Real-Time Monitoring in Vivo

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Real-Time Monitoring In Vivo Behaviors of Theranostic Nanoparticles by Contrast-Enhanced T1 Imaging Xianglong Zhu, Xiaoqin Chi, Jiahe Chen, Lirong Wang, Xiaomin Wang, Zhong Chen, and Jinhao Gao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02095 • Publication Date (Web): 07 Aug 2015 Downloaded from http://pubs.acs.org on August 12, 2015

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Analytical Chemistry

Real-Time Monitoring In Vivo Behaviors of Theranostic Nanoparticles by Contrast-Enhanced T1 Imaging # # Xianglong Zhu,†, Xiaoqin Chi,‡, Jiahe Chen,§ Lirong Wang,† Xiaomin Wang,‡

Zhong Chen,§ and Jinhao Gao*,†

†

State Key Laboratory of Physical Chemistry of Solid Surfaces, The MOE Key Laboratory of

Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China ‡

Fujian Provincial Key Laboratory of Chronic Liver Disease and Hepatocellular Carcinoma,

Zhongshan Hospital, Xiamen University, Xiamen 361004, China §

Department of Electronic Science and Fujian Key Laboratory of Plasma and Magnetic Resonance,

Xiamen University, Xiamen 361005, China #

These authors contributed equally to this work

*Email: [email protected]

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ABSTRACT The innovative applications of engineered nanoparticles (NPs) in medicine, such as diagnosis and therapy, have attracted considerable attention. It is highly important to predict the interactions between engineered NPs and the complex biological system as well as the impacts on the subsequent behaviors in living subjects. Herein, we report the use of T1 contrast-enhanced magnetic resonance imaging (MRI) to monitor the in vivo behaviors of NPs in a real-time manner. We chose ultra-small Pd nanosheets (SPNSs) as the object of NPs because of their promise in theranostics and fitness for diverse surface chemistry. SPNSs were modified with different surface coating ligands (e.g., polyethyleneglycol, zwitterionic ligands, polyethylenimine) and functionalized with Gd-chelates to render T1 contrast-enhanced capability. MRI real-time monitoring recorded the location and accumulation of SPNSs in small animals, and revealed the prominent roles of surface coating ligands in pharmacokinetics. These results highlighted the significance of selecting proper surface coating for particular biomedical assignment. Moreover, we demonstrated a powerful and noninvasive means to predict and detect the behaviors of NPs in living subjects, which may be helpful for rational design and screening of engineered NPs in biomedical applications.

Keywords: MRI; zwitterionic coating; PEG; real-time monitoring; in vivo behaviors

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INTRODUCTION Over the past two decades, nanoparticles (NPs) have attracted tremendous interest for their potential wide-ranging applications in biology and medicine:1,2 (i) their unique intrinsic properties which are hardly substituted by bulk materials or small molecules;3,4 (ii) their surface can be decorated as a platform in nanoscale with varied species to integrate multi-functionalities, such as targeting, bio-responding, diagnosis and/or therapy.5,6 However, once injected into bodies, NPs always exhibit ambiguous in vivo behaviors which are dramatically different from those in vitro and difficult to predict or determine. For example, when NPs are exposed to biofluids, such as plasma and serum, proteins and other biomolecules adsorb onto the surface and form “protein corona”, which masks the engineered surface modification and intensely changes the in vivo behaviors of these materials.7-9 It is reported that nanomaterials readily adsorb plasma proteins, which compromise the targeting efficacy of antibody-functionalization,10 even make them interact strongly with tissue-resident macrophages in the mononuclear phagocytic system (MPS), leading to rapid blood clearance and accumulation in liver and spleen.11,12 Approaches have been exploited to prevent the formation of protein corona to achieve biocompatibility of NPs with non-fouling properties.13-15 Poly(ethyleneglycol) modification (PEGylation) is one of the most common and successful methods for reducing nonspecific protein adsorption.16-18 The strong hydration of PEG via hydrogen bonding prolongs the blood circulation of NPs so that they can target tumors through the well-known enhanced permeability and retention (EPR) effect.19-21 Recently, the use of zwitterionic coating as an alternative strategy for suppressing the non-specific adsorption of biomolecules to NPs has attracted much interest.22,23 Zwitterionic ligands are a family of molecules that possess moieties with both cationic and anionic groups, but

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still macroscopically charge neutral. The high dipole moments of zwitterionic coating induce strong hydration, which is similar to PEG that provides resistant surface to non-specific binding, and remains the hydrodynamic diameters (HDs) significantly small due to short molecule length.24 However, there is still lack of essential understanding about the roles of these surface coating ligands in vivo. Particularly, it is urgent to comprehensively investigate the roles of ligands and especially, “see” the in vivo behaviors of NPs with different surface coating in a real-time manner. Nowadays, various well-established imaging modalities, such as photoacoustic25,26, Raman27, near-infrared fluorescence (NIRF)28,29, positron emission tomography (PET)30,31 and single-photon emission computed tomography (SPECT)32,33, are utilized to monitor the in vivo behaviors of NPs and reveal their interactions with biological system. However, due to the relatively high optical extinction and scattering by tissues, most of the illuminative imaging modalities are limited to detect objects in a few millimeters depth under skin with reasonable resolution and signal-to-noise ratio. PET and SPECT are prevailing techniques that can provide real-time screening of the in vivo behaviors of NPs because of superb tissue penetration of signal as well as highly sensitive and quantitative resolution.34 Nevertheless, PET and SPECT hold ionizing radiation risk and inconvenience of extempore preparation, which restrict its applicable range. Furthermore, nuclides with short radioactive half-lives are not amenable to long-time monitoring. Magnetic resonance imaging (MRI) is one of the most powerful and noninvasive imaging techniques with superior resolution in anatomical details.35 Various magnetic NPs are employed as MRI contrast agents to improve the sensitivity and reliability because they are able to alter the relaxation time of nearby water protons, without being affected by tissue depth or shelf-time.36,37 Hence, MRI possesses great suitability for real-time monitoring the in vivo behaviors of NPs and drugs in biomedical

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applications.38 Ultra-small Pd nanosheets (SPNSs) were chosen as the object due to their well-defined hexagonal morphology, size, thickness, and tunable surface plasmon resonance (SPR) in the near-infrared region. SPNSs exhibit high near-infrared absorption and photo-thermal conversion effects, which is favorable for theranostics combining photoacoustic imaging (PAI) and photo-thermal therapy (PTT).39 Moreover, the diverse surface modification of Pd nanoparticles can be easily achieved due to the well-explored metal surface chemistry.40 First, SPNSs were modified with three representative coating ligands which are popular in biomaterials: electropositive polyethylenimine (PEI), electroneutral zwitterionic ligands, and uncharged PEG (Figure 1). Then gadolinium chelates (DTPA-Gd) were functionalized to render SPNSs with T1 contrast-enhanced capability for MRI. Dynamic monitoring by MRI recorded the location and accumulation of SPNSs in a real-time manner. The results demonstrated distinct behaviors of SPNSs with different modifications and revealed the significant roles of surface coating ligands in pharmacokinetics. Furthermore, Gd-functionalized SPNSs could act as both PTT agents for cancer ablation and MRI contrast agents for tumor diagnosis.

EXPERIMENTAL METHODS Chemicals. All reagents were purchased from Acros, Sigma-Aldrich, Alfa-Aeser and J&K, and used without further purification. HeLa and S180 cell lines were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Synthesis of Pd@PVP. Briefly, 10 mg of Pd(acac)2, 30 mg of PVP and 30.6 mg of NaBr were dissolved in a mixed solvent of DMP (2 mL) and water (4 mL). The resulting yellow solution was

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left undisturbed at room temperature overnight and then transferred to a glass pressure vessel. After being charged with CO to 1 bar, the vessel was heated at 100 °C and kept at this temperature for 100 min with stirring before being cooled to room temperature. Surface Modification with Ligands. The as-prepared Pd@PVP solution (1mL) was diluted in the mixture solution of ethanol (1 mL) and acetone (8 mL), and then collected by centrifugation to remove excessive surfactants and ions. For PEG and PEI coating, the precipitate was suspended in containing 3 mg HS-PEG1k-NH2 or 10 mg branched PEI (MW = 60000) and shaken for 2 h, followed by ultra-filtration. For zwitterion coating, prior to ligand exchange of SPNSs, 5 mg LA-ZW and 0.85 mg LA-PEG3-NH2 (molar ratio 5:1) were reduced by 20 mg NaBH4 into DHLA ligands. Then repeat the procedure of ultra-filtration. Functionalize DTPA-Gd onto SPNSs. Functionalization of DTPA-Gd was performed by adding p-SCN-DTPA into SPNSs solutions after surface modification. We stirred for 2 h followed by ultra-filtration to remove the excess p-SCN-DTPA. Then Gd3+ was chelated by adding GdCl3·6H2O and stirring for 1 h. Ultra-filtration was performed to remove the excess Gd3+. Size Exclusion Chromatography. We performed the SEC analysis on a Superose-6 10/300 GL column (GE Healthcare Life Sciences) by high-performance liquid chromatography (HPLC) (Ultimate 3000, Dionex) monitored at the absorbance of 365 nm, using PBS buffer (1×, pH 7.4) as the mobile phase. Flow rate was 0.5 mL/min. MRI Phantom Study. The samples for MRI phantom study were prepared separately. Pd@PEG-Gd, Pd@ZW-Gd and Pd@PEI-Gd were prepared with concentrations of 0.32, 0.16, 0.08, 0.04, 0.02, 0.01 µM of Gd3+ ions in 1× PBS buffer. The control sample denoted as 0 µM. The longitudinal relaxation times were measured (at 298 K) and used for calculating the relaxation rate of

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the samples. T1-weighted MR images of all the samples were acquired under the following parameters: TR/TE = 300/12 ms, 256×256 matrices, repetition times = 4. In Vivo MRI Study. Before in vivo experiments, the Pd@PEG-Gd, Pd@ZW-Gd and Pd@PEI-Gd samples were filtered through sterilized membrane filters (pore size 0.22 µm). The mice were then intravenously injected with SPNSs solution (2.0 mg Pd kg-1 body weitht). Time-scale acquisition of postinjection images at 1, 8, 24, 32 h (for Pd@PEG-Gd) or 1, 2, 4, 8 h (for Pd@ZW-Gd and Pd@PEI-Gd) were obtained with the same slices. All the images were acquired using fSEMS sequence under the following parameters: TR/TE = 300/10 ms, 256 × 256 matrices, slices = 5, thickness = 2 mm, averages = 2, FOV = 80 × 80. Photo-thermal Therapy. To assess the photo-thermal effect on cells, HeLa cells were seeded in the 3.5 cm culture dish at a density of 1×105 cells, incubated with 12.5 mg Pd/mL Pd@PEG-Gd and partly covered with 808 nm laser spot. After co-stained with both acridine orange (AO) and propidium iodide (PI) and rinsed with PBS three times, live and dead cells were imaged using Axio Observer as a boundary between green and red could be obtained. S180 tumor-bearing mice were separated into four groups (n = 4). The Pd@PEG-Gd + Laser group mice were intravenously injected with 2.0 mg kg-1 body weight via tail vein and irradiated with the 808 nm laser at 1 W/cm2 for 5 min. The other three groups of mice were used as controls. The laser spot was adjusted to cover the entire region of the tumor. Then the sizes of tumors were measured by a caliper every other day and the tumor volume was calculated according to equation: Tumor volume = (tumor length)×(tumor width)2/2. Relative tumor volumes were calculated as V/V0 (V was the tumor volume calculated after treatment, while V0 was the initiated tumor volume before treatment). The photographs were also taken to record the changes of tumors.

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RESULTS AND DISCUSSION Construction of SPNSs with Different Surface Coatings. In a typical synthesis of SPNSs according to previous reports,40 palladium(II) acetylacetonate, poly(vinylpyrrolidone) (PVP) and sodium

bromide

were

dissolved

in

a

mixed

solvent

consisting

of

H2O

and

N,N

-dimethylpropionamide (DMP). After being charged with CO to 1 bar into the glass vessel, the mixture was heated to 100 °C and kept for 2 h with stirring. The resulting blue colloidal products were collected by centrifugation, and washed several times with ethanol and acetone. The average plane diameter of the SPNSs was 4.4 nm with thickness of 1.8 nm (Figure S1), which was below the glomerular filtration-size threshold (~10 nm) and might be eliminated from the body through renal clearance.41 To investigate the effects of coating ligands on the in vivo behaviors of SPNSs, we modified the as-prepared SPNSs with three different coating ligands, HS-PEG-NH2, dihydrolipoic acid-zwitterion (DHLA-ZW), and PEI to render SPNSs surface with different electrical charges: uncharged (Pd@PEG), electroneutral (Pd@ZW), and electropositive (Pd@PEI), respectively. For zwitterionic coating, DHLA-ZW was mixed with DHLA-PEG3-NH2 in a 5:1 ratio, to provide amino groups for further functionalization (details of ligands synthesis and characterization, see Figure S2). Then DTPA-Gd was conjugated onto SPNSs through the coupling between amino groups and isothiocyanates. Ultra-filtration was performed after each step to remove the excess coating ligands, DTPA, and Gd3+. The ratios of Pd/Gd in these Gd-functionalized SPNSs were controlled in a range of 16-17 under inductively coupled plasma atomic emission spectroscopy (ICP-AES) analysis. These SPNSs conjugates exhibited different surface electro-charges with -4.1, -9.8, and +36.0 mV for

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Pd@PEG-Gd, Pd@ZW-Gd, and Pd@PEI-Gd, respectively (Figure S3). Stability of SPNSs with Different Coatings in Serum. Although nanomaterials hold promise in biomedical applications as multifunctional diagnostic and therapeutic agents, the interaction of nanomaterials with biological systems always bring uncertainty to their in vivo behaviors.42,43 In particular, when nanomaterials are covered by protein corona, their HDs and interfacial properties would be changed, which might initiate physiological reactions, such as enzymatic cascades and immune response.44-46 to determine the protein corona formation of SPNSs in biological media, size exclusion chromatography (SEC) was employed because this system allowed online and specific analysis of multicomponent samples with high reliability and repeatability. The SEC profiles ranged the HDs of NPs with the retention times inversely. Under the same conditions, Pd@PEG-Gd, Pd@ZW-Gd and Pd@PEI-Gd exhibited respective retention peaks (Figure 2a), which approximated to the corresponding SPNSs before Gd-functionalization (Figure S4), indicating negligible influence in surface properties after Gd-functionalization. We then tested a group of standards in PBS as markers to calibrate the HDs: blue dextran (M1, 19.0 min, 29.5 nm), thyroglobulin (M2, 31.7 min, 18.8 nm), alcohol dehydrogenase (M3, 41.6 min, 10.1 nm), ovalbumin (M4, 45.0 min, 6.1 nm), and vitamin B12 (M5, 55.6 min, 1.5 nm). According to the curve of markers (Figure S5), the HDs of Pd@PEG-Gd, Pd@ZW-Gd and Pd@PEI-Gd were calibrated at 20.6, 10.9 and 23.8 nm, respectively. The dynamic light scattering (DLS) results (Figure S6) showed comparable HDs of about 19.3, 11.7 and 23.6 nm for Pd@PEG-Gd, Pd@ZW-Gd and Pd@PEI-Gd, respectively, which were consistent with the SEC analysis. We then studied the SEC profiles of SPNSs after incubation with 20% fetal bovine serum (FBS) for 4 h (Figure 2b). For Pd@PEG-Gd and Pd@ZW-Gd, the retention peaks were still kept at their

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original positions, with the addition of FBS peaks (42.4 min) in the spectra. However, no peaks were observed for Pd@PEI-Gd at its original position, instead, strong absorption immerged at shorter retention time, indicating protein corona formation on PEI surface, which enlarged its HDs. These results suggested the resistant property of PEG and ZW coating from protein adsorption. The non-fouling surface characteristics of PEG coating derived from their ability to bind large amounts of water by hydrogen-bonding to ether groups which acted as a water barrier preventing proteins from approaching the surface.47 Zwitterionic coating obtained its protein-resistant properties through strong ionic structuring, creating a highly hydrophilic surface. In addition, the net charge of zwitterion was internally balanced and no ions were likely to release from the surface as the formation of ion pairs with electrostatic forces.22 Relaxivity Measurements. We performed the relaxivity test and phantom imaging on a 7 T MRI scanner. Pd@PEG-Gd, Pd@ZW-Gd and Pd@PEI-Gd showed r1 values of 15.90 ± 0.24, 11.53 ± 0.22 and 14.64 ± 0.31 mM-1s-1 (Gd) in phosphate buffered saline (PBS) solution, respectively (Figure 3a). This almost 4-fold increase of the r1 values over commercial contrast agent Magnevist (DTPA-Gd, r1 = 3.30 ± 0.25 mM-1s-1) was attributed to the prolonged correlation time (τR) of multiple Gd-chelates, as their tumbling were confined after anchoring onto the surface of nanostructures.48

It is worth noting that Pd@ZW-Gd exhibited the relatively small r1 value among

three Gd-functionlized SPNSs, which might be due to the relatively short τR as its small HD. T1-weighted phantom images showed that Gd-functionalized SPNSs have much stronger positive contrast effect than Magnevist (Figure 3b), indicating that Gd-functionalized SPNSs can serve as high-performance T1-enhanced MRI contrast agents. In Vivo MRI Study. MRI provided noninvasive and real-time monitoring, which facilitated us

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to “see” the in vivo behaviors of SPNSs with different coating ligands. Before MRI study, cytotoxicity test was performed on HeLa and RAW 264.3 cell lines at various Pd concentrations (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 125, and 250 µg/mL) for 24 h (Figure S7). The results proved no cytotoxicity of Pd@PEG-Gd and Pd@ZW-Gd. However, SPNSs with PEI coating exhibited evident toxicity at high concentrations, which might be attributed to membrane damage caused by the strong electro-positivity.49,50 The even worse toxicity on RAW 264.3 cells might be caused by their efficient endocytosis of positive-charged (i.e., Pd@PEI-Gd) nanoparticles. Tumor vessels are known to have high vascular leakiness and lack functional lymphatics due to the uncontrolled growth rate and defects in angiogenesis, resulting in NPs accumulation in tumor based on the EPR effect. To better understanding the in vivo behaviors of engineered NPs, we used tumor-bearing small animals for in vivo study. SPNPs solutions of different surface coatings were intravenously injected at a dose of 2.0 mg Pd per kg of body weight. We acquired T1-weighted MR images before and after the injection (1, 8, 24, 32 h for Pd@PEG-Gd, 1, 2, 4, 8 h for Pd@ZW-Gd and Pd@PEI-Gd) on a 7 T MRI scanner. To monitor the behaviors of SPNSs with different surface modifications, especially their tumor targeting capability and clearance routes, we focused on tumor, liver, kidney and bladder as the targeting regions by taking both coronal and transverse images. In vivo MRI monitoring of Pd@PEG-Gd (Figure 4a) illustrated obvious signal enhancement in tumor area in late periods (blue arrows). To quantify the contrast, we calculated the signal-to-noise ratio (SNR) by finely analyzing region of interests (ROIs) of the MR images and calculated the values of SNRpost/SNRpre to represent the signal changes. The SNR values were calculated according to SNRROIs = SIROIs/SDnoise (SI stands for signal intensity and SD stands for standard deviation). The signal changes (Figure 4b) in tumor area were 1.11 ± 0.06, 1.44 ± 0.32, 2.57 ± 0.29 and 2.83

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± 0.31 at 1, 8, 24, and 32 h p.i., respectively, indicating the gradual tumor targeting through EPR effect. Liver uptake of PEG coated NPs was reported previously,51 but no apparent signal enhancement in liver was found in our experiments. Besides the suppressed MPS uptake by the corona-resistance of PEG, it might be ascribed that Gd-chelates were stripped by the digestion of macrophages in liver, causing molecular tumbling recovery and then lower T1-enhancement.48 Though PEGylation increased the effective HDs of SPNSs, we still observed small amount of Pd@PEG-Gd filtrated into urine probably due to the unique morphology of SPNSs. Accumulation in tumor substantiated Pd@PEG-Gd as promising diagnostic agent for in vivo tumor targeted imaging. In contrast, we observed no tumor targeting for Pd@ZW-Gd and Pd@PEI-Gd. As shown in Figure 5, intense signal enhancement in bladder at 1 and 2 h (red dashed circle, SNRpost/SNRpre = 5.66 ± 1.52 and 6.18 ± 0.47), as well as the pelvises of kidney pointed to rapid clearance of Pd@ZW-Gd via renal filtration. Zwitterionic coating rendered SPNSs with small HDs and resistance from serum protein adsorption, making them passing through the glomerular filtration-size threshold easily.52 Efficient urinary excretion of zwitterionic modified nanomaterials permits their applications for angiography, which has been widely explored.53 Further, we supposed that zwitterionic coating would also endow passive tumor targeting capability to various NPs with bigger sizes above the glomerular filtration-size threshold.21,41 Pd@PEI-Gd illustrated accumulation in liver with signal increase (Figure 6a, red dashed circle) in this area (SNRpost/SNRpre = 1.83 ± 0.23 and 1.84 ± 0.20 at 4 and 8 h, respectively). High liver uptake (Figure 6b) indicated that Pd@PEI-Gd was easily recognized by MPS in living subjects, as a result of protein corona formation on PEI surface.54 Signal vanished in heart area at 4 h for both Pd@ZW-Gd and Pd@PEI-Gd validated their fast clearance from blood circulatory system, and no signal increase in tumor area over time suggested

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their incapability for tumor targeting. Pharmacokinetics, such as blood circulation (Figure S8) and biodistribution (Figure S9), were provided to describe the fate of SPNSs after administration. Pd@PEG-Gd demonstrated relatively long half-life in blood (15.5 h) and high accumulation in tumor (14.9 ID%/g at 24 h). In contrast, Pd@ZW-Gd and Pd@PEI-Gd were rapidly cleared from blood with short half-lives (52 min and 117 min) through renal filtration (16.6 ID%/g in kidney at 8 h) and MPS uptake (19.5 ID%/g in liver at 8 h), respectively. The pharmacokinetic study was highly consistent with the MRI monitoring, further substantiated the reliability of Gd-functionalization for MRI monitoring in vivo behaviors. Notably, liver and spleen exhibited obvious distinction between MRI and biodistribution. As the biggest digestive and immune organs, liver and spleen preserve multiple digestive enzymes, macrophages and lymphocytes in large quantity, which can accumulate, digest, and process the nanoparticles. So we ascribed the weak signal of liver and spleen in MRI to the discharged Gd-chelates, which were stripped from SPNSs, causing molecular tumbling recovery and then deceasing T1 contrast ability.55 Moreover, MRI monitoring with noninvasive and real-time features overcame the obstacles of pharmacokinetic detection, including ex vivo experiments and delayed information. Photo-thermal Therapy Using Pd@PEG-Gd. Since Pd@PEG-Gd represented high accumulation in tumor, their therapeutic capability as photo-thermal therapeutic agents was evaluated both in vitro and in vivo. Benefited from the two dimensional sheet structures, Gd-chelates functionalized SPNSs displayed well-defined and strong SPR absorption with a peak above 700 nm, which extended into the NIR region (Figure S10). The photo-thermal effect of Pd@PEG-Gd was investigated by recording the temperature changes at an 808 nm NIR laser irradiation (Figure 7a). After 10 min irradiation (1 W/cm2), temperature elevated as high as ~25 °C even at low

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concentration of 12.5 µg Pd/mL, indicating excellent photo-thermal conversion efficiency of SPNSs. Therapy effect at cellular level was assessed by co-stain with acridine orange (AO) and propidium iodide (PI). AO enters the live cells and binds to DNA, emitting green fluorescence, while PI only penetrates through the dead cells membrane, emitting red fluorescence. The cells partly exposed by 808 nm laser showed a boundary between green and red fluorescence in accordance with the laser spot (Figure 7b), further confirming the PTT effect of [email protected] To further investigate the in vivo therapeutic effect, we randomly divided BALB/c mice with xenografted S180 tumors into four groups (n = 4) as follows: untreated (Blank), irradiated for 5 min (Laser only), intravenous injected with Pd@PEG-Gd solutions (2.0 mg Pd per kg of body weight) but without irradiation (Pd@PEG-Gd only), intravenous injected with Pd@PEG-Gd followed by irradiation (Pd@PEG-Gd + Laser). During NIR irradiation, temperature changes in the tumor area were acquired by infrared thermal mapping apparatus. Thermographic images (Figure 7c) showed that the temperature at tumor site rose rapidly for Pd@PEG-Gd + Laser group within 5 min. In comparison, the maximum temperature of the Laser only group was just about 40 °C under the same irradiation condition. To verify the PTT efficacy of Pd@PEG-Gd, photos were taken to record tumor growth (Figure S11). The tumors after single treatment of Pd@PEG-Gd injection or NIR irradiation both continuously grew and finally caused mice death no more than 30 days (Figure 7d,e), showing no significant difference compared to Blank group. However, the Pd@PEG-Gd + Laser group showed seriously empyrosis in tumor areas and resulted in a substantial suppression of tumor growth. The black burning scab detached in 15 days (Figure 7f), and the mice completely recovered without recurrence over 60 days. These results proved that Pd@PEG-Gd would cause tumor injury through the conversion of NIR irradiation into thermal energy, as efficient PTT agents with tumor targeting

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capability.

CONCLUSION In summary, we successfully investigated and monitored the in vivo behaviors of SPNSs with three representative coating ligands (PEG, ZW and PEI) by MRI in a real-time manner. Coating ligands rendered individual surface properties to SPNSs, making considerable influence to their in vivo behaviors. Pd@PEG-Gd showed relative long half-life in blood, and achieved passive targeting capability in tumor. Moreover, the excellent photo-thermal effect of SPNSs caused tumor ablation suggested the promise of Pd@PEG-Gd for cancer diagnosis and therapy. Pd@ZW-Gd exhibited fast clearance via renal filtration while Pd@PEI-Gd accumulated in liver due to MPS uptake. The distinct in vivo behaviors from different coating ligands helped us to make further understanding and control over the interactions between NPs surface and complex biological systems. Moreover, the strategy of contrast-enhanced T1 imaging can act as a powerful and noninvasive means for real-time monitoring in vivo behaviors of NPs, which is believed to be helpful and instructive for rational design and screening of NPs in biomedical applications and clinical translations.

ASSOCIATED CONTENT Supporting Information Detailed experimental sections and Figures S1-S11. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author

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Fax: (+) 86-592-2189959, Tel: (+) 86-592-2180278. E-mail: [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the National Key Basic Research Program of China (2013CB933901, 2014CB744502 and 2014CB932004), National Natural Science Foundation of China (21222106, 81370042, 81430041, 81472231 and 81201805), Natural Science Foundation of Fujian (2013J06005 and 2013D014), Fok Ying Tung Education Foundation (142012), and National Key Sci-Tech Special Project of China (2012ZX10002-011-005). We thank Prof. N. Zheng and Dr. S. Tang for providing Pd nanosheets.

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Figure 1. Schematic representation of SPNSs modification with three representative coating ligands: PEG, zwitterionic ligands, and PEI. Each ligand brings unique surface properties to SPNSs, and may results in distinct in vivo behaviors. After conjugation of DTPA-Gd chelates on SPNSs, the in vivo behaviors were monitored by T1-weighted MRI in real-time.

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Figure 2. Size-exclusion chromatography (SEC) profiles and size analysis of (i) Pd@PEG-Gd, (ii) Pd@ZW-Gd and (iii) Pd@PEI-Gd in (a) PBS buffer and (b) 20% FBS solution after 4 h incubation. Note that the HDs of NPs were inversely related to the retention times. Arrows in (b) indicate the retention peaks of FBS. Protein markers M1 (blue dextran, 29.5 nm HD), M2 (thyroglobulin, 18.8 nm HD), M3 (alcohol dehydrogenase, 10.1 nm HD), M4 (ovalbumin, 6.1 nm HD), and M5 (vitamin B12, 1.5 nm HD) are shown by arrows.

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Figure 3. (a) T1 relaxivity and (b) phantom images of Pd@PEG-Gd, Pd@ZW-Gd, Pd@PEI-Gd, and Magnevist at different Gd concentrations on a 7 T MRI scanner. Gd-functionalized SPNSs exhibited almost 4-fold higher r1 values and more significant contrast enhancement than Magnevist.

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Figure 4. (a) T1-weighted MR imaging for monitoring the in vivo behaviors of Pd@PEG-Gd at different times after intravenous injection into tumor-bearing mice. Blue arrows indicate the signal enhancement at tumor sites due to the passive tumor targeting by EPR effect. (b) MR SNR analysis of liver, bladder, and tumor areas after administration (n = 3).

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Figure 5. (a) T1-weighted MR imaging for monitoring the in vivo behaviors of Pd@ZW-Gd at different times after intravenous injection into tumor-bearing mice. Red dashed circles indicate the signal enhancement at bladder because of rapid renal clearance. (b) SNR analysis of liver, bladder, and tumor areas after administration (n = 3).

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Figure 6. (a) T1-weighted MR imaging for monitoring the in vivo behaviors of Pd@PEI-Gd at different times after intravenous injection into tumor-bearing mice. Red dashed circles indicate the signal enhancement at liver because of the uptake by MPS. (b) SNR analysis of liver, bladder, and tumor areas after administration (n = 3).

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Figure 7. (a) The temperature elevation curves of Pd@PEG-Gd solutions with different concentration (0, 12.5, 25, 50 µg Pd/mL) as a function of irradiation time by 808 nm laser (power density = 1 W/cm2). (b) The merged fluorescence image of HeLa cells after treatment with 12.5 µg/mL Pd@PEG-Gd and laser irradiation. The boundary between green (AO) and red (PI) fluorescence responds to the laser irradiation spot (red circle in the left). (c) IR thermographs at the time points of 0, 1, 2, 3, 4, 5 min present the photo-thermal conversion efficacy of the accumulated Pd@PEG-Gd in tumor under NIR irradiation. Experiments were performed at 24 h after intravenous injection. (d) Relative tumor volume changes and (e) the survival rate curves of different groups after treatments (n = 4). (f) Representative photographs of tumor-bearing mice taken at Day 0 and Day 15 after Pd@PEG-Gd + Laser treatment. 25



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TOC

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Real-Time Monitoring in Vivo

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