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Albumin-mediated Biomineralization of Paramagnetic NIR AgS QDs for Tiny Tumor Bimodal Targeted Imaging in vivo 2

Jing Zhang, Guang-Yu Hao, Chenfei Yao, Jiani Yu, Jun Wang, Weitao Yang, Chunhong Hu, and Bingbo Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04738 • Publication Date (Web): 14 Jun 2016 Downloaded from http://pubs.acs.org on June 19, 2016

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ACS Applied Materials & Interfaces

Albumin-mediated Biomineralization of Paramagnetic NIR Ag2S QDs for Tiny Tumor Bimodal Targeted Imaging in vivo

Jing Zhanga, Guangyu Haoa, Chenfei Yaoa, Jiani Yub, Jun Wangb, Weitao Yangc, Chunhong Hua*, and Bingbo Zhangb*

a

Imaging Center, the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province

215006, China. b

Institute of Photomedicine, Shanghai Skin Disease Hospital; The Institute for Biomedical

Engineering & Nano Science, Tongji University School of Medicine, Shanghai 200443, China. c

School of Materials Science and Engineering, School of Life Science, Collaborative Innovation

Center of Chemical Science and Engineering, Tianjin University, Tianjin 300072, China

Corresponding authors: Chunhong Hu: [email protected]; Tel: +86 0512-67780422; Fax: +86 0512 67780633 Bingbo Zhang: [email protected]; Tel: +86 21 65988029; Fax: +86 2165983706-0

1

ACS Paragon Plus Environment


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Abstract Bimodal imaging has captured increasing interests due to its complementary characteristics of two kinds of imaging modalities. Among the various dual-modal imaging techniques, MR/fluorescence imaging has been widely studied owing to its high 3D resolution and sensitivity. There is, however, still a strong demand to construct biocompatible MR/fluorescence contrast agents with near-infrared (NIR) fluorescent emissions and high relaxivities. In this study, BSA-DTPAGd derived from bovine serum albumin (BSA) as a novel kind of biotemplates is employed for biomineralization

of

Ag2S@BSA-DTPAGd

paramagnetic

NIR

Ag2S

quantum

dots

(denoted

as

pQDs). This synthetic strategy is found bioinspired,

environmentally benign and straightforward. The obtained Ag2S@BSA-DTPAGd pQDs have fine sizes (ca. 6 nm) and good colloidal stability. They exhibit unabated NIR fluorescent emission (ca.790 nm) as well as high longitudinal relaxivity (r1=12.6 mM-1s-1) compared to that of commercial Magnevist (r1= 3.13 mM-1s-1). In vivo tumor-bearing MR and fluorescence imaging both demonstrate Ag2S@BSA-DTPAGd pQDs have pronounced tiny tumor targeting capability. In vitro and in vivo toxicity study show Ag2S@BSA-DTPAGd pQDs are biocompatible. And biodistribution analysis indicates they can be cleared from body mainly via liver metabolism. This protein-mediated biomineralized Ag2S@BSA-DTPAGd pQDs presents great potentials as a novel bimodal imaging contrast agent for tiny tumor diagnosis. Keywords: Biomineralization; Silver Sulfide; Bovine Serum Albumin; Fluorescence Imaging; Magnetic Resonance Imaging; 2


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1. Introduction In the latest studies on tumor theranostics, molecular imaging techniques have being played growing vital roles in improving the precision of early diagnosis.1-4 To date, various kinds of imaging techniques have been developed for clinic applications, including computed tomography (CT), 5 magnetic resonance imaging (MRI), 6-8 proton emission tomography/computed tomography (PET/CT),

9-11

and optical imaging.

12-14

Single-modality imaging, however, either performs as a kind of functional imaging (e.g. optical imaging, PET) or structural imaging (e.g. MRI, CT), lacking in comprehensive imaging information for diagnosis. Thus, the combination of two or multiple molecular imaging moieties, including functional and structural imaging, is emerging for this demand. In recent years, multifunctional nanoprobes, usually consisting of two or three imaging moieties, have been carried out for tumor precise diagnosis applications. 15-17 Among bimodal imaging techniques, MR/fluorescence dual-modal imaging has harvested intensive interests due to: (1) Non-invasive and non-radiative characteristics of both MR and fluorescent imaging allow its frequent use if needed for body scan without safety concerns; (2) No limit in tissue penetration and high 3D anatomical resolution qualify MR imaging technique extensive applications in disease detection; (3) Fluorescent imaging, a functional imaging technique, with inherent properties of high detection sensitivity, fast feedback and multiplexing, can compensate MRI for its low sensitivity at subcellular levels. 18 MR/fluorescence dual-modal imaging, based on 3



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the complementary characteristics of two kinds of imaging modalities, is therefore promising to provide more imaging information and then improve the accuracy of tumor diagnosis. 19-23 Contrast agent based imaging can increase the contrast ratio between normal tissues and suspicious regions. In the aspect of MR/fluorescence dual-modal imaging, nanostructures, composed of fluorescent and paramagnetic modules, are getting rapid popularity since its easy assembling and versatile decoration on nanoarchitectures. 21-22

19,

For fluorescent imaging moieties, semiconductor QDs have attracted intensive

attentions on their biomedical applications since their unique optical properties comparing with the traditional organic dye molecules, such as excellent photo-stability and size-dependent emissions with broad excitation spectra.

24-25

Recently, studies

suggest Ag2S or Ag2Se, as the latest type of green QDs, are very promising Cd-free candidates for in vivo imaging applications, thanks to their low toxicity and near-infrared (NIR) emissions. 26-30 For MR imaging modules, superparamagnetic iron oxide (SPIO) and paramagnetic ions (Gd3+ and Mn2+) are both commonly used.

31-33

The blooming effect of SPIO in T2-weighted MRI however has been considered amplifying the signal decrease due to affected protons at distant sites. This effect exaggerates the size of labeled area and blurs the image and makes the distinction of suspected lesions difficult from normal tissues nearby.

34-35

effect of SPIO could reduce fluorescent emissions nearby.

Moreover, the quenching 36

Considering the above

issues, paramagnetic Gd3+ is employed for MRI modality in this study. Consequently, magnetically Gd3+ engineered Ag2S-based paramagnetic QDs are designed and 4


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expected for producing MR/fluorescence dual-modal imaging signals with decent NIR emission, high relaxivity and greatly suppressed toxicity for in vivo imaging of tumor.37 Thermal decomposition in organic phase at high temperature (generally over 200 °C) and coprecipitation technology in aqueous solution are mainly two methods reported for synthesis of Ag2S QDs.

26-27, 38

Thermal decomposition approach contributes high

crystallinity of Ag2S QDs, while it is obviously complicated in the process of hydrophobic Ag2S QDs synthesis and the following water-solubilization for their biomedical applications. 39 There is therefore a great need for fabrication of Ag2S QDs with high quality in a convenient manner. Recently, biomineralization of inorganic nanoparticles or nanostructures are found efficient and straightforward in ambient environment. 40-41 Gd3+ based nanoparticles, for instance, inspired by biomineralization for magnetic resonance angiography (MRA) was reported in our group early.

42

Wang

et al synthesized BSA stabilized Ag2S QDs for in vivo tumor fluorescence imaging, but this single optical imaging modality is insufficient for accurate tumor diagnosis. 28 Herein, we design magnetically engineered Ag2S pQDs with Gd3+ ions, aiming to providing MRI in addition to the NIR optical imaging, and synthesize it by Gd3+-labeled BSA as the biotemplates at 37 °C through an ambient biomineralization reaction for bimodal tumor targeted imaging. BSA is known easily decorated with other small molecules for multi-functions. Based on this principle, in this study, BSA protein has played double roles, namely, the Gd3+ carrier besides the template for 5



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biominerlization. To the best of our knowledge, albumin-biomineralized paramagnetic Ag2S pQDs has not been reported. Ag2S@BSA-DTPAGd pQDs fabricated by this one-step aqueous synthetic strategy show both good NIR fluorescent effect and high relaxation rate, favoring for biomedical imaging, and particularly for tiny tumor diagnosis with its ultrasmall hydrodynamic diameter feature.

2. Experimental section 2.1 Materials Silver nitrate (AgNO3, 99.99%), gadolinium (III) chloride hexahydrate (GdCl3·6H2O, 99.9%), dimethyl sulfoxide (DMSO, 99.8%), Diethylene triamine pentacetic acid dianhydride (DTPAA, 95%) were purchased from Alfa Aesar. Sodium sulfide nonahydrate (Na2S·9H2O, 98%) was purchased from Sigma-Aldrich. Sodium hydroxide (NaOH, 96%) was purchased from Aladdin Reagent Company. Bovine serum albumin (BSA, 96%) was purchased from Genview. Phosphate Buffered Saline (PBS, 0.01 M, pH 7.4) was purchased from Beijing Dingguo Biotechnology. Trisodium citrate (C6H5Na3O7·2H2O, 99.0%), sodium hydrogen carbonate (NaHCO3, 99.5%) were purchased from local suppliers. All the chemicals were used without further purification. Deionized water (18.2 MΩ•cm resistivity at 25 °C) was used throughout the experiments.

2.2 Preparation of BSA-DTPAGd Complex 6


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The BSA-DTPAGd complex was synthesized according to the strategy reported previously with slightly modifications.43 Briefly, 2 g of BSA was dissolved in 30 mL of borate saline buffer (50 mM, pH 8.2). And then 2 g of DTPAA dissolved in 10 mL of anhydrous DMSO was added dropwise to the above BSA solution, and the pH was immediately adjusted to 8-8.5 by using 0.5 M NaOH. Keep the mixed solution stirred for 4 h. Then the mixture was purified by dialyzing against 5 × 4 liter of citrate buffer (0.1 M, pH 6.5). Subsequently, GdCl3·6H2O (3 mmol) dissolved in 10 mL of Na-acetate buffer (0.1 M, pH 6.5) and was dripped slowly to the mixture mentioned above. Keep it reacting for another 24 h on a stirrer and then dialyze against 5 × 4 liter of citrate buffer (0.1 M, pH 6.5) for 3 days and then against deionized water. The final product was lyophilized to give BSA-DTPAGd as white powder and keep it at 4 °C for further use. The whole process was conducted at room temperature.

2.3 Preparation of Ag2S@BSA-DTPAGd pQDs Albumin-biomineralized

Ag2S

pQDs

was

synthesized

through

a

one-pot

straightforward synthetic protocol. Typically, 1 g of BSA-DTPAGd complex was first dissolved in 10 mL of deionized water in a one-neck flask (25 mL), and the pH was adjusted to 11-12 by 0.5 M NaOH. Then 5 mL of AgNO3 (10 mM) was added and vigorously stirred. After 5 min 1 mL of Na2S (50 mM) was slowly injected into the mixture. Leave this reaction under fiercely stirring overnight to promote the growth of QDs at 37 °C in an ambient solution. The color of the solution was changed from transparent light yellow to dark red-brown. Then the mixture was dialyzed against 5 × 7



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4 liter PBS (0.01 M, pH 7.4) to remove unreacted ions. The resulting purified Ag2S@BSA-DTPAGd pQDs was stored in dark at 4 °C for further analysis and use.

2.4 Materials Characterization The size, morphology, structure and composition of the prepared Ag2S@BSA-DTPAGd pQDs were characterized by transmission electron microscopy (TEM, G2 F20, Tecnai), energy dispersive X-ray spectroscopy (EDX, S-4800, Hitachi), X-ray photoelectron spectroscopy (XPS, Escalab 250Xi, Thermo), and Fourier transform infrared spectrometry (FTIR, Tensor 27, Bruker). The hydrodynamic diameters (HDs) of Ag2S@BSA-DTPAGd pQDs were determined with a dynamic laser scattering (DLS, NanoZS90, Malvern). The fluorescent spectrum of Ag2S@BSA-DTPAGd pQDs was required on an F-4500 spectrophotometer (Hitachi) at the excitation wavelength of 570 nm. The relaxation rate of Ag2S@BSA-DTPAGd pQDs, BSA-DTPAGd and commercial Magnevist were measured on a 1.41 T minispec mq 60 NMR analyzer (Bruker, Germany). The concentrations of the investigated ions were measured by an inductively coupled plasma mass spectrometry (ICP-MS, Hitachi Ltd. Japan).

2.5 Relaxivity Characterization and MR Imaging in vitro The relaxation times of Ag2S@BSA-DTPAGd pQDs, BSA-DTPAGd and Magnevist were measured using a 1.41 T minispec mq 60 NMR analyzer (Bruker, Germany), respectively. Their corresponding relaxation rates were assessed from the slope of a linear relationship between the reciprocal of T1 relaxation rate (1/T1) and the Gd3+ 8


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concentration. The in vitro T1-weighted images of as-prepared Ag2S@BSA-DTPAGd pQDs at various Gd3+ concentrations were acquired using MesoMR23-060H-I (Shanghai Niumag Corporation, China, 0.5 T). The spin echo sequence (SE sequence) parameters were as follows: TR = 80 ms, TE = 18.2 ms, Matrix = 256 × 192, FOV = 80 mm × 80 mm, slice width = 5.0 mm, 32 °C.

2.6 In vitro Toxicity Test An MTT colorimetric assay was taken to evaluate the toxicity of Ag2S@BSA-DTPAGd pQDs on Hela cells. Briefly, Hela cells were seeded into a 96-well plate at a density of 5×103 cells per well and cultured at 37 °C in a humidified 5% CO2 atmosphere. Sterile PBS was filled in the side holes to prevent the evaporation of moisture resulting in death of cells. After incubation for 24 h, the existing medium was replaced with fresh medium containing 100 µL of pQDs with different Ag+ concentrations, and incubated for 4 h and 24 h under the same condition. One row of 96-well plate was set as controlled trial. The cells were washed twice with PBS, and then were added with fresh medium. Then 20 µL per well of MTT (5 mg/mL) was added and the mixture was incubated for further 4 h at 37 °C. The remaining formazan crystallization was dissolved into 100 µL DMSO after the excess medium was carefully removed. Finally, the absorption of each well at 570 nm was measured by an enzyme-linked immunosorbent instrument. The cell viability was obtained by drawing a cell growth curve.

2.7 MR/Fluorescence Tumor-Targeted in vivo Imaging 9



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The tumor imaging was performed on seven-week-old female athymic nude mice (ca. 25 g each). The animal procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee of Tongji University. The xenograft tumor model was established by subcutaneous injection of U87 MG cells (ca. 5×106 per 50 µL PBS) into the front flank of the mice. The mice were subjected to imaging studies when the tumor size reached ca. 2.0 mm after inoculation. The bosselated tissues were checked by histological study to confirm they were cancerous. The in vivo MR images were acquired on a 0.5 T magnetic resonance machine (MesoMR23-060H-I, NIUMAG, China) before and after the injection of Ag2S@BSA-DTPAGd pQDs at the dosage of 0.05 mmol Gd3+/Kg body weight via the tail vein. The SE sequence parameters were as follows: TR = 300 ms, TE = 18.2 ms, Matrix = 256 × 192, FOV = 80 mm × 80 mm, slice width = 2.5 mm, 32 °C. The in vivo fluorescent images were obtained on a VisEn FMT 2500 in vivo imaging system after

the

mice

were

intravenously

injected

with

the

same

dosage

of

Ag2S@BSA-DTPAGd pQDs.

2.8 Biodistribution of Ag2S@BSA-DTPAGd pQDs Organ distributions of Ag2S@BSA-DTPAGd pQDs were determined by ICP-MS analysis of Ag+ at 2 h, 5 h, 12 h, 24 h, 48 h, and 7 d, respectively. The experimental groups were injected with pQDs at the same dosage with imaging via the tail vein. The mice were sacrificed at different intervals and their main organs including heart, liver, spleen, lung, kidney, intestine and tumors were removed and specially treated for 10


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ICP-MS analysis of Ag+ concentrations. Gd

2.9 In vivo Toxicity Analysis of Ag2S@BSA-DTPA

pQDs

Hematoxylin andeosin (H&E) staining was conducted to test in vivo toxicity of Ag2S@BSA-DTPAGd pQDs. Main organs including heart, liver, spleen, lung, kidney and

intestine

were

excised

from

a

group

of

mice

post-injected

with

Ag2S@BSA-DTPAGd pQDs after 7 days and another group of normal mice without any injection. The injected dosage was consistent with that of biodistribution study mentioned above. The organs were sliced, fixed with 4% paraformaldehyde and dyed with H&E. The obtained tissue slices were observed and analyzed under an optical microscope.

3. Results and discussion

3.1 Synthesis strategy and characterizations of Ag2S@BSA-DTPAGd pQDs

11



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Scheme 1. Schematic illustrations of passive tumor targeting imaging via EPR effect (a) and the typical synthetic process of Ag2S@BSA-DTPAGd pQDs (b). Ag2S QDs synthesized in this study are inspired by a biomineralization strategy, whereas in previous studies, Ag2S QDs were usually fabricated at high temperatures in organic solvents and phase transfer should be done before their biomedical applications.39 To simplify the synthesis process, biomineralization in a biocompatible manner is introduced in synthesis of Ag2S QDs. BSA with abundant amino acid residues and sulfhydryl bonds which can capture metal ions, and its unique molecular space structure for crystal grain growths, has been increasingly used as a natural 12


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biotemplate in biomineralization of various nanoparticles.

40

Nevertheless, the other

inherent functions of BSA are not well exploited. In this study, utilizing easy molecular modifications of BSA with other small compounds, BSA is covalently modified with DTPA, a metal ion chelating agent for carrying Gd3+, in order to afford NIR Ag2S QDs MR imaging modality.

43

The derivative BSA-DTPAGd as a new

biotemplate for the first time in this study was synthesized and employed for biomineralization of Ag2S@BSA-DTPAGd pQDs (Scheme 1).

13



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14


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Figure 1. (a) TEM image of Ag2S@BSA-DTPAGd pQDs; (b) EDX spectra of Ag2S@BSA-DTPAGd pQDs; XPS spectra for Ag2S@BSA-DTPAGd pQDs (c)，Ag 3d (d) and S 2p (e); (f) Hydrodynamic diameters (HDs) of Ag2S@BSA-DTPAGd pQDs; (g) FTIR spectra of BSA-DTPAGd, DTPA and BSA; and (h) Fluorescent emission of Ag2S@BSA QDs and Ag2S@BSA-DTPAGd pQDs in PBS buffer. The size, morphology, structure and composition of Ag2S@BSA-DTPAGd pQDs were characterized and displayed in Figure 1a-g. The prepared Ag2S@BSA-DTPAGd pQDs show monodisperse spheres without aggregates on TEM (Figure 1a) and a mean diameter of 6.0 nm (Figure 1f), which is smaller than that of Ag2S@BSA (ca. 13 nm) (Figure S1). This difference in size can be clearly distincted by DLS analysis. The presence of Gd components on BSA templates could account for this slight decrease in size. Gd3+, as a positively charged ion, has contribution on convergence of BSA macromolecules. These ultrasmall particles below 10 nm favor for in vivo imaging, since they could be escaped from reticuloendothelial system (RES) and cleared via kidneys, whereas larger particles limit their biological functions, as they are easy to be recognized and removed from the circulation by RES.

44

EDX result shows that the

pQDs were composed of Ag, S and Gd elements with the Ag: S atomic ratio being about 0.4: 1 (Figure 1b), which is less than the theoretical stoichiometry of Ag2S mainly because BSA contains S element. The XPS peaks at 367.7 eV and 373.7 eV from Ag 3d5/2 and 3d3/2 of pQDs, and the S 2p peak at 162.8 corresponds to S in Ag2S@BSA-DTPAGd pQDs (Figure 1c, d, e). FTIR spectra reveal the integration of DTPA and BSA (Figure 1g). The above results from multiple perspectives prove the 15



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successful synthesis of Ag2S@BSA-DTPAGd pQDs. Furthermore, the fluorescent performance of Ag2S@BSA-DTPAGd was evaluated together with that of Ag2S@BSA, and their emission profiles are shown in Figure 1h. It is found almost 70% of the fluorescence intensity of Ag2S@BSA-DTPAGd can be preserved, and the emission peak is still single and nearly symmetrical at maximum emission at about 790 nm excited at 570 nm. This slight decrease in emission is likely attributed to the microenvironmental change as the occupation of Gd components near the Ag2S QDs.

For comparison, in this study, Ag2S QDs was first prepared according

to the previous report,45 and DTPAA and Gd3+ were then coupled and chelated on BSA successively. Unfortunately, it was found that although the resulting relaxation rate (12.3 mM-1S-1) was as high as that of Ag2S@BSA-DTPAGd, the fluorescence was gradually quenched in the process of chelating with Gd3+. This quenching could be attributed to the long period exposure to the buffer with low pH of 6.5 during Gd3+ chelating and purification. On the contrary, the aforehand modification on BSA is experimentally shown more effective. 3.2 Relaxation Charaterizations

16


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Figure 2. Linear relationship between longitudinal (a), transverse (b) relaxation rate (1/T1, 1/T2) and the concentration of Gd3+ of Ag2S@BSA-DTPAGd pQDs, BSA-DTPAGd and Magnevist; (c) T1-weighted images of Ag2S@BSA-DTPAGd pQDs at different Gd3+ concentrations and their corresponding relative signal intensities (d). The relaxivity of BSA-DTPAGd bioinspired Ag2S@BSA-DTPAGd pQDs is shown the highest (12.6 mM-1S-1) in Figure 2a, in comparison with those of BSA-DTPAGd (9.9 mM-1S-1) and Magnevist (3.1 mM-1S-1, a frequently used commercial MRI contrast agent). In this study, in order to apply MR/fluorescence imaging in vivo, Gd3+ is ingeniously incorporated into Ag2S QDs via BSA molecules, as Gd3+ can promote 17



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longitudinal relaxivity of hydrogen protons and generate obvious contrast where the pQDs gather. The improvement on relaxivity of Ag2S@BSA-DTPAGd pQDs is mainly in virtue of the increase of molecule rigidity.46 The r2/r1 ratio of 1.34 plays an essential role to determine the comparative efficiency of Ag2S@BSA-DTPAGd pQDs as a T1 positive contrast agents. Figure 2c shows T1-weighted images of Ag2S@BSA-DTPAGd pQDs in vitro at different Gd3+ concentrations. It is obviously seen that signal intensity is enhanced with the increasing of Gd3+ content in samples, which is further demonstrated intuitively in Figure 2d. It is therefore can be concluded that on account of high relaxivity, Ag2S@BSA-DTPAGd pQDs can be a potential candidate as a MRI contrast agent. 3.3 Stability Analysis of Ag2S@BSA-DTPAGd pQDs

18


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Figure 3. Stability studies on fluorescence (a and b), hydrodynamic diameters (HDs, c), dispersion capability in PBS buffer (d) and T1, T2 relaxation times (e) of Ag2S@BSA-DTPAGd pQDs under room temperature at various time intervals. Figure 3 presents the stability of Ag2S@BSA-DTPAGd pQDs on colloid storage, fluorescent emission and relaxivity dispersed in PBS (1×) at various time. All of the above characteristics are kept stable well, supported by solid data (Figure 3a-e). 19



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Particle agglomerations or precipitations, or quenching did not occur during the investigated time (Figure 3a-d). Relaxation time monitoring further demonstrates Ag2S@BSA-DTPAGd pQDs are stable in structure and suggests there is no Gd3+ leakage observed (Figure 3e). Stability of nanoparticles is crucial for biomedical applications. Especially, the bi/tri/multi-modal nanoprobes should have stable structures; otherwise they could be separated during circulation in bloodstream, Covalently engineered BSA-DTPAGd

resulting in diverse pharmacokinetics.

macromolecules used in this study themselves are very stable and found suitable for biomineralization of Ag2S QDs. All of the stability test results indicate Ag2S@BSA-DTPAGd pQDs can be used as a stable bimodal molecular probe for in vivo imaging. 3.4 MTT Assay

Figure 4. Cell viability of Hela cells incubated with different concentrations of 20


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Ag2S@BSA-DTPAGd pQDs for 4 h and 24 h. Cells that not exposed to nanomaterials were set as control. The results were performed as mean ± SD (n = 5). Cytotoxicity of Ag2S@BSA-DTPAGd pQDs at different concentrations was evaluated on Hela cells using MTT assay (Figure 4). It shows cell viability remains more than 80 % at all investigated concentrations. Even at the highest concentration of 0.461 mM of Ag+, 95% and 93% of the Hela cells survive relative to the control group after incubation with Ag2S@BSA-DTPAGd pQDs for 4 h and 24 h, respectively, indicating the low cytotoxicity and good biocompatibility of pQDs. This low toxicity could be attributed to the safe raw materials and serum protein component used in this study. 3.5 In Vivo MR/fluorescence Imaging on Tiny Tumor Mice Model

21



Pseudo-color

MRI

a.

b.

c.

Signal Enhancement (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. In vivo T1-weighted MR images of tiny tumor-bearing nude mice which were taken pre and post injection of Ag2S@BSA-DTPAGd pQDs at different time points (a) and their corresponding quantified signal-to-noise ratio (SNR) of intensity (post)-to-intensity (pre) contrast in tumor (b); And fluorescent image of tiny tumor-bearing nude mice was taken after 2 hours injected intravenously with Ag2S@BSA-DTPAGd pQDs (c), excited at 670 nm. Tumor areas are confirmed cancerous by histological analysis and circled in red. In vivo MR/fluorescence bimodal imaging was conducted on tiny tumor bearing mice by Ag2S@BSA-DTPAGd pQDs. For evaluation of the tumor detection capability 22


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of Ag2S@BSA-DTPAGd pQDs, a tiny tumor model, less than 2.0 mm, was used in this study. In vivo T1-weighted MR images were acquired before and after injection of Ag2S@BSA-DTPAGd pQDs at different time points shown in Figure 5a. It is clearly seen that the tiny tumor disclosed with the most significant enhanced contrast at 1.5hour time point postinjection. And pQDs-enhanced MRI pattern on tiny tumor is found time dependent and becoming weak at three-hour postinjection and disappeared at 12hour postinjection. The corresponding quantified signal-to-noise ratio (SNR) of intensity (post)-to-intensity (pre) contrast in tumor was further summarized in Figure 5b. In vivo fluorescence imaging was also carried out on the same tumor model (Figure 5c). Thanks to the NIR emission at 790 nm of Ag2S@BSA-DTPAGd pQDs excited at 670 nm, the tiny tumor lesion is remarkably contrasted at two- hour time point postinjection of Ag2S@BSA-DTPAGd pQDs, and the background signal on the living mouse is significantly suppressed in this study on account of NIR excitation. It is worth noting that the enhanced area tissues were removed and stained to analyze their histological appearance, and they were found cancerous (Figure S2), suggesting Ag2S@BSA-DTPAGd pQDs can effectively and selectively home to the tumor. Nanoparticles with proper sizes have been demonstrated to be capable of targeting tumors by taking advantage of the leaky vasculature of tumors, known as passive tumor targeting. Moreover, the landed nanoparticles could be hooked by enhanced permeability and retention effect (EPR effect) since the lymphatic drainage in tumor area is impaired.

37

However, it becomes challenging when it comes to imaging tiny

tumors in vivo. 47 Contrast agents or nanoprobes should be synthesized in high quality 23



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and with properly surface engineered. Nanoparticles with big sizes, usually found trapped in RES system organs, are not suitable for tiny tumor imaging. The surface microenvironment of nanoparticles, determined by ligand component, density, rigidity, flexibility, charge, and etc., also plays an essential role in their biodistribution and half-life profiles in body. 48 Furthermore, the imaging performance, i.e. the relaxivities and quantum yields of probes, undoubtedly should be paid attentions to. In this study, albumin-biomineralized Ag2S@BSA-DTPAGd pQDs, with ultrasmall HD (ca. 6.0 nm) and protein capping, hold high relaxivity and decent fluorescent feature, presenting a potent tiny tumor bimodal in vivo imaging. This advanced biomineralization strategy to synthesis of in vivo imaging probes is found promising and effective. It's worth noting that imaging contrast at tumor can be further enhanced by specific bioligands mediated active targeting, leading to increased accumulation of nanoparticles at targeted areas.

8, 49-50

Ag2S@BSA-DTPAGd pQDs with BSA protein

have abundant active chemical groups favoring for covalent coupling with bioligands, such as antibody, aptamer, peptide and oligonucleotide.

51

The synthesis and imaging

work done at this stage in this study is found effective and encouraging, persuading it could be instructive and a good start for the future biomedical translational applications. 3.6 Organ Biodistribution in Vivo

24


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Figure 6. Biodistribution of Ag2S@BSA-DTPAGd pQDs at different time points in vivo. To determine the clearance pattern of Ag2S@BSA-DTPAGd pQDs, mice were administrated and sacrificed at different time points to study this process. Ag+ content was detected in major organs and tumor of mice exposed to Ag2S@BSA-DTPAGd pQDs via ICP-MS analysis. Figure 6 shows that the Ag+ ions are observed in all investigated organs, with a little more found in liver owing to the macrophages uptake of nanoparticles. In particular, Ag+ ions are presented in kidneys, indicating Ag2S@BSA-DTPAGd pQDs are small enough to quickly get through glomerulus, even though the more are located in liver. And the digestion of Ag2S@BSA-DTPAGd pQDs in liver leads their small parts to the bile and intestine. After 7 days postinjection, the 25



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injected pQDs were gradually metabolized in vivo, resulting in significant decreases of Ag+ content in all organs investigated. Ag2S@BSA-DTPAGd pQDs were found accumulated in tumor with a peak at 2 h followed by a gradual decline, which is consistent with that revealed by in vivo MR imaging. The biodistribution result further confirms the pQDs can target the tiny tumor. According to this time-dependent biodistribution pattern in Figure 6, it could be concluded that the administrated pQDs are cleared from body in a hepatic and renal combined manner. 3.7 In Vivo Toxicity Study by Histological Analysis

Figure 7. H&E stained pictures (200×) of major organs of mice, including the blank without administration and the ones after 7 days postinjection. Tissues were collected from heart, liver, spleen, lung, kidney and intestine. The tissue staining of the subcutaneous tumor is provided in Figure S2. Besides the cytotoxicity test, in vivo toxicity of Ag2S@BSA-DTPAGd pQDs was further investigated with H&E staining examination (Figure 7). The mice were sacrificed, and major organs, including heart, liver, spleen, lung, kidney and intestine, were collected and treated to evaluate the histological changes of tissues. Compared 26


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with the control group, there is no notable edema, inflammation or necrosis observed in the experimental group after exposure to pQDs. The tumor was also taken out to perform H&E staining and its tissue histology was shown in Figure S2. All the mice were found without any weight loss, illness or abnormal behaviors during the investigated period. Evidence from in vitro cytotoxicity and in vivo histological study suggests Ag2S@BSA-DTPAGd pQDs are low toxic.

4. Conclusion

In summary, BSA proteins coupled with DTPA-Gd3+ small molecules are used as biological scaffolds for bioinspired synthesis of paramagnetic Ag2S NIR QDs therefore affording MR imaging modality, in addition to their inherent fluorescence imaging. This protein-mediated biomineralization is found effective, environmentally benign

and

straightforward.

The

obtained

MR/fluorescence

dual-modal

Ag2S@BSA-DTPAGd pQDs hold decent NIR fluorescent property and high longitudinal relaxivity, which favors the application of in vivo tumor imaging with high sensitivity. In vivo MR and fluorescence imaging indicate Ag2S@BSA-DTPAGd pQDs are promising candidates for tumor-targeted imaging. In particular, the tiny tumor is contrasted effectively by taking advantage of these well-constructed nanoparticles with ultrasmall hydrodynamic diameter and biocompatible protein capping. No obvious toxicity is found in vitro and in vivo toxicity tests. And biodistribution suggests Ag2S@BSA-DTPAGd pQDs are cleared from body in a 27



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hepatic- and- renal hybrid

pattern.

Prospectively,

Page 28 of 32

the

quantum yield

of

Ag2S@BSA-DTPAGd pQDs should be concerned and improved. Moreover, it suggests pQDs could be further linked with specific bioligands and therapeutic moieties, in order to achieve tumor active targeted imaging with theranostic characteristics. Based on the evidence demonstrated in this study, it can be concluded that albumin-triggered biomineralization is of remarkable advantages, and the resulting Ag2S@BSA-DTPAGd pQDs are promising in tiny tumor bimodal imaging. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Further size distribution comparison of Ag2S@BSA-DTPAGd pQDs and Ag2S@BSA; H&E stained picture of subcutaneous tumor, supplied as Supporting Information.

Acknowledgements This work was supported by the National Natural Science Foundation of China (81171393, 81571742, 81371618), Shanghai Innovation Program (14ZZ039), Program for Outstanding Young Teachers in Tongji University, and the Fundamental Research Funds for the Central Universities.

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Graphic Abstract

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Albumin-Mediated Biomineralization of ... - ACS Publications

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