 μg TMV) was injected intravenously to each mouse. Figure 1 shows the percentage of 125I-TMV of initial value in

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RESULTS AND DISCUSSION To evaluate the biobehavior, TMV particles were radiolabeled with 125I using Iodogen method, which is widely used in protein labeling of radioimmunoassay (RIA) and nuclear medicine imaging.33 Compared with other commonly used isotopes, 125Iradiolabeling has many advantages including simple and rapid operation, mild reaction condition, and it possesses almost no effect on the protein properties. For the 125I-labeling procedure, 125 I need to be attached to the phenolic hydroxyl group. A significant advantage of TMV is 2130 phenolic hydroxyl groups of tyrosine (Y139; Scheme 1b) exposing on the outer surface of capsid, which can facilitate the 125I-labeling procedure (Scheme 1c). The labeling reaction was implemented by adding the TMV and Na125I solution to a vial precoated with Iodogen. This reaction lasted 10 min at room temperature. After labeling, the product was purified using MidiTrap G-25 column to eliminate the free Na125I. The quantity of 125I attaching onto TMV capsid was evaluated by measuring the radiochemical purity (RCP) with instant thin layer chromatography (ITLC). For each preparation, an average number ranged from 150 to 190 125I per TMV particle was obtained. The stability of 125I on the surface of TMV is a crucial parameter for our study. Therefore, the stability tests were executed in saline and 10% FBS solutions at different time points. Obtained RCP was still above 95% after 96 h incubation in both saline and 10% FBS solutions (Figure S1). It is clear that radio-iodinated TMV remained stable for at least 96 h in saline and 10% FBS conditions. Additionally, to verify the integrity of TMV after labeling reaction, a parallel test was performed with no-radioactive NaI using the same protocol. Both TEM and fast protein liquid chromatography (FPLC) results (Figure S2) confirm that TMV particles remained intact after labeling. It is well-known that VNP structure is held by various weak interactions, for example, electrostatic interactions, hydrogen bonds, and hydrophobic interactions. After intravenously administration, VNPs will confront a complex environment including the enzymes, various proteins, macrophages, and so on. Therefore, the in vivo stability of TMV was initially investigated by visualization using TEM. A total of 2 mg of

Figure 1. Blood clearance kinetics of 125I-TMV in BALB/c normal mice. About 10 μCi of 125I-TMV (∼1 μg TMV) was injected intravenously to each mouse. Blood was harvested at 1, 5, 7, 10, 15, 20, 30, 60, 90, 120, 240, and 360 min, weighed, and measured for radioactivity in a γ-counter. The radioactivity in blood was calculated as a percentage of the injected dose per gram (%ID/g). The initial value was determined by curve-fitting and set as 100%. The inset shows the selected zone from 0 to 50 min p.i.

the plasma over time. According to the results of multiple experiments, a quick clearance from bloodstream resulted in a half-time of 3 min of 125I-TMV in blood. The blood clearance half-time of TMV was determined as the radioactivity accumulation in blood decreased to the half of the initial value. This half-time is close to that of CPMV, which was ranged from 4 to 7 min determined by labeling with lanthanide metals.27 About 97% of TMV particles were washed out from blood at 40 min p.i. and this was consistent to the blood clearance time of CPMV (>95% within 30 min).27 In general, VNPs are eliminated from the bloodstream relatively rapidly by the reticulo-endothelial system (RES).27 Surface modification with masking agent such as PEG could effectively avoid the recognition of immune system to prolong the circulation time in vivo.34,35 PVX, a filamentous plant virus (515 × 13 nm), possesses a similar shape with TMV and had a longer blood circulation time (half-time of 12.5 min in tumor mice) after surface-PEGylation compared to TMV.29 In addition, a surface modification with single-walled carbon nanotubes (SWNTs) provided to bacteriophage M13, also a filamentous structure (880 × 6.5 nm), a long blood circulation of approximately 60 min in mice.36 On the other hand, the RES eliminates the particles whose size is greater than 200 nm by filtration. Therefore, it could be conjectured that the size of TMV (300 nm length) is probably another cause of the rapid elimination from blood. In vivo biodistribution of 125I-TMV was determined within various mouse organ tissues including blood, heart, liver, spleen, lung, kidney, intestine, stomach, bone, and muscle. A 4034

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Figure 2. 125I-TMV biodistribution in (a) BALB/c normal mice (n = 4) and (b) U87MG tumor-bearing mice (n = 4). About 10 μCi 125I-TMV (∼1 μg TMV) was intravenously injected to each mouse. Selected tissues were harvested at different time points, weighted and measured for the radioactivity in a γ-counter.

Figure 3. Selective γ-imaging of 125I-TMV in (a) BALB/c normal mice and (b) U87MG tumor-bearing mice. A total of 500 μCi of 125I-TMV (∼50 μg TMV) in 0.1 mL of saline was administered intravenously to each mouse. Anterior images were acquired at different time points and stored digitally in a 128 × 128 matrix. The acquisition count limits were set at 200 k.

total 20 BALB/c normal mice were randomly divided into five groups, each of which had four animals. A total of 10 μCi of 125 I-TMV (∼1 μg TMV) was intravenously injected to each mouse. Tissue samples were taken at 1, 4, 24, 48, and 72 h p.i. Figure 2a shows the 125I-TMV uptake in different organs as a function of time p.i (detailed in Table S1). It is evident that 125 I-TMV dispersed quickly and broadly to all tested tissues within 1 h p.i. Matching with previous data, a rapid clearance from blood circulation resulted in a small amount of 125I-TMV remaining in blood at the first hour p.i. Moreover, more than 90% of 125I-TMV particles were accumulated in the liver and spleen at all the tested time points. The uptake of 125I-TMV particles in these organs increased within 4 h, and then the particles were progressively cleared from liver and spleen 24 h later. A small amount of 125I-TMV was found in other tested organs. Figure 3a presents the typical scintigraphic images of normal white mice at 4 and 24 h after administration of 500 μCi 125I-TMV (∼50 μg TMV per mouse). It confirms that 125ITMV particles mainly located in the liver and spleen tissues. This is coherent to the biodistribution result observed with M13-SWNT. The fluorescent-labeled M13-SWNT was mostly observed in the immune system of mice including liver and spleen.36 In addition, the biodistribution results (Table S1) indicate that the uptake of 125I-TMV in the liver was less than that in the spleen at all the tested time points, which is different to the result observed in the case of PEGylated PVX. The PEGylated PVX accumulated in the liver of mice with a higher amount (55%) than in the spleen (23%).29 We suppose that this different observation of accumulations between 125I-TMV and PEGylated PVX particles is very likely due to the different surface properties, such as PEGylated surface, surface charge (negatively charge for TMV and positively charge for PVX).

Furthermore, the intense accumulation of 125I-TMV in the spleen is opposite to the observations with the spherical VNPs, such as CPMV27 and CCMV,28 which were mostly located in the liver of mice. Overall, the difference of in vivo behavior between TMV and spherical VNPs27,28 demonstrates the shape effect on the biodistribution in term of VNPs. The extensive accumulations of 125I-TMV in the immune system of mice make the understanding of the TMV-associated toxicity more important. A histological examination of the tissues including liver, spleen, and kidney was conducted with phosphate buffer and native TMV inoculated animals (0.2 mg of TMV per mouse, equal to 10 mg/kg). Microscopic findings (Figure 4) were similar between the control and TMVinoculated animals, with no signs of overt toxicity, for example, tissue degeneration and cell necrosis, in examined tissues at 72 h p.i. However, an increase of B-cell number was observed by FACS following intravenous administration of native TMV. BALB/c normal mice were injected with 0.2 mg of TMV per mouse (equal to 10 mg/kg). The peripheral blood was harvested, treated, and analyzed at 24 and 72 h p.i. respectively. Figure 5 shows that the number of B cell progressively increased within 72 h p.i. The proliferation of B cell suggests the TMVs-induced immune response. Meanwhile, CD4+ and CD8+ cells had no significant change in the number. The same phenomenon has been observed for the CPMV-incubated mice within one day following intravenously administration.27 In addition, although the liver of TMV-incubated mice appeared to be normal with no evidence of pathology, a liver function test may be necessary to get more details on the TMVassociated toxicity. 4035

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accumulation in liver and spleen is consistent with the biodistribution data and similar of that observed for BALB/c normal mice. Figure 3b shows that the U87MG tumor was barely seen at early time points. However, it is clearly visualized at later time points. Mostly due to massive radioactivity was washed out from liver after 24 h p.i., which lead to an increase of tumor-to-liver ratio over time in the γ-imaging (Figure 3b). In addition, the massive radioactivity washed out from the liver and spleen may circulate to tumor and contribute to tumor accumulation. The distribution data shown in Table S2 confirm that the uptake of 125I-TMV in the tumor increased to around 0.38 %ID/g at 24 h p.i, which is greatly different with PEGylated PVX. In the case of PEGylated PVX, 15% of administered dose accumulated in the tumor tissue at 24 h p.i.29 Moreover, to further improve the uptake in tumor, it is necessary to attach targeting molecule, for example the folic acid4 onto the surface of TMV protein coat in order to improve the recognition of tumor cell. In vitro cytotoxicity of TMV was valuated with HeLa and L929 cells using CCK8 assay. To exclude the possible influence of TMV on cell viability, various concentrations of TMV were incubated ranged from 5 to 1000 μg/mL for 24 h before the cell viability was assessed. No obvious adverse effect of TMV was found on both HeLa and L929 cells viability, even the concentration reaching up to 1 mg/mL. Cell viabilities at 24 h maintained above 85% (Figure 6).

Figure 4. Histological examination of liver, spleen, and kidney tissues at 3 days p.i. (at 200x magnification). Native TMV (0.2 mg in 0.1 mL phosphate buffer, equal to 10 mg/kg) and phosphate buffer (0.1 mL) were injected intravenously in BALB/c normal mice, respectively. The sections were stained with hematoxylin and eosin.

Figure 5. Cell analyses following native TMV administration determined by flow cytometry. BALB/c normal mice (n = 3) were intravenously injected with 0.2 mg (equal to 10 mg/kg) of TMV in 0.1 mL saline. The mice were sacrificed at 24 and 72 h p.i. respectively. The peripheral blood was harvested, treated with red blood cell lysis buffer, and incubated with APC-conjugated rat antimouse CD4, APCCy7-conjugated rat antimouse CD8, and PE-conjugated rat antimouse CD19.

Figure 6. Viability of HeLa and L929 cells against different concentrations of TMV solution for 24 h incubation at 37 °C in a humidified atmosphere of 5% CO2.

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CONCLUSIONS In conclusion, TMV shows a rapid blood clearance, an extensive accumulation in immune system in either BALB/c mice or U87MG tumor-bearing mice, and no overt toxicity for in vivo and in vitro uses, while a further study on the immune responses for TMV is probably necessary. The understanding of how the TMV behaves in vivo provides valuable information on their potential uses and limitations, and contributes to the further research of rod-like nanomaterials. Moreover, TMV has advantages such as easy production in large quantity, possible modification via genetic or chemical techniques, and particularly flexible design to obtain a desired size, particularly aspect ratio. Therefore, there will be a favorable prospect for TMV in biomedical applications. Further studies are in progress to understand the influences of size and surface properties on in vivo biobehavior of TMV, in order to minimize the uptake in liver and spleen and to ameliorate the blood circulation time of TMV.

In vivo biodistribution of 125I-TMV within different organs of U87MG tumor-bearing mice was also evaluated in the present work. Mice were also injected intravenously with 10 μCi of 125ITMV (∼1 μg TMV) in saline solution. Tissue samples were taken at 4, 24, and 72 h p.i. The uptake of 125I-TMV particles was determined in the interested tissues such as heart, liver, spleen, lung, kidney, and particularly, the U87MG tumor. The biodistribution results (Figure 2b) show that the highest uptake of 125I-TMV was in liver and spleen (Table S2), which is consistent to the results obtained in BALB/c normal mice. However, the total uptake was less relative to that of BALB/c normal mice. It is probably due to the immune deficiency of athymic nude mice, which we used for establishing tumor model. The scintigraphic images of U87MG tumor-bearing mice were presented in Figure 3b at 4, 24, 48, and 72 h after administration of 500 μCi 125I-TMV (∼50 μg TMV). Extensive 4036

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ASSOCIATED CONTENT

S Supporting Information *

The in vitro and in vivo stability results of labeled-TMV and the data of the biodistribution results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; [email protected]. Author Contributions †

Both authors contributed equally to the work (M.W. and J.S.).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the 973 Program (2013CB933800), National Natural Science Foundation of China (Grant Nos. 21304103, 21074143, 91027030, and 30930030), and Hundred Talents Program of the Chinese Academy of Sciences and the Outstanding Youth Fund (81125011). We are grateful to Dr. Jie Li (Chinese PLA General Hospital, Pathology Department) for histological analysis and Dr. Na Li (Beijing Institute of Heart Lung and Blood Vessel Disease, Molecular and Cellular Biology Research Lab) for flow cytometry analysis.

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dx.doi.org/10.1021/bm401129j | Biomacromolecules 2013, 14, 4032−4037