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Blood Circulation, Biodistribution and Pharmacokinetics of Dextran Modified Black Phosphorus Nanoparticles Zhen Li, Caixia Sun, yifan Xu, Lijuan Deng, Hao Zhang, Qiao Sun, and Chongjun Zhao ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00150 • Publication Date (Web): 14 Aug 2018 Downloaded from http://pubs.acs.org on August 19, 2018

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ACS Applied Bio Materials

Blood Circulation, Biodistribution and Pharmacokinetics of Dextran Modified Black Phosphorus Nanoparticles Caixia Sun, † Yifan Xu, †, ‡ Lijuan Deng, † Hao Zhang, † Qiao Sun, † Chongjun Zhao, ‡* and Zhen Li † * †

Center for Molecular Imaging and Nuclear Medicine, State Key Laboratory of Radiation

Medicine and Protection, School for Radiological and Interdisciplinary Sciences (RAD-X), Soochow University, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Suzhou 215123, China. ‡

Key Laboratory for Ultrafine Materials of Ministry of Education, Shanghai Key Laboratory of

Advanced Polymeric Materials, School of Material Science and Engineering, East China University of Science and Technology, Shanghai, 200237, China. * E-mail: [email protected] (Prof. C. Zhao), [email protected] (Prof. Z. Li)

ACS Paragon Plus Environment

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ABSTRACT

Black phosphorus (BP) nanostructures have been received enormous attention in diverse bioapplications owing to their unique physicochemical properties. However, it is still challenging to investigate their blood circulation, bio-distribution and pharmacokinetics after administration due to the difficulty in quantifying them. Here, we report the quantification of blood circulation, bio-distribution and pharmacokinetics of dextran modified BP nanoparticles (i.e., BP-DEX NPs) in balb/c mice through the highly sensitive noninvasive photoacoustic (PA) imaging and singleemission computed tomography/computed tomography (SPECT/CT) imaging. We first prepare small water-soluble and biocompatible BP-DEX NPs, and label them with radioisotopes

99m

Tc.

We then quantify their half-lives of distribution- and elimination phases to be 0.2 h and 9.5 h by counting the γ-emissions in the blood of mice administrated with BP-DEX NPs. We also show the dynamic variation of BP-DEX NPs in tumor, liver, spleen and kidney through in-vivo PA imaging, and illustrates the accumulation of nanoparticles in major organs by SPECT/CT imaging, which follows an order of spleen > liver ˃ lung ˃ kidney. Our work demonstrates the renal clearance and hepatobiliary excretion of BP-DEX NPs, which could be potentially translated for clinical in-vivo imaging and therapy of cancer. KEYWORDS Black phosphorus, Pharmacokinetics, Blood circulation, Photoacoustic imaging, SPECT/CT imaging


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Introduction In the last few years, great attention has been paid to black phosphorus (BP) nanostructures owing to their intrinsic properties1-2 (e.g., layer-dependent band gap) and various applications in electronic devices,3-7 rechargeable batteries,8-10 nano-catalysts,11-12 as well as chemical and biological sensors. Recently, BP nanostructures have been also extensively exploited for imaging and therapy of cancer because of their biodegradability and biocompatibility. There are a number of reports on these applications.13-22 For bio-labeling and bio-imaging, 10 nm fluorescent BP nanodots were adopted to label the Hela cells.13 PEGylated ultra-small BP nanoparticles (NPs) have been used for photoacoustic (PA) imaging because of their excellence in photothermal conversion.16-17 Moreover, BP nanostructures were also successfully used for photodynamic therapy (PDT)18, drug delivery19-20 and photothermal therapy (PTT)21. For example, BP nanosheets can efficiently generate singlet oxygen for PDT against cancer under near-infrared (NIR) irradiation.18 The liquid-exfoliated ultra-small BP quantum dots also displayed a high photothermal conversion efficiency.21 They were embedded in poly(lactic-co-glycolic acid) (PLGA) to form larger uniform nanospheres, which show a better antitumor efficacy, a slower degradation in the physiological environment, and better biocompatibility, in comparison with uncoated BP nanodots.22 The above works illustrate the promise of BP nanomaterials in imaging and therapy of cancer. However, the fate, metabolism and biological effects of nanoscale BP in animals have not been fully investigated and understood, which are extremely important for their future clinical translation. One

fundamental

issue

is

their

blood

circulation

profile,

biodistribution,

and

pharmacokinetics, which are very difficult to determine by direct quantification of phosphorus via the common methods such as inductively coupled plasma optical emission spectrometry


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(ICP-OES), because of abundant phosphorus in the body.22 An alternatively noninvasive and real-time imaging approach was reported. For example, the bio-distribution of core-shell BP@PLGA nanoparticles in mice was monitored and quantified by the fluorescence of NIR dye Cy5.5 encapsulated in the core-shell nanoparticles.22 The bio-distribution of PEGylated BP NPs and their tumor accumulation were also assayed by the dynamic change of fluorescence intensity through a whole-animal NIR imaging approach.20 These examples demonstrate the feasibility of molecular imaging techniques in quantifying in-vivo distribution of BP NPs. Compared with conventional methods, molecular imaging approaches not only provide the dynamic biodistribution of BP NPs, but also save lots of experimental animals. It is known that fluorescence is very sensitive to the surroundings23 and could be partially quenched by BP NPs themselves, development of complementary approaches to assess their bio-distribution is highly significant. In this article, the blood circulation time, bio-distribution and pharmacokinetics of dextran modified BP NPs (referred as BP-DEX NPs) in mice bearing tumors are revealed by using PA imaging and SPECT/CT imaging (Scheme 1). PA imaging could overcome the drawbacks of low spatial resolution of ultrasound imaging and limited penetration of optical imaging.24-26 It possess high sensitivity (~10-9 mol/L) and capability of deep penetration (up to 5 - 6 cm), and provides inherently background free detection.27-29 SPECT/CT imaging integrates the high resolution of X-ray computed tomography (CT) and high sensitivity of SPECT (10-10 - 10-12 mol/L), which could provide quantitative three dimensional (3D) information about biological processes (e.g., blood circulation, biodistribution, degradation) of BP nanoparticles.30-32

Experimental section


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Materials: Red phosphorus (high purity chemicals, >99%) and dextran (Mw = 20,000 Da) were purchased from Aladdin. Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were received from Sigma-Aldrich. Ultra-filter tubes were bought from Millipore. Preparation of BP-DEX NPs: BP-DEX NPs were obtained by a ball milling method as described elsewhere.16-17 In a typical synthesis, red phosphorus was milled in a vial by stainless steel balls to form BP. Then, dextran (DEX) was loaded into the vial and ground for another 12 h. The resultant black product was dissolved in Milli-Q water and then centrifuged to remove large particles. The supernatant was concentrated by ultrafiltration to separate free DEX. The purified black solution was stored for use. Characterization of BP NPs: A Shimadzu XRD-6000 X-ray diffractometer was used to determine the crystal structure of BP NPs. The used X-rays were from Cu Kα1 radiation with a wavelength of 0.15406 nm. The scanning range and rate were set to be 20-80˚ for 2θ and 0.05˚s1

, respectively. A PerkinElmer Lambda 750 UV-vis-NIR spectrophotometer was utilized to

measure the absorbance of BP NP solutions. A FEI Tecnai G20 transmission electron microscope (TEM) working at 200 kV was used to characterize the particle size and morphology. A small droplet of BP-DEX colloidal suspension (10 µL, 10 µg/mL) was deposited onto a carbon film coated copper grid, which was dried in a vacuum oven after water evaporation. A Veeco atomic force microscope (AFM) was used to measure the size and thickness of BP NPs through its tapping mode at a scanning rate of 1 Hz under ambient conditions. A photoelectron spectroscopy (XPS) instrument equipped with Mg Kα X-ray radiation was used to determine the binding energies of elements in BP NPs, which were calibrated with C1s at 284.6 eV. A Zeta sizer Nano-ZS90 (Malvern, UK) was adopted for the


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measurement of hydrodynamic size and zeta potential of BP NP solution. A Gamma counter (Science and Technology Institute of China in Jia Branch Innovation Co., Ltd.) was utilized to record the intensity of γ-emissions. Cytotoxicity assay of BP-DEX NPs: The viability of 4T1 cells cultured with BP-DEX NPs was evaluated with a MTT assay. First, 5 × 104 4T1 cells were planted into each well of a culture plate, which contained 100 µL RPMI-1640 culture media that has 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were cultured at 37 ˚C for 12 h under a humidified atmosphere with 5% CO2, and then incubated with different concentrations of BP-DEX NPs in the fresh culture media for 12 h, followed by addition of 20 µL MTT solution (5 mg/mL). The cells were cultured for another 4 h, and then 100 µL DMSO were added for dissolving the purple formazan formed in living cells. Finally, the absorbance value of each well at 570 nm was recorded with a PerkinElmer EnSpire® Multimode Plate Reader. Apoptosis of cells: 1 × 106 4T1 cells were planted into each well of 6-well plates and incubated with different concentrations of BP-DEX NPs for 12 h. The concentrations of BP-DEX NPs were 0, 6.25, 50, and 100 µg/mL, respectively. After that, the cells were stained with two dyes (i.e., Annexin-V and 7-AAD), and then measured with a flow cytometer (BD FACSVerse). Tumor model of mice: BALB/c female mice [Specific pathogen free (SPF) grade, 18-20 g] were bought from Changzhou Cavensla Experimental Animal Technology Co. Ltd. The tumors were implanted by subcutaneous injection of 2 × 106 4T1 cells in 50 µL of phosphate buffered saline solution (PBS, pH = 7.2 ~ 7.4) into the right back of each mouse. After the tumor grew to 60 mm3, the mice were selected for the in-vivo imaging experiments. The use of animals followed the standard procedures authorized by the Soochow University Laboratory Animal


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Center. Photoacoustic (PA) imaging: All the PA imaging were tested with a multispectral optoacoustic tomography scanner (MSOT, iThera Medical). For in-vitro imaging, BP-DEX NP solutions with different concentrations (e.g., 0, 12.5, 25, 50, 100 and 200 µg/mL) were excited with different wavelengths (e.g., 680, 750, 800, 850 and 900 nm) to obtain their PA images. Regarding the invivo imaging, a tumor mouse was intravenously injected with BP-DEX nanoparticle solution (200 µL, 1.0 mg/mL of the P corresponding to 10 mg/kg) after it was anesthetized with 1.5 % isoflurane. PA images were collected before injection and post injection of 0.5, 1, 2, 4, 6, 8, and 24 h. Radioactive labeling of BP-DEX NPs: Radioactive technetium-99m (99mTc) was used to label BP-DEX NPs through the chelating effect of the phosphate groups on the surface of nanoparticles. Briefly, 10 µL SnCl2 solution in 0.1 M HCl (1.0 mg/mL) was added into Na99mTcO4 solution with the radioactivity of 500 µCi. After reaction for 5 min, BP-DEX NPs (200 µL, 1 mg/mL) were introduced and further reacted for half an hour under ambient conditions. The reaction mixture was purified by using 100 kDa MWCO centrifugal filter to get rid of free 99mTc, and to yield a radioactivity of 80 ~ 90% and a labeling yield of 83.8%. The labeled nanoparticles were denoted as 99mTc-BP-DEX NPs. Radiolabeling stability test: To detect the dissociation of 99mTc from the labeled nanoparticles, the purified

99m

Tc-BP-DEX NPs were divided into three parts, and then diluted to 2 mL with

Milli-Q water, PBS, and 10% FBS, respectively. The solutions were stirred for 1, 2, 4, 6, and 24 h, then 200 µL of each solution was filtered by a centrifugal filter (MWCO = 10 kDa). The intensity of γ-emissions in the filtrate was quantified by a Gamma counter to characterize the


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release of radioactive 99mTc. The radiolabeling stability (η%) was calculated using the Eq 1. η% = ቀ1 −

େ౜

େ౩

ቁ ∗ 100%

(1)

where Cf is the intensity of γ-rays of filtrate, Cs is the intensity of γ-rays of 200 µL original solution. In-vivo circulation and distribution of

99m

Tc-BP-DEX NPs: To investigate the blood

circulation profile and biodistribution of 99mTc-BP-DEX NPs, we injected them into Balb/c mice via tail vein. Blood samples were collected from mouse eyes and their time-dependent γemissions were determined by the Gamma counter. The mice were killed to harvest the major organs for radioactivity measurement (Eq 2) after they were administrated with

99m

Tc-BP-DEX

NPs for 24 h. ID%/g = ቀ

େఽ౮ େా

ቁ /݉஺௫ ∗ 100%

(2)

CAx means the intensity of γ-rays from each organ, CB means the intensity of γ-rays of injected labeled nanoparticles, and mAx means the weight of organ. Similar to the radiolabeling stability, there is no influence of the natural decrease of radioactivity of the 99mTc. SPECT/CT imaging: The tumor-bearing mice were used for SPECT/CT imaging, which was did by using an animal SPECT/CT scanner (MI Labs, the Netherlands) and the following parameters. The scan time was 12 min; frame was 40; field of view (FOV) was 26 × 26 × 70 mm3; and resolution was 0.4 mm. The administrated dose was 200 µL

99m

Tc-BP-DEX NPs (1

mg/mL) per mouse, which corresponded to 400 µCi per mouse. The obtained SPECT/CT images were rebuilt by a software package provided by MI Labs and then fused with PMOD software.


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The organs or tissue of interest were selected and quantified by using the tools in the PMOD software. The contents were expressed by the percentage injected dose per gram (% ID/g), based on the assumption that the density of tissue was 1 g/cm3. The radioactivity of major organs was imaged for evaluating the accumulation or clearance of BP-DEX NPs.

Results and discussion BP-DEX NPs were prepared from commercial red phosphorus in the presence of dextran by a high energy mechanical milling method.16-17 The optical image in Figure S-1 in the supporting information clearly shows the colour change of red phosphorus after ball milling. The crystal structures of BP and purified BP-DEX nanopowder were measured by the powder X-ray diffraction, and compared in Figure S-2, both which exhibits the characteristics of orthorhombic BP (JCPDS No.76-1957). They were then analyzed with X-ray photoelectron spectroscopy (XPS). Figure S-3 displays the sample XPS spectrum after calibration with C 1s at 284.6 eV. The whole survey spectrum of BP-DEX NPs only presents C, N, O, P elements and no other elements, which indicated their high purity. In addition, three typical Raman peaks of BP and BP-DEX were clearly observed (Figure S-4), which are ascribed to the out-of-plan vibration mode (Ag1) at 360.62 cm-1, and the in-plan vibration mode B2g and Ag2, which were located at 436.2 and 464.4 cm-1, respectively. It has been known that BP nanoparticles can be covalently modified with molecules containing hydroxyl groups during ball milling.16-17 Dextran was selected due to its abundant hydroxyl groups, and the Fourier transform infrared (FTIR) spectrum of product in Figure S-5 illustrates the presence of P-O-C bonds. The characteristic stretching vibration of P=O at ~1200


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cm-1 and ~1620 cm-1,33-35 and that of P-O-C at 990 cm-1 33, 36 demonstrate that dextran molecules are covalently grafted on the surface of BP NPs. The FTIR results also demonstrate the surface oxidation of BP nanoparticles, which is further supported by the binding energy of P 2p at 133.2 eV from the oxidized phosphorus (P5+) in their XPS spectrum (Figure S-6). The spectrum of P 2p clearly shows the characteristic binding energies of P0 and P5+, where the former one is located at 129.3 eV and 130.2 eV, and the later one is located at 133.2 eV.16 Since BP NPs are unstable in the atmosphere of oxygen and moisture,18, 37 their degradation in water were investigated. Figure S-7 shows the XPS spectra of BP-DEX NPs degraded for different days. The fraction of BP decreases gradually with degradation, which clearly indicates the degradation property of BPDEX NPs. Figure1a and 1b show the size and height of the BP-DEX NPs determined by TEM and AFM, respectively. It should be noted that the highly bright spots in the AFM image are the aggregates of BP nanoparticles. The size and size distribution of BP-DEX nanoparticles (8.5 ± 1.5 nm) were measured from TEM images by using imaging analysis software. Around 100 nanoparticles were counted (Figure S-8). The thickness of BP-DEX nanoparticles (4.0 ± 1.0 nm) is measured from the AFM image in Figure 1b by nanoscope analysis (Figure S-9). The covalent conjugation of dextran on the surface of BP NPs makes these nanoparticles water soluble. The solution zeta potential is -36.6 mV, and its hydrodynamic size remains around 21 nm within 24 h (Figure S-10). Negligible increase in the hydrodynamic size demonstrates that BP NPs are quite stable within short time owing to the surface abundant hydroxyl groups and negative charges. The negative zeta potential indicates the presence of phosphate groups on the particle surface.9,18 Due to their negative surface charge and the adsorption of proteins, mixing BP-DEX NPs with the serum media leads to an increase of hydrodynamic size to 38 nm (Figure


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S-11). Similar to other layered nanomaterials such as bismuth selenide and bismuth sulfide,[38-39] BP NPs have broad NIR absorbance (Figure S-12), which endows them excellent photothermal conversion performance for PA imaging and PTT. Figure 1c shows the performance of BP-DEX NPs in PA imaging obtained with different wavelengths. The PA signal decreases with the laser wavelength from 680 to 900 nm, due to the gradual decrease in absorbance of BP NPs. Therefore, 680 nm excitation was selected for the subsequent PA imaging experiments. The dependence of PA performance of BP-DEX NPs on different concentrations of nanoparticles (0, 25, 50, 100, 200, and 400 µg/mL) shows a linear increase of PA signal with particle concentration (Figure 1d). BP-DEX NPs were labeled with radioactive isotopes, such as

99m

Tc nuclides, which have

strong γ-rays and short half-life (6.02 h) for SPECT imaging.40-43 Free

99m

Tc nuclides were

thoroughly removed by washing and filtration to obtain a radiolabeling yield of 83.8%. Figure S13 displays the similar hydrodynamic size of labeled and unlabeled BP NPs, indicating their good stability during labeling. The zeta potential of labeled NPs was -27 mV, which is slightly higher than that of unlabeled nanoparticles (-36.6 mV) (Figure 1e). The slight variation of zeta potential further supports the coordination of 99mTc cations with phosphate anions on the surface of BP-DEX NPs. To further demonstrate the stability of labeled nanoparticles, the dissociation of 99m

Tc from 99mTc-BP-DEX NPs was investigated by counting the γ emissions of filtrate. Figure

1f and Figure S-14 show that the overall relative radioactivity of BP-DEX NPs in pure water, PBS, and 10% FBS can be maintained around 85% after stirred for 24 h, which indicates the tight binding of

99m

Tc with BP-DEX NPs and their excellent stability in these media. These

results prove that BP-DEX NPs are suitable for in-vivo PA imaging, and for SPECT/CT imaging


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after they were labeled with radioisotopes. As a return, these two imaging methods can be used to evaluate the in-vivo behaviors of BP-DEX NPs due to their high sensitivity. Prior to these investigations, their potential cytotoxicity was assessed by a MTT assay, which was carried out after 4T1 cells were incubated with BP-DEX NPs at various concentrations for 12 h. Figure 2a demonstrates the dependence of cell relative viabilities on the concentration of BP NPs. The cell viability remained above 80% when the cells were cultured with BP-DEX NPs with a concentration smaller than 100 µg/mL, demonstrating BP-DEX NPs have a good biocompatibility. It has been known that fluorescence-activated cell sorting (FACS) technique is particularly useful for sorting cells, and can be used to assess the potential genotoxicity of nanoparticles to cells.44 The cytotoxicity of various concentrations of BP-DEX NPs toward 4T1 cells was further assessed with FACS by detection of cell apoptosis after the cells were double labelled with annexin-V-APC/7-ADD-A. The results obtained from FACS technique were shown in Figure 2b, where the area of UR indicates the apoptosis of cells at a later stage induced by the toxicity of nanoparticles. There are around 11.21% and 21.17% of cells apoptosed in the absence and presence of 100 µg/mL BP-DEX NPs (Figure 2c). Figure 2d shows the necrosis of 4T1 cells induced by various concentrations of BP-DEX NPs. The necrosis is less than 5% when the concentration of BP-DEX NPs is below 100 µg/mL. Both results of FACS and MTT illustrate that BP-DEX NPs have no serious cytotoxicity to 4T1 cells. The blood circulation time was investigated by intravenous injection of

99m

Tc-BP-DEX (10

mg/kg, 200 µL, 400 µCi) nanoparticles into mice through the tail vein. After intravenous injection,

99m

Tc-BP-DEX NPs adsorbed different proteins in blood due to their surface negative

charges (-27 mV), which would significantly influence their blood circulation behavior. It has been known that when nanoparticles are mixed with protein-rich physiological fluids, their


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surface is usually adsorbed with proteins to form a “protein corona”, the nature of which determine the circulation and bio-distribution of nanoparticles.45 Blood was withdrawn to measure its time-dependent intensity of γ emissions (Table 1). The radiotracers were rapidly reduced from the blood, and their contents decreased from 8.2 ± 0.7 %ID/g at 0.25 h to 1.1 ± 0.1 %ID/g at 4 h post injection. The blood circulation curve follows a typical two-compartment model46-47, which can be expressed by Eqs. (3-5) (Figure 3a). C = A·e-αt + B·e-βt

(3)

t1/2α = 0.693/α

(4)

t1/2β = 0.693/β

(5)

α is the first phase and represents the distribution of nanoparticles with a rapid decline. The halflife of the distribution phase is only (0.22 ± 0.02) h. The second phase (β) represents the elimination of nanoparticles from blood, which is the predominant process with a half-life of (9.47 ± 0.0013) h. The volume of distribution (V) and the area under curve (AUC) are quantified to be (0.52 ± 0.15) mL and (28.4 ± 1.9) %ID·h/mL, respectively. Table 2 shows the pharmacokinetic parameters of BP-DEX NPs, which have a long blood circulation time due to their small particle size. They can be delivered to whole body through the blood circulation. It has demonstrated that photoacoustic imaging could be used for semi-quantifying the pharmacokinetics of drugs.48 It can provide a preliminary evaluation of the pharmacokinetics of BP-DEX NPs in mice. The distribution and clearance of BP-DEX NPs in tumor and in other major organs was firstly studied by the PA imaging. Figure 3b shows the superposed crosssectional PA images of tumor acquired at different times. The overall contrast of tumor in the


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first 8 h was gradually enhanced, suggesting that nanoparticles were continuously accumulated in the tumor through the well-known enhanced permeability and retention (EPR) effect, which has been considered as a common mechanism for the accumulation of nanoparticles in tumor.49 After 8 h, the PA signal intensity in tumor decreased due to the elimination of nanoparticles. Figure 4a shows the bio-distribution of BP-DEX NPs in the major organs monitored by PA imaging. Similar to the tumor, weak signals are clearly observed in the pre-contrast images of liver, kidney, and spleen, which are considerably increased after intravenous injection of BPDEX NPs, reach their maximum within 4 - 6 h post injection, and then gradually decreased. The time-dependent variation of PA signals of different organs is displayed in Figure 4b, which demonstrates the dynamic accumulation and elimination of BP nanoparticles. The strong PA signal in the reticuloendothelial system (RES) is due to the rich macrophages. The drastic decreases of PA intensity in the liver, kidney and spleen after 24 h demonstrate the degradation and clearance of BP-DEX NPs. To further quantify the distribution and clearance of BP-DEX NPs, SPECT/CT imaging was carried out, and Figure 5a displays the typical whole-body SPECT/CT images of a mouse collected in a period of 0 - 8 h, after it was intravenously administrated with 99m Tc-BP-DEX NPs (400 µCi, 200 µL). A very similar uptake of the nanoparticles to that observed from the PA imaging was obtained. The nanoparticles were mainly uptaken by the liver and spleen. The quantification of radioactivity of the injected 99m Tc-BP-DEX NPs in major organs is presented in Figure 5b. The nanoparticles in liver were increased to 35% ID/g within 5 min after intravenous injection and remained for 3 h, and then gradually reduced to 15% ID/g after 8 h post injection. In contrast, the accumulations of nanoparticles in spleen and bladder in this period were steadily increased, i.e., from 5% to 20% ID/g for spleen, from 5% to 20 ID% for bladder. The results


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show that BP NPs were excreted via the hepatobiliary transport pathway, as liver is the key organ for detoxification of xenobiotic by metabolism and biliary excretion. The metabolism of nanoparticles is significantly influenced by their size, composition, and surface characteristics.50-52 Most large nanoparticles are accumulated in liver in comparison with smaller ones. When BP nanoparticles were systemically administered, various serum proteins bound to their surface to form large particles, which were recognized by the scavenger receptors on the macrophages. BP NPs were mainly enriched in liver and spleen, because they were exogenous and easily captured by the macrophages in these organs.53-54 In contrast to liver and spleen, the uptakes of BP-DEX NPs in heart, lung and kidney are much smaller. Larger microsized particles are usually captured by lung, and ultra-small nanoparticles (less than 6 nm) are directly excreted via kidney.55-56 The distribution of BP-DEX NPs in major organs at 24 h post injection is shown in Figure S-15, which follows an order of spleen ˃ liver ˃ lung ˃ kidney. Major nanoparticles were accumulated in spleen (19.7% ID/g) and liver (7.6% ID/g) after 24 h post injection, further illustrating the major metabolism of BP-DEX NPs via hepatobiliary excretion.

Conclusions In summary, we prepared biocompatible BP-DEX NPs with excellent photothermal conversion property by the ball milling approach. Their blood circulation, bio distribution and clearance were determined by highly sensitive PA imaging and SPECT/CT imaging. These BPDEX NPs have a long blood circulation time of 9.47 h. Quantification results illustrates the major accumulation of BP-DEX NPs in the reticuloendothelial system organs, and their


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degradation and clearance via the hepatobiliary excretion and kidney clearance. Our research provides insights and guidance for clinical translation of BP nanoparticles as a novel theranostic agent. ASSOCIATED CONTENT Supporting Information. XRD, Raman, FTIR and XPS patterns of black phosphorus nanoparticles; statistical histogram of the size distribution, hydrodynamic size, stability of BPDEX nanoparticles; In-vitro retention of radioactivity of 99mTc-BP-DEX NPs; bio-distribution of 99m

Tc-labeled BP-DEX NPs.

This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * C. Zhao (email: [email protected]); Z. Li (email: [email protected])

Author Contributions Caixia Sun did the experiments and wrote the manuscript; Yifan Xu, Lijuan Deng, and Hao Zhang assisted in doing the experiments; Qiao Sun, Chongjun Zhao and Zhen Li supervised students in doing experiments and revised the manuscript. Notes There are no conflicts to declare. ACKNOWLEDGMENTS Z. Li is grateful for the support from the National Natural Science Foundation of China


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(81471657, 81527901), the 1000 Plan for Young Talents, Jiangsu Specially Appointed Professorship, and the Program of Jiangsu Innovative and Entrepreneurial Talents. The authors also are grateful for support from the Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection, the Priority Academic Development Program of Jiangsu Higher Education Institutions (PAPD). The authors would like to thank Dr. Tania Silver for critical reading of the manuscript. REFERENCES (1) Sofer, Z.; Bouša, D.; Luxa, J.; Mazanek, V.; Pumera, M. Few-Layer Black Phosphorus Nanoparticles. Chem. Commun. 2016, 52 (8), 1563-1566. (2) Gusmao, R.; Sofer, Z.; Pumera, M. Black Phosphorus Rediscovered: From Bulk Material to Monolayers. Angew. Chem. Int. Ed. 2017, 56 (28), 8052-8072. (3) Li, L.; Yu, Y.; Ye, G. J.; Ge, Q.; Ou, X.; Wu, H.; Feng, D.; Chen, X. H.; Zhang, Y., Black Phosphorus Field-Effect Transistors. Nat. Nanotechnol. 2014, 9 (5), 372-377. (4) Koenig, S. P.; Doganov, R. A.; Schmidt, H.; Castro Neto, A. H.; and Oezyilmaz, B., Electric Field Effect in Ultrathin Black Phosphorus. Appl. Phys. Lett. 2014, 104 (10), 103106. (5) Buscema, M.; Groenendijk, D. J.; Blanter, S. I.; Steele, G. A.; van der Zant, H. S. J.; Castellanos-Gomez, A., Fast and Broadband Photoresponse of Few-Layer Black Phosphorus Field-Effect Transistors. Nano Lett. 2014, 14 (6), 3347-3352. (6) Mayorga-Martinez, C. C.; Mohamad Latiff, N.; Eng, A. Y. S.; Sofer, Z.; Pumera, M. Black Phosphorus Nanoparticle Labels for Immunoassays via Hydrogen Evolution Reaction Mediation. Anal. Chem., 2016, 88 (20), 10074-10079. (7) Li, D.; Castillo, A. E. D. R.; Jussila, H.; Ye, G.; Ren, Z.; Bai, J.; Bonaccorso, F. Black Phosphorus Polycarbonate Polymer Composite for Pulsed Fibre Lasers. Appl. Mater.


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into Bile. Hepatology, 1989, 9 (3), 380-392.


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Scheme 1. PA imaging and SPECT/CT imaging by using BP-DEX NPs


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Figure 1. The characterization of as-prepared BP-DEX NPs: (a) TEM image and (b) AFM image of BP-DEX NPs, (c) excitation-dependent PA images and the corresponding signal intensity of BP-DEX NP solution with a concentration of 100 µg/mL (the excitation wavelength is 680, 750, 808, 850, and 900 nm, respectively), (d) concentration-dependent PA images and the corresponding signal intensity of BP-DEX NP solution with different concentrations of 0, 25, 50, 100, 200, and 400 µg/mL excited with 680 nm, (e) Zeta potential of BP-DEX NPs before and


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after labeled with

99m

Tc, and (f) In-vitro retention of radioactivity of

99m

Tc-BP-DEX NPs in

water.


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Figure 2. In-vitro cytotoxicity of BP-DEX NPs towards 4T1 cells: (a) relative cell viabilities after 4T1 cells were incubated with various concentrations of BP-DEX NPs for 12 h, (b) fluorescein APC-A/7-ADD-A double labeling flow cytometry analysis of 4T1 cells incubated with BP-DEX NPs at different concentrations of 0, 6.25, 50, and 100 µg/mL, and (c-d) apoptosis and necrosis of cells based on the results of flow cytometry.


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Figure 3. (a) Blood circulation profile of

99m

Tc-BP-DEX NPs expressed as a percentage of the

injected dose (200 µCi, 2 mg/mL, 200 µL) per gram of blood (ID%/g) and presented as mean ± standard deviation (n = 3), (b-c) the superposed PA images and corresponding signal intensity of tumor obtained at different time intervals after intravenous injection of BP-DEX NPs


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Table 1．．Time-dependent concentration of 99mTc-BP-DEX collected from blood pool

Time (h)

0.25

0.5

1

2

4

6

8

21

%ID/g （SD）

8.2035 (0.717)

5.3124 (0.929)

2.92764 (0.8632)

1.46359 (0.1723)

1.1047 (0.119)

1.10162 (0.063)

0.91324 (0.163)

1.08105 (0.116)

%ID/g, percentage injected dose per gram; SD, standard deviation (n=3).

Table 2. Pharmacokinetic parameters for BP-DEX NPs after intravenous injection into mice a

AUC HD (nm)

t1/2α (h)

(%ID

t1/2β (h)

VC (mL)

CL (mL/h)

0.52 ± 0.148

0.35 ± 0.038

h/mL)

21 ± 2.0

a

0.22 ± 0.02

9.47 ± 0.001

28.43 ± 1.86

Values are means ± standard deviation. Abbreviations: t1/2α, blood distribution half-life; t1/2β,

blood terminal elimination half-life; AUC, area under the blood activity-time curve; VC, volume of distribution in centre compartment; CL, total body clearance.


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Figure 4. Distribution and clearance of BP-DEX NPs investigated by PA imaging after they were intravenously injected into the mice: (a) PA images of liver, kidney, and spleen of mouse collected at different time intervals after injection, (b) variation of PA signals of liver, kidney, and spleen with time after injection of BP-DEX NPs.


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Figure 5. Distribution and clearance of BP-DEX NPs investigated by SPECT/CT imaging after they were intravenously injected into the mice: (a) time-dependent SPECT/CT images of the mice injected with 99mTc-labeled BP-DEX NPs, (b) quantification of 99mTc-labeled BP-DEX NPs in liver, spleen, bladder, heart, kidney, and lung, respectively.


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Blood Circulation, Biodistribution, and ... - ACS Publications

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