ZnS QDs with Tumor

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Water-solubilizing Hydrophobic ZnAgInSe/ZnS QDs with Tumor-targeted cRGD-Sulfobetaine-PIMA-Histamine Ligands via a Self-assembly Strategy for Bio-Imaging Tao Deng, Yanan Peng, Rong Zhang, Jie Wang, Jie Zhang, Yue-Qing Gu, Dechun Huang, and Dawei Deng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16639 • Publication Date (Web): 15 Mar 2017 Downloaded from http://pubs.acs.org on March 17, 2017

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ACS Applied Materials & Interfaces

Water-solubilizing Hydrophobic ZnAgInSe/ZnS QDs with Tumor-targeted cRGD-SulfobetainePIMA-Histamine Ligands via A Self-assembly Strategy for Bio-imaging Tao Deng,a,b Yanan Peng,b Rong Zhang,b Jie Wang,b Jie Zhang,b Yueqing Gu,a,b Dechun Huang*a and Dawei Deng*a,b

a

Department of Pharmaceutical Engineering, and b Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, Nanjing 210009, China

Keywords: Quaternary quantum dots, Self-assembly, QDs-clusters, Tumor targeting Polymer, Optical imaging

ACS Paragon Plus Environment

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Abstract:

Exploring the organic-to-aqueous phase transfer of quantum dots (QDs) is significant for achieving their versatile applications in biomedical fields. In this thematic issue, surface modification, size control and biocompatibility of QDs and QDs-based nanocomposites are core problems. Herein, the new highly fluorescent tumor-targeted QDs-clusters consisting of ZnAgInSe/ZnS (ZAISe/ZnS) QDs and sulfobetaine-PIMA-histamine (SPH) polymer with the ανβ3 integrin receptor cyclic RGD (c-RGD) were developed via ligand exchange and an accompanying self-assembly process. It was found that the structure of RGD-SPH QDs-clusters was propitious to reduce the capture of reticulo-endothelial system (RES) in virtue of external stealth ligands, and benefit to selectively accumulate at the tumor site after intravenous injection via active tumor targeting cooperated with the enhanced permeability and retention (EPR) effect. In the meantime, those clusters also recognized and enriched to cell surface when co-cultured with the ανβ3 integrin receptor overexpressed malignant cells (U87MG tumor). Based on the results, fabricating mutil-functional nanocomposites integrated with the long-term circulation and dual-targeting effects should be an interesting strategy for imaging cancer in vitro and in vivo.


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1. Introduction Biomedical optical imaging techniques can be applied across multi-spatial scales and provide key information for the preclinical and clinical assessments of biological and biochemical processes, compared with other imaging modalities.1‒3 Specifically, the visible light can be used for in vitro cell microscopic imaging, while the near-infrared (NIR) light (700-900 nm), allowing deeper penetration in tissue and lower autofluorescence, is suitable for in vivo small animal imaging.4‒7 Semiconductor nanocrystals, also called quantum dots (QDs), along with metal and metal oxide nanoparticles, possess unique size- and/or composition-dependent physical and chemical properties, which are promising alternatives to organic fluorophores for bio-imaging.8‒11 Surface properties, size control and biocompatibility are prerequisites for QDs-based nanocomposites used in any long-term bio-imaging experiments.12‒15 On the one hand, typical synthetic strategies provide oil-soluble fluorescent QDs capped with hydrophobic ligands,16‒18 whereas their next bio-applications need

excellent water-solubility.16,19‒22 In fact, the

hydrophilic part of nanoparticles is directly contacted with biological environments, which is the major factor for their ADME (absorption, distribution, metabolism, excretion) in vivo. For instance, QDs capped with biocompatible hydrophilic stealth corona, involving in PEG19,20 and zwitterion21,22, could escape effectively the capture of reticulo-endothelial system (RES) (e.g., the liver and spleen) to further prolong the circulation time in vivo. For the further modification with physiologically active molecules, likely c-RGD and folic acid, nanocomposites could be


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developed to largely enhance the delivery efficiencies of therapeutic drugs or imaging probes to tumors.23 On the other hand, the size of nanoparticles is also found to greatly affect their biodistributions, even tumor accumulation.24‒26 Nanocomposites with the size between 50~200 nm could show high tumor targeting efficiency via the disorganized and defective vascular architecture in tumor tissue (namely, the EPR effect),24,25 while for those with size less than 5.5 nm would easily excrete through kidney and larger than 200 nm would plug smaller vessels and lead to decrease blood flow with ischemia.26 Consequently, it has inspired the search for the biocompatible QD nanocomposites with excellent surface capping and appropriate size for tumor imaging in vitro and in vivo. Polymeric ligands, which possess the anchoring groups to facilitate a strong coordination with the QD surface as well as the hydrophilic tails enabling QD solubility in aqueous media, have provided an excellent platform to simultaneously achieve surface modification, biocompatibility and size control of QD nanocomposites via ligand exchange procedure. With the diverse modifiability, polymer could introduce specific functionalities, such as targeted ability27,28 and electric charge29,30 to the inorganic nanoparticles of interest.31-33 Several effective polymer ligands of phase transfer have been devised, but some of them have serious drawbacks for bioimaging, especially for in vivo application. The size of QDs capped with electroneutral ligands, likely PSMA and DHLA-PEG, was smaller than 15 nm.20 In addition, their colloidal stability is enslaved to the medium conditions, such as pH, salinity, dilution, and biological environment.33 Recently, some studies have shown that nanoparticles may organize into superlattices or


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superparticles with collective physical and chemical properties, as well as specific morphology, size distribution and colloidal stability; this also allows the rational control of the interparticle electronic coupling.34,35 Hence, endowed tendency of self-assembly to the obtained water-soluble QDs after ligand exchange is a feasible route to control the size of QD nanocomposites, and make further efforts to improve their application prospect in bio-imaging. We reported here to fabricate highly fluorescent biocompatible tumor-targeted QDs-clusters with excellent water solubility and suitable size for tumor accumulation combined with ligand exchange and self-assembly strategy. Firstly, we transferred the oil-soluble highly luminescent quaternary ZAISe/ZnS QDs into water by ligand exchanged with RGD-SPH polymer, which was designed to combine the benefits of the strong metal coordination ability of histamine, hydrophily of zwitterionic sulfobetaine, initiative tumor targeting ability of c-RGD peptide and biocompatibility of PIMA. Then, the intermediate water-soluble QDs (RGD-SPH-ZAISe) selfassembled into QDs-clusters, and the size also can be adjusted by changing the ratio of sulfobetaine. In addition, we further confirmed that the preparation route for QDs-clusters is quite universal and can be extended to other quaternary QDs, like ZnCuInSe/ZnS (These quaternary QDs do not contain highly toxic metal elements, compared with CdSe, CdTe and PbS QDs: reducing the use of the toxic component element is the other efficient way to decrease the cototoxicity of QDs (or QDs-based nanocomposites),17,18 besides controlling the QDs surface properties6,7,14). Eventually, by means of in vitro cell and in vivo NIR imaging techniques, the


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self-assembly RGD-SPH QDs-clusters with the mean size of ~55 nm were proved to be a potential nanocomposite for tumor imaging in vitro and in vivo.

2. Experimental Materials Zinc acetate (Zn(Ac)2, 99.99%), silver nitrate (AgNO3, 98+%), cupric acetate (Cu(Ac)2, 99.99%), indium acetate (In(Ac)3, 99.99%), Se powder (100 mesh, 99.5%), sulfur powder (S, 99.5%), 1-dodecanethiol (DT, 98%), oleylamine (OLA, 97%), 1-octadecene (ODE, 90%), oleic acid (OA, 90%), poly(isobutylene-alt-maleic anhydride) (PIMA, 96%),

1,3-propanesultone

(99%), histamine (96%), di-tert-butyldicarbonate (BOC, 90%), N,N-dimethylamino proylamine (DMPDA, 99%), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). Human malignant glioma cell line (U87MG) and human breast cancer cell line (MCF-7) (American Type Culture Collection, Manassas, VA, USA). Athymic nude mice bearing U87MG tumors or MCF-7 tumors under armpit (male, 18-20 g; KeyGEN Biotech. Co. Ltd, Nanjing, China). Synthesis of Oil-Soluble Quaternary Core/Shell QDs Oil-soluble ZnAgInSe/ZnS core/shell QDs were synthesized according to the previously reported method.17,18 The detailed synthesis procedure was given in Supporting Information. In


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addition, the similar procedure also was used to prepare oil-soluble ZnCuInSe/ZnS core/shell QDs, besides replacing the AgNO3 with Cu(Ac)2. Synthesis of RGD-SPH polymer Sulfobetain-PIMA-histamine (SPH): 0.385 g of PIMA (MW ≈ 6000 g/mol, 2.5 mmol of monmer units) was dissolved in 10 mL of DMSO for 0.5 h at 45 °C full with nitrogen. Histamine (0.186 g, 1.67 mmol) in DMSO (1 mL) was added dropwise to the above PIMA solution via an addition funnel. Subsequently, sulfobetaine (0.19 g, 0.83 mmol) in DMSO (1 mL) was added, which is obtained by alkalified sulfobetaine hydrochloride using triethylamine. The reaction solution was left to stir at 45 °C for 12 h until the dots of reactant disappeared by TCL monitor, and the chromogenic agent is ninhydrin. After that, the product was precipitated by addition of acetone and dried under vacuum as white powder (0.6 g, ~84% yield). 1H-NMR (D2O, Bruker 500MHz); δ 3.18 (s, 3H), 7.32 (s, 1H), and 8.62 (s, 1H). (List characteristic H peaks of each monomer only, and normalized with respect to the height of proton in imidazole ring.) cRGD-sulfobetain-PIMA-histamine (RGD-SPH): 0.25 g of SPH polymer was dissolved in 10 mL of DMSO for 10 min at RT, and the –COOH group of SPH polymer was catalyzed by EDCI/NHS (SPH polymer/NHS/EDCI molar ratio of 1/1.7/1.5) for 2 h at 45 °C. Then, cyclic RGD (20 mg, 0.03 mmol) with free -NH2 was added dropwise to react with the intermediate activated product. After further purification by recrystallized with a large excess of acetone, RGD-SPH polymer was prepared as white powder with hygroscopicity (0.22 g, ~82% yield). 1HNMR (D2O, Bruker 500MHz); δ 3.18 (s, 48H), 6.82 (m, 1H), 7.35 (s, 16H), 7.13(m, 1H) and


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8.62 (s, 16H). (List characteristic H peaks of each monomer only, and normalized with respect to the height of proton in benzene ring.) Water Solubilization of oil-soluble QDs by Ligand Exchange Core/shell ZAISe/ZnS QDs were precipitated by ethanol addition followed by centrifugation (4000 r/min, 5 min) and redispersed in 1 mL chloroform after removal of the supernatant. Simultaneously, SPH polymer (15 mg) was dissolved in warm DMSO (500 µL) with sonication for 5 min. The two kinds of precursor solution were mixed in round-bottom flask, and were left to stir at 50 °C for 5 h in nitrogen atmosphere. After that, the mixture was precipitated and removed the native ligand by adding a large excess of intermixture formed with hexane and acetone (the volume ratio is 1/1), followed by centrifugation for three times. The final precipitate could be readily dispersed in phosphate buffer (pH 5-12) with sonication for 2-3 min. The products were freeze-dried for subsequent research and further characterization. Remark: The procedure of transferring ZAISe/ZnS QDs into water by ligand exchange via RGD-SPH was similar to the above operation, and so does to transfer ZCISe/ZnS QDs. Characterization Perkin-Elmer UV/Vis spectrometer and S2000 eight-channel optical fiber spectrophotometer (Ocean Optics corporation, USA) (X-Cite Series 120Q broadband light source (Lumen Dynamics Group Inc., Canada) and FC-660-2W semiconductor laser (λ=660 nm, Enlight, China)) were combined to characterize the optical properties of the samples at room temperature. Dynamic light scattering (DLS, Mastersizer 2000, Malvern, British), transmission electron


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microscope (TEM, JEOL JEM-2100, Japan), zeta potential analyzer (Brookhaven, USA), termogravimetric analysis (TGA, Perkin-Elmer, USA), 1H-NMR spectroscopy (Bruker 500 MHz) and X-ray photoelectron spectroscopy (XPS) (PHI5000 VersaProbe, ULVAC-PHI Inc.) were used to investigate the physic-chemical characters of the obtained water-soluble QDs. In vitro Cell Study Cell culture The cells (U87MG, MCF-7 and L02) were cultured in DMEM medium supplemented with 10% (v/v) calf serum, penicillin (100 U/mL) and streptomycin (100 mg/mL) at 37 °C in a humidified atmosphere containing 5% CO2. Cytotoxicity evaluation The in vitro cytotoxicity of SPH-QDs was assessed preliminarily by the colorimetric MTT assay using L02, U87MG and MCF-7 cells. In brief, the cells were seeded onto 96-well plates (1× 104 cells per well) and cultured for 24 h. Next, the cells were incubated with samples at a wide concentration range from 10 µg/mL to 300 µg/mL for 24 h at 37 °C, and then 10 µL of MTT solution was added. After further incubation (4 h), the medium was gently discarded. Subsequently, DMSO (150 µL) was added into each well and the plates were gently shaken for 15 min for dissolution. At last, the absorption of sample at 570 nm was measured. According to the principle of MTT assay, the cell viability was calculated. In vitro Cell Imaging


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ανβ3-positive U87MG cells and ανβ3-negative MCF-7 cells were seeded in laser scanning confocal microscope (LSCM) culture dishes with a density of 5× 105 cells per well. Subsequently, the dishes were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 24 h until the whole cells occupied about 70% of the space at the bottom of culture dishes. For staining, 200 µL RGD-SPH-QDs buffer solution was added to cells for co-incubation 2 h. Then, the cells were washed by PBS for three times and imaged by LSCM. The red fluorescence of QDs was measured under laser light (λ=488 nm) excitation. In vivo Imaging in Tumor-bearing mice When the tumors (U87MG or MCF-7) grew to 100-150 mm3 in volume, 0.9% NaCl solution containing RGD-SPH-QDs (200 µL, ~10 µg/g of body weight) was injected into tumor-bearing nude mouse through the vena caudalis. After that, NIR fluorescence images were collected at certain time points within 72 h post-injection (P.I.) (before injection, initial background image of the nude mouse was taken). By using region of interest (ROI) functions of Scion Image software, the tumor to normal tissue (T/N) contrast ratio at each time points was calculated and used for the comparison of the targeting ability of the QDs-based nanocomposite in different tumorbearing mice further. All animal experiments were carried out in compliance with the Laboratory Animal Management Rules of the Jiangsu Provincial People’s Government (Document No. 45, 2008). The in vivo experimental protocols were approved by the Department of Science and Technology of Jiangsu Province and Jiangsu Association for Laboratory Animal.


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3. Results and discussion This work focuses on phase transfer and bio-application of the low cytotoxic oil-soluble quaternary ZAISe/ZnS QDs. As depicted in the overall route in Scheme 1, the design and synthesis of SPH and RGD-SPH polymers are presented in section 3.1, in which the main structural characterizations of polymers were measured. Next, ZAISe/ZnS QDs were transferred into water by ligand exchange with the SPH and RGD-SPH, and following self-assembled into QDs-clusters (section 3.2). The main physical and chemical properties of the as-prepared watersoluble QDs-clusters, including optical property, morphology characteristics and colloidal stability, were collected in this section. In section 3.3, using in vitro and in vivo optical imaging techniques, the targeting capability of QDs-clusters capped with RGD-SPH to tumor cells and tissues was further investigated. 3.1. Design and Synthesis of Ligand Exchange Agents Transferring oil-soluble QDs into water by ligand exchange procedure with well-designed molecules is an effective way to fulfill QDs with specific physiological functions and long-term colloidal stability in complex biological systems. Here, promising RGD-SPH polymer was designed as ligand exchange agent based on the facts that (i) histamine, which contained the imidazole ring, is an excellent anchor group for coordination to QDs with ZnS shell.36‒38 In addition, it is not prone to oxidation, less detrimental to QDs fluorescence, and do not interfere with standard bioconjugation reactions compared with traditional thiols anchors, (ii) sulfobetaine, which is the alternative of PEG, has been used as hydrophilic part to stabilize


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nanoparticles in water and shows excellent antifouling performance, such as more compact size, longer circulation time and better stability in undiluted blood serum.37‒41 Moreover, as inner-salt, sulfobetaine would drive nanoparticles for the subsequent self-assembly process, (iii) c-RGD peptide, which has high affinity for the ανβ3 integrin receptor, was chosen as the targeting molecule to promote the accumulation of the obtained nanocomposites in U87MG tumors,42,43 and (iv) poly (isobutylene-alt-maleic anhydride) (PIMA), was an excellent biocompatible backbone,44,45 which has a lot of active carbonyl groups to couple with the foregoing anchor groups, hydrophilic parts and targeted biomolecules together. Figures 1A and 1B summarize the general schemes employed to prepare two sets of imidazoleand sulfobetaine-modified polymers: one set is made of PIMA simultaneously coupled to sulfobetain groups and histamine anchoring groups, which is used as the transfer reagent to study the physic-chemical characters of the obtained water-soluble QDs. The other is modified with the targeted groups into the RGD-SPH polymer for biological experiments. (i) As shown in Figure 1A, SPH was easily synthesized by one-pot nucleophilic addition-elimination reaction between PIMA, sulfobetaine and histamine without catalysts. And sulfobetaine was synthetized according to the published method38 with a little modification (see the Experimental Section in Supporting Information and Figure S1, and mass spectrum and 1H-NMR spectrum were shown in Figures S2 and S3). In order to analyze the composition of polymer, SPH was characterized by 1H-NMR and the rough proportion of each monomer can be calculated by the integral value of H peak areas. As shown in Figure 1C, the molar ratio of sulfobetaine and histamine was about 1/2,


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which was speculated from H peak areas of the N-methyl (δ = 3.18) and imidazole (δ = 7.32 or 8.62). (ii) With the SPH in hand, we investigated its reaction with c-RGD peptide with the aim of producing the tumor targeting polymer. The c-RGD with the active amino was bonding with SPH via acetylation reaction (Figure 1B). 1H-NMR spectra in Figure 1D shows the characteristic peaks at 6.82 and 7.13 ppm of the protons of benzene ring, which indicated that the c-RGD was successfully linked into SPH polymer. From peak integration ratio between benzene, imidazole and N-methyl units, the experimental content ratio of c-RGD, sulfobetaine and histamine was 1/7.9/16.4, which is reasonably close to the theoretical values that originate from the initial molar ratio of precursors. The close matches of these percentages suggest that the numbers of functional units attached to the PIMA backbone can be simply controlled by the amount of precursors used within the coupling reactions. In addition, SPH and RGD-SPH were found to be easily soluble in water and warm dimethyl sulfoxide (DMSO), which make the possibility for the following phase transfer procedure. 3.2. Water Transfer of Hydrophobic Quaternary QDs via Ligand Exchange The low cytotoxic quaternary ZAISe/ZnS QDs have been synthesized by following published protocols.17,42,43 These QDs, exhibiting wide emission from 550 to 1000 nm, should have the potential in use of one probe for multi-scale bio-imaging. Because of the distinct structure between ZAISe/ZnS and traditional CdSe/ZnS QDs, the operation of directly exchanging the native QD surface ligands (1-dodecanethiol) was a little modified in this paper. The watersolubilization of oil-soluble QDs depends strongly on the ligand and the specific experiment


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method used.46 To circumvent the poor solubility of sulfobetaine-based polymers in majority of solvents other than water, the SPH polymer, as well as RGD-SPH polymer, was dissolved in small amount of DMSO prior to mixing with the CHCl3 solution of oil-soluble ZAISe/ZnS QDs. The mixture was stirred in round-bottom flask filled with nitrogen at 50 °C for 5 hours. After ligand exchange, the reaction mixture was blended with acetone/hexane (1/1) to precipitate the obtained hydrophilic QDs, and the initial native ligands also can be detected in the supernatant by the further mass spectrum analysis (data not shown). The obtained water-soluble QDs could be directly dispersed in phosphate buffer used for biological imaging without dialysis as usual, which greatly reduced the time and the cost of phase transfer. Subsequently, the obtained QDsbased nanocomposites were characterized by optical techniques, TEM, DLS, zeta potential analyzer, 1H-NMR and TGA. The absorption and photoluminescence (PL) spectra of ZAISe/ZnS QDs before and after water solubilization via ligand exchange with SPH polymer were measured and shown in Figures 2A and 2B. As shown in Figure 2A, the absorption spectrum of the obtained water-soluble QDs almost coincides with the initial one (oil-soluble QDs). The relative PL intensity of the aqueous QDs with respect to the initial hydrophobic dispersion was ~85%, and the emission peak of water-soluble QDs was at 740 nm, which has a little bathochromically shift (initial QDs, at 725 nm) (Figure 2B). The optical images of the corresponding samples under different excitation light sources are taken and shown in Figures 2C to 2E. These data not only demonstrate that the


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oil-soluble QDs can be transferred effectively into water by ligand exchange with SPH polymer, but also confirm that the main PL properties of initial QDs could be retained after phase transfer. Next, the shapes and sizes (or size distributions) of obtained SPH-QDs were further characterized by TEM and DLS. As shown in Figure 3A, the hydrophilic QDs self-assembled into spheroidal clusters in the buffer, and the average diameter of QDs-clusters is about 55 nm (Figure 3B), which is an effective size for the EPR effect in tumor treatment and imaging. Noticeably, we found that the molar ratio of sulfobetaine in polymer is the major determinant for the particle size in buffer of the obtained QDs-clusters. And the size of QDs-cluster can be tuned from 50 to 100 nm (Table S1). In order to study the dynamic process of self-assembly, the morphologies of initial hydrophobic QDs and obtained hydrophilic QDs dispersed in buffer at intermediate state were imaged by TEM. As shown in Figure 4A, initial oil-soluble QDs were monodispersed in hexane. However, Figure 4B showed two kinds of coexisted nanostructures: one is the nearly monodisperse water-soluble QDs and the other is QDs-clusters, which manifest stepwise growth into quasi-spherical QDs-clusters. As sulfobetaine endowing certain charge and water-solubility to QDs, the adjacent intermediate QDs will spontaneously assemble gradually, resulting in the formation of qusi-spherical QDs-clusters, which is the request of lowest energy principle. As mentioned above, the size of QDs-clusters capped with SPH is conducive to passive targeting for tumor cells by EPR effect. In order to further increase the tumor targeting, we introduced c-RGD to SPH polymer by chemical reaction. And then, the obtained RGD-SPH


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polymer was used as water transfer agent. Figure 5 showed the absorption and PL spectra of ZAISe/ZnS QDs before and after water transfer by RGD-SPH polymer. The data suggested that low content of c-RGD have little influence on the fluorescence properties of obtained QDs when it was linked with SPH polymer. In addition, TEM and DLS measurements of RGD-SPH QDs displayed the similar results with QDs capped with SPH (Figure S4). To characterize the contents of activated ligands on the particle surface, TGA analyses for SPH QDs-clusters and RGD-SPH QDs-clusters were performed to detect significant weight loss of the ligands from 30 °C to 350 °C at a rate of 10 °C/min under air (Figure S5). The first stage, the weight loss of ~16.71 wt% at ~57.5 °C of SPH QDs-clusters, can be attributed to removal the crystal water from the QDs-clusters, as well as a weight loss of 15.23 wt% at ~54 °C of RGDSPH QDs-clusters. The second stage, showing a weight loss of 3.62 wt% at 78.2 °C of SPH QDs-clusters and 4.11% of RGD-SPH QDs-clusters, can be attributed to the fracture of polymer from the QD-cluster surface, which could infer the approximate proportion of c-RGD (~0.48 wt%). Combined with the height of the H peak of the N-methyl (δ = 3.18), imidazole (δ = 7.32 or 8.62) and benzene (δ = 6.82 and 7.13) in 1H-NMR spectrum of polymer (Figure 1D), the roughly percentage of monomer in polymer can be calculated. As a result, each QD-cluster conjugated ~1.41 wt% of histamine and ~1.37 wt% of sulfobetaine. In line with our aim of producing robust nanocomposites to be employed in biological applications, the influence of several parameters, including storage time and pH values, on the colloidal stability and PL intensity was carefully inspected. The size distributions of RGD-SPH


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QDs-clusters were tested by DLS at various time points for 5 days storage (Figure 6A), and integrated zeta potential of QD dispersions in buffer spanning a 2-12 pH range was measured (Figure 6C). As the result, the particle size has a little fluctuation between 50-60 nm for 5 days storage. And the absolute value of QDs-clusters’ zeta potential was greater than 30 at the pH range 6-12, which indicated that the QDs-clusters capped with RGD-SPH were stable at phosphate buffer. Simultaneously, the influence of storage time and pH for relative PL intensities of the QD dispersions was collected. As presented in Figure 6B, the relative PL intensity of the obtained QDs-clusters dispersed in phosphate buffer maintained at ~70% steadily for 5 days storage. Meanwhile, the relative PL intensity decreased with the lowering of the pH values (Figure 6D). These data revealed that the colloidal stability and PL intensity of obtained QDsclusters capped with RGD-SPH have great tolerance to long time storage and widely pH range, such as the physiological environment. And it provides the possibility for the further imaging in vitro and in vivo. Mechanism of phase transfer: To explore the mechanism of water transfer, the freeze-dried powder of QDs-clusters capped with SPH polymer, as well as RGD-SPH polymer, was dissolved in D2O for the further 1H-NMR testing. The 1H-NMR spectrum collected from SPH QDs-clusters (Figure 7B) shows two distinct peaks at 7.18 and 8.49 ppm characteristic of the two protons in the imidazole ring of SPH polymer (Figure 7A), and these peaks are slightly shifted compared with those measured for the pure ligand. The similar results of RGD-SPH-QDs were observed in Figure 7C. These results were responsible for the change in environment following coordination


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to the QDs surface by dative bond between the lone pair electrons of nitrogen-atoms at the 1position of imidazole and unoccupied orbitals of zinc-atoms in the shell of ZAISe/ZnS core/shell QDs, which indicate that the phase transfer in this paper is ligand exchange rather than surface encapsulation. Universality of RGD-SPH as phase transfer agent: For further confirming the applicability of RGD-SPH for water-solubilizing oil-soluble quaternary QDs by ligand exchange, hydrophobic ZnCuInSe/ZnS (ZCISe/ZnS) QDs with the similar structure to ZAISe/ZnS QDs were used. The similar data were observed and given in Figures S6 and S7. Noteworthily, in this study, RGDSPH ZCISe/ZnS QDs-clusters were not used for bio-imaging. Mechanism of self-assembly: We speculated the possible causes of the self-assembled hydrophilic QDs-clusters and summarized them below. (i) Surface charge: As the different molar ratio of sulfobetaine in polymer, the diameter of QDs-clusters existed obvious difference. Specifically, with the ratio of sulfobetaine decreased, the mean diameter of QDs-clusters is becoming smaller because of the weaker electrostatic interaction between each QDs, as well as QDs and solution. (ii) Solubility: The acidity and basicity of the solution was the second reason for the dispersion performance of the obtained QDs-clusters. The QDs-clusters capped with SPH polymer, as well as RGD-SPH polymer, were hardly soluble in the buffer at pH lower than 2. And the absolute value zeta potential of those QDs in the solution at pH range from 2-5 were below 20 (Figure 6C), which is not conducive to the colloidal stability. However, when in the buffer at pH more than 6, the particles were dispersed in solvent rapidly, as the carboxyl groups


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of PIMA will deprotonate under such condition. (iii) The structure of the core/shell QDs: The quaternary ZAISe/ZnS QDs, as well as ZCISe/ZnS QDs, used in this paper have many distinctions compared with the conventional CdSe/ZnS. These differences are not only reflected in the elemental, but also endow those QDs with specific structure. More concretely, the traditional CdSe/ZnS QDs has complete core/shell structure, and ZnS as a shell in the outside of the QDs thoroughly isolated and enwrapped the interior CdSe. However, ZnS in the quaternary ZAISe/ZnS QDs is probably just an extension part of ZAISe, which makes inside ZAISe has an opportunity to contact external solvent. 3.3. Multi-scale Bio-Imaging with Water-Soluble RGD-SPH QDs-clusters. The cytotoxicity of QD-based nanocomposites is extremely important for their future bioapplications.1,13,14 To evaluate the cytotoxicity at cell level, L02, U87 and MCF-7 were incubated with RGD-SPH ZAISe/ZnS QDs-clusters, and then the cell viability was investigated by MTT assay (Figure S8). As shown, these cell lines all retained high survival rate after longterm (24‒48 h) incubation with QDs-clusters (the cell viability at the highest concentration (300 µg/mL), more than 60%). Subsequently, using the RGD-SPH ZAISe/ZnS QDs-clusters for in vitro cell and in vivo small animal imaging was investigated further to evaluate the tumor targeting capability. After intravenous injection of QDs-clusters, the mice did not show obvious unusual behavior, and had the normal survival state even after one week feeding. These observations demonstrated that RGD-SPH ZAISe/ZnS QDs-clusters are relatively safety in biological applications, while more detailed experiments to assess their cytotoxicity are needed47.


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To explore the cell affinity and the potential for in vitro optical imaging of RGD-SPH QDsclusters within various cell lines, ανβ3 integrin receptor-positive U87MG cells and ανβ3 integrin receptor-negative MCF-7 cells were selected for co-incubation with these nanocomposites. And the red fluorescence (λex = 488 nm) from the QDs-clusters was captured by LSCM respectively (Figure 8). After 2h of incubation, pale red fluorescence is observed in MCF-7 cells as the lower expression of the ανβ3 integrin receptor (Figure 8B), while the stronger signal exhibits in U87MG cells at the same time (Figure 8A: cRGD co-mediated endocytosis enhanced the cell uptake of the QDs-clusters). At the same time, these images also indicate the obtained highly fluorescent QDs-clusters should be an interesting targeted probe for in vitro cell microscopic imaging. Owning to NIR fluorescence with the deeper penetration in tissue, the broad spectrum fluorescent RGD-SPH ZAISe/ZnS QDs-clusters were explored for in vivo imaging. By the aid of tumor-bearing mice, the tumor specificity of QDs-clusters with different sizes was studied. Figure S9 reveals that QDs-clusters with the average size of ~55 nm possess a relatively significant tumor-targeting ability. In order to evaluate tumor imaging ability further, RGD-SPH QDs-clusters were injected in nude mice via vein, which were born with the ανβ3 integrin receptor-positive U87MG tumor or the ανβ3 integrin receptor-negative MCF-7 tumor. Next, for tracking the biodistribution of the nanocomposites, the NIR fluorescence images at different times were taken and shown in Figure 9. After 5 min P.I., bright fluorescence arose in the whole body of the mice. In Figure 9A, at 1h P.I., the recognizable fluorescence was observed in U87MG-tumor; the fluorescence signal reached optimal T/N contrast ratio at 4‒8h P.I. However,


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the recognizable fluorescence in tumor of the MCF-7 mice present until 8h P.I., which could be attributed to the EPR effect and the appropriate particle size of QDs-clusters (Figure 9B). Those all revealed that RGD-SPH QDs-clusters have the fast and effective tumor-targeting ability for the ανβ3 integrin receptor-positive U87MG tumor, which should give the credit to the active targeting effect of c-RGD and passive targeting effect (EPR), namely dual-targeting effects. And it also can be inferred through the significant difference at the T/N(muscle) ratio in different tumor-bearing nude mice (Figure 9C). At the same time, the obvious fluorescence at chest side could be seen, which is due to the liver enrichment of fluorescent RGD-SPH QDs-clusters (i.e., the uptake by RES20,47,48). The ex-vivo fluorescence imaging of different organs harvested from the tumor-bearing mice in Figure S10 also support the above observations obtained by in vivo noninvasive method. Here, we observed that surface modification and size optimization of nanocomposites are helpful to enhance the T/N ratio, although it is difficult to remove the liver enrichment of nanocomposites.20,48 For confirming the long-term imaging for tumor, the observation time of U87MG tumorbearing mice was extended. The fluorescence signal remains bright for 60h P.I. to distinguish the tumor tissues in mice obviously (Figure S11), which is helpful to long-term detection for tumor in vivo. In other word, the RGD-SPH QDs-clusters will be able to work as a tumor-targeted drug carrier with the function of NIR imaging for tumor treatment with sustaining over a longer period at high concentration.


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4. Conclusions In summary, we have developed biocompatible QDs-clusters with excellent water-solubility and precise size control through two processes: ligand exchange and self-assembly. The phase transfer agent was designed combining the imidazole anchoring group with the hydrophilic sulfobetain and synthesized by nucleophilic addition-elimination reaction. After ligand exchange, the obtained QDs kept the optical properties of initial QDs and self-assembled into QDs-clusters based on electronic coupling between QDs, and their size can be tuned from 50 to 100 nm by using the polymer with the different content of sulfobetaine. By multi-scale optical imaging techniques, these QDs-clusters have been confirmed to be a potential fluorescent probe for versatile tumor bio-imaging with good cellular uptake and high tumor accumulation due to the active targeting effect of c-RGD and passive targeting effect of EPR effect. This study also is interesting for exploiting a stagey to fabricate biocompatible QDs-clusters to improve the watersolubility of hydrophobic quaternary QDs and overcome the drawback of the existing ligand exchange method for biomedical applications.


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Figure 1. Synthetic schemes for (A) SPH polymer and (B) RGD-SPH polymer. 1H-NMR spectra of (C) SPH polymer and (D) RGD-SPH polymer. The inset of panel D was the enlarged image of the characteristic H peak of benzene ring in c-RGD and imidazole ring in histamine.


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The initial oil-soluble QDs The resultant QDs capped with SPH

0.8 0.6 0.4 0.2 0.0 500

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Wavelength (nm)

1000

1100

B1000 PL Intensity (a.u.)

A1.0 Absorbance

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The initial oil-soluble QDs The resultant QDs capped with SPH

800 600 400 200 0 500

600

700

800

Wavelength (nm)

900

Figure 2. (A) Absorption and (B) PL spectra (λex = 450 nm) of ZAISe/ZnS QDs before and after phase transfer by ligand exchange using SPH polymer. The true-color photographs of initial hydrophobic QDs and as-prepared hydrophilic SPH QDs captured under (C) room light and (D) UV light (λmax = 365 nm) excitation, and (E) the pseudocolored photographs captured under NIR laser light (λmax = 660 nm). These data confirm all samples before and after water transfer having the bright PL emission.


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B100 Particle Counts

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80 60 40 20 0 0

20

40

60

80

100

Hydrodynamic Diameter (nm)

Figure 3. (A) TEM image of obtained SPH ZAISe/ZnS QDs-clusters. (B) Size distribution of SPH ZAISe/ZnS QDs-clusters measured by DLS.

Figure 4. (A) TEM image of oil-soluble ZAISe/ZnS QDs. (B) TEM image of the intermediate state of the SPH ZAISe/ZnS QDs-clusters dispersed in buffer before reaching steady-stage. The incubation time is about 30 min.


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The initial oil-soluble QDs The resultant QDs capped with RGD-SPH

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The initial oil-soluble QDs The resultant QDs capped with RGD-SPH

800 600 400 200 0 500

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Figure 5. (A) Absorption and (B) PL spectra (λex = 450 nm) of ZAISe/ZnS QDs before and after

Hydrodynamic Diameter (nm)

water transfer by RGD-SPH polymer. 100

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B PL Intensity (%)

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0

-10 -20 -30 -40 -50

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3

4

5

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2

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D

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Figure 6. Temporal dynamics of (A) size distribution and (B) relative PL intensity of RGD-SPH ZAISe/ZnS QDs-clusters stored in phosphate buffer. (C) Zeta potential and (D) relative PL


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intensity of RGD-SPH ZAISe/ZnS QDs in PBS (pH range, 2 to 12). Remark: the relative PL intensity was normalized with respect to the value of the initial oil-soluble QDs.

Figure 7. 1H-NMR spectra of initial (A) SPH polymer and resultant hydrophilic ZAISe/ZnS QDs-clusters capped with (B) SPH or (C) RGD-SPH. The inset of each panel was the structure of SPH polymer, as well as the QDs-clusters capped with SPH and RGD-SPH corona. And the inset of panel B was the enlarged image of the characteristic H peak of imidazole ring.


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Figure 8. The typical LCSM photographs of (A) αυβ3-positive U87MG cells and (B) αυβ3negative MCF-7 cells co-incubated with RGD-SPH ZAISe/ZnS QDs-clusters (λex = 480 nm). These photographs were captured after 2h of co-incubation.

C 3.0 Tumor/Normal Ratio

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U87 MG MCF-7

2.5

2.0

1.5

1.0 5min

1h

2h

4h

Time

8h

12h

Figure 9. Dynamic distributions of RGD-SPH ZAISe/ZnS QDs-clusters in nude mice bearing (A) αυβ3-positive U87MG tumor or (B) αυβ3-negative MCF-7 tumor, detected by the NIR imaging system (λex = 660 nm), and the tumor site is marked by a white circle. (C) Corresponding evolution of T/N(muscle) ratios of RGD-SPH QDs-clusters in U87MG and MCF7 tumor-bearing nude mice with time.


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Scheme 1. Overall synthetic scheme for water-soluble RGD-SPH ZAISe/ZnS QDs-clusters, and their applications for bio-imaging. ASSOCIATED CONTENT

Supporting Information: Detailed experimental procedures and additional data (synthesis and structure characterization of sulfobetain, TEM images, TGA analysis, MTT assay, etc.) are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (D. Deng)

*E-mail: [email protected] (D. Huang) ORCID：Dawei Deng: 0000-0002-1391-1845

Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (81371627 and 81220108012), the Program for New Century Excellent Talents (NCET-12-0974) in University of the Ministry of Education of China, A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Graduate Innovation Foundation of Huahai Pharmaceutical (1010110002).


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Abstract Graphic:


39

ZnS QDs with Tumor

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