Quantum Dots-Based Multifunctional Nano-Prodrug Fabricated by

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Biological and Medical Applications of Materials and Interfaces

Quantum Dots-Based Multi-Functional Nano-Prodrug Fabricated by Ingenious Self-Assembly Strategies for Tumor Theranostic Tao Deng, Jie Wang, Yunyan Li, Zhihao Han, Yanan Peng, Jie Zhang, Zhen Gao, Yueqing Gu, and Dawei Deng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08512 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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ACS Applied Materials & Interfaces

Quantum Dots-Based Multi-Functional NanoProdrug Fabricated by Ingenious Self-Assembly Strategies for Tumor Theranostic

Tao Deng, † Jie Wang, † Yunyan Li, † Zhihao Han, † Yanan Peng, ‡ Jie Zhang, † Zhen Gao,*, § Yueqing Gu, †,‡ and Dawei Deng *,†,‡

†

Department of Pharmaceutical Engineering, and ‡ Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, Nanjing 211198, China §

College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211816, China.

Keywords: Quantum dots, water transfer, self-assembly, Host-Guest chemistry, nanomedicine.

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ABSTRACT: The rapid developments of quantum dots (QDs) based nano-agents for imaging tumor and tracking drug delivery have been proven to be reliable nano-diagnostic techniques. Although, abundant types of QD nano-agents have been developed for fighting against cancer, it still is a challenge to control their quality and achieve prefect repetition, due to the complicated synthetic steps. The precise intermolecular self-assembly (SA) may afford a facile and low-cost strategy for this challenge. Herein, a pH and H2O2 dual-sensitive Sb-CDDOX molecule was designed to construct QD-based theranostic prodrug (named as Sb-CDDOX-ZAISe/ZnS) via Host-Guest strategy (1st SA strategy), in which QDs water-transfer and drug-uploading were integrated well. That is, the nano-prodrug (NPD) inherited highly luminescent properties from “Host” QDs for bio-imaging, as well as environment sensitivities from “Guest” Sb-CD-DOX for drug-release. Experimental results indicate that the Sb-CDDOX-ZAISe/ZnS exhibited effectively passive tumor-targeting and could provide clear imaging for malignant tumors in metaphase or advanced stages; meanwhile, after coating with folic acid (FA) through electric attraction (2nd SA strategy), the final Sb-CD-DOXZAISe/ZnS@FA NPD showed expected pH-controlled negative-to-positive charge reversal ability, and better curative effect compared with free DOX. Hence, fabricating nanocomposites by highly efficient self-assembly strategies is favorable towards inorganic nanoparticles-based prodrug delivery system for tumor-targeting theranostic.


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1. Introduction In the last two decades, nanotechnology has tremendously impacted the field of medicine through advances in drug delivery. Nanotechnology-based drug delivery aims to target the drug payload to the right place, at the right time, at the right (optimal) dose. However, the poor quality-control and low repetition of nanoparticles hindered their clinical applications.1,2 Some nature nanocomposites, such as proteins and DNAs, exhibited complex and elaborate tertiary structures formed via self-assemblies driven by hydrogen bonding, which inspired an interesting way for controlling the quality of artificial nanoparticles.3 As gradually deepening the understanding of nanomaterials, researchers found that other intermolecular forces, such as “Van der Waals' force” and “electrostatic interaction”, etc., could also mediate selfassemblies to fabricate high-quality nanoparticles.4,5 With the development of nanotechnology, scientists attempted to construct nanodrug via inorganic-organic co-assembly, in which inorganic nanoparticles could endow as-prepared nanocomposites with multifunction besides drug loading, including magnetic, heating and fluorescence effects.6-8 Among various inorganic nanoparticles (metal, semiconductor and metallic oxide), quantum dots (QDs) have arguably affected bioimaging or drug delivery research more than any other nanomaterials, due to their outstanding optical and physicochemical properties.9 However, poor water-solubility and disappointing biocompatibility of the high-quality hydrophobic QDs limited their biological applications.10-12 It has been reported that water transfer with hydrophilic polymer encapsulation11,

13

and ligand exchange12,

14

will provide promising

potential to overcome the hydrophobicity barrier and improve the biological properties of bare uncoated QDs. Among diverse hydrophilic molecules, the active moieties-decorated surface coronas will endow QD-based nanocomposites with many attractive biological functions, such as excellent biocompatibility, long-circulation, and active tumor-targeting.15,16 However,



the generally used surface modifications will to a certain extent reduce the fluorescence intensity of QDs, and their particle sizes were often increased considerably.17 In addition, it is extremely difficult to build QD-based nano-prodrug (NPD) with a fixed repetition and controlled quality using the as-prepared water-soluble QDs because of the following tedious and uncertain drug uploading processes.15, 18 Ingenious self-assembly strategies, driven by controllable chemical or physical interactions, provide promising platforms to build multi-functional nanoparticles in many fields.19 For the construction of medicine, using specific hierarchical self-assembly methodologies, such as host-guest20 and electrostatic interaction21,22, etc., might help to achieve the quality control of NPDs from an implementation standpoint. Host-Guest chemistry is a rising surface modification method between the surface ligands of nanoparticles (Host) and the hydrophilic polycyclic macromolecules (Guest), which is associated with richer amphiphile topologies and more facile chemical synthesis than traditional covalent amphiphiles.23-25 More importantly, Host-Guest chemistry could only modify the “Guest” molecule, which has few effects on the physical and chemical properties of “Host” nanoparticles generally.20, 26 That is, this repeatable water-solubilizing strategy could provide the compact biocompatible QDs with strong fluorescence for the following bio-applications. It is worth noting that phasetransferring QDs using “Guest” prodrug molecules, which are constructed by linking the drugs to polycyclic molecule via sensitive chemical bonds, is an effective method to control drug loading process on molecular level. Meanwhile, electrostatic (ion-ion, charge−charge) interaction strategy is also an interesting way to fabricate nanocomposites with desired surface charge properties.21,22,27 Moreover, supramolecular nanostructures constructed by these reversible noncovalent interactions have the abilities to undergo dynamic switching of structure, morphology, and function in response to various external stimuli, which endow them with outstanding potential applications in drug delivery. 4 ACS Paragon Plus Environment

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Herein, we reported the successful construction of a pH and H2O2 dual-sensitive QD-based NPD via ingenious hierarchical self-assembly (SA) strategies, which was formed from biocompatible “Guest” α-cyclodextrin (α-CD) based prodrug and alkyl chain-modified “Host” ZnAgInSe/ZnS (ZAISe/ZnS) QDs (the 1st SA strategy), and then coated with anionic folic acid (FA) (the 2nd SA strategy). That is, the obtained NPD achieved water-solubilizing of QDs and uploading of anticancer drug (DOX), synchronously. (Scheme 1) The obtained Sb-CDDOX-ZAISe/ZnS NPD inherited highly luminescent properties and environment sensitivities, which was able to image tumors and release DOX at malignancy sites in vivo, efficiently. Notably, tumor imaging experiments in mice indicated that tumor size (or tumor course) affected the passive tumor-targeting of NPD (the early tumor with small size showed nonaccumulation). Furthermore, after overcoating with FA through electric attraction, the final Sb-CD-DOX-ZAISe/ZnS@FA NPD exhibited the admirable surface charge reversal capability, and showed the better curative effect than free DOX in animal models. Hence, hierarchical self-assembly strategies provided an interesting way to fabricate multi-functional NPDs for tumor theranostic.

2. Experimental Section 2.1. Materials. Oil-soluble ZnAgInSe/ZnS core/shell QDs was prepared according our previous reports (For this quaternary nanostructure, Ag, In, and Se are the basic elements, in which Zn doping affords QDs with higher PL quantum yields (QY) and broader PL spectral range. Meanwhile, ZnS shell overcoating will improve PL QY and photostability further).28-30 α-cyclodextrin (α-CD, 98%), triethylamine (TEA, 99.5%), 4-nitrophenyl chloroformate (4-NC, 98.0%), hydrazine hydrate aqueous solution (80%), doxorubicin hydrochloride (DOX·HCl, 98%), trifluoroacetic acid (TFA, 99.5%), 1,3-propane sultone (99%), ditert-butyldicarbonate (BOC, 99%), N,N-dimethylamino proylamine (DMPDA, 99%), and 3-(4,5-dimethylthiazol-25 ACS Paragon Plus Environment


yl)-2,5-diphenyltetrazolium bromide (MTT), dimethylformamid (DMF, 99.5%) and dimethyl sulfoxide (DMSO, 99%) were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. Human malignat glioma cell line (U87MG) and mouse hepatoma H22 cell line were obtained from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Healthy male ICR mice (18-20 g) were purchased from KeyGEN Biotech. Co., Ltd., Nanjing, China. 2.2. Synthesis of Sb-CD-DOX Segment. Sulfobetaine-α-cyclodextrin (Sb-CD): Sulfobetaine (0.9 g, 4 mmol) and EDCI (0.79 g, 6 mmol) were dissolved in DMSO (20 mL) for 15 min; DMAP (0.067 g, 6 mmol) was added for another 15 min to catalyze. Then, 1 mL DMSO solution containing α-cyclodextrin (1.35 g, 1.38 mmol) was added to the reaction system and stirred for 24 h under nitrogen. After reaction, the product was precipitated by addition of acetone and dried under vacuum as white powder with viscosity (1.7 g, ~75% yield). Sb-CD activated by 4-nitrophenyl chloroformate: Sb-CD (1g, 0.6 mmol) and triethylamine (0.32g, 3.2 mmol) were dissolved in dry DMF (20 mL), and then the mixture was charged into a 100 mL three necked flask and stirred for 30 min in ice bath. 10 mL dry dichloromethane (DCM) solution of 4-nitrophenyl chloroformate (0.67g, 3.3 mmol) was added into a 25 mL dropping funnel. Subsequently, the DCM solution was dropped into the DMF mixture slowly under nitrogen. The resulting mixture was stirred in ice bath for 2 h, and then allowed to stir at room temperature for 24 h. After reaction, the product was precipitated by addition of aether and dried under vacuum as white powder (1.2 g, ~76.9% yield). Sb-CD-hydrazide: Hydrazine hydrate aqueous solution (0.102g, 2 mmol) was added into DMF solution of the activated Sb-CD (0.3g, 0.15 mmol). The mixture was left to stir for 24 h at 50 °C. After the dot of activated α-cyclodextrin disappeared by TCL monitor, the resultant 6 ACS Paragon Plus Environment

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mixture was concentrated and the product was obtained by addition of aether (0.23 g, ~76.7% yield). Sb-CD-doxorubicin: Sb-CD-hydrazide (65.9 mg, 0.03 mmol) and doxorubicin (100mg, 0.18 mmol) were dissolved in 5 mL of DMSO. After stirring for 5 min, a drop of trifluoroacetic acid was added into the flask. Then, the mixture was allowed to react in the dark for 48 h at room temperature. The crude product was isolated by extensive dialysis against distilled water (MWCO 1000) for 2 days and lyophilization. NOTE: The similar procedure was used to prepare CD-DOX, besides replacing the Sb-CD with α-cyclodextrin. 2.3. Phase Transfer of Oil-Soluble ZnAgInSe/ZnS QDs by Host-Guest Chemistry. The phase transfer was conducted by ultrasound assisted stirring the mixture, namely, the chloroform suspension of oil-soluble ZnAgInSe/ZnS QDs (50 µL) and the aqueous solution of Sb-CD-DOX (α-CD, or CD-DOX) (the concentration of α-CD-based molecule was equivalent with 10 mM/L α-CD) under room temperature. After chloroform volatilization, the aqueous layer was collected and centrifuged twice to obtain a transparent suspension, which was used in the characterization and the further application. 2.4. Preparation of FA Coating Complex. FA (5 mg) was dissolved in Tris buffer (pH = 9.0, 10 mL). After dissolving completely, FA solution was mixed with the Sb-CD-DOX-ZAISe/ZnS complex solution by pipetting thoroughly and stirring for 10 min at room temperature (Scheme 1). In this work, the FA coating complex was constructed at various theoretical charge ratio, such as FA/Sb-CD-DOXZAISe/ZnS = 10:1. 2.5. Drug Release from the Prodrug Nanocomposite. Sb-CD-DOX-ZAISe/ZnS@FA NPD was dissolved in DMEM medium (pH = 7.4). Then, the nanocomposite solution (2 mL) was transferred to a dialysis bag (MWCO = 3.5 kDa) and 7 ACS Paragon Plus Environment


immersed in a centrifuge tube containing 10 mL DMEM medium (pH = 7.4 or 5.0) without or with 1 mmol H2O2 under constant shaking at 37 °C in dark. At each designated time point, 1 mL of liquid was sampled from the out solution and then replaced with the same volume of DMEM medium. The collected release medium was used for fluorescence measurement to determine DOX concentration against calibration curve. Experiments were performed in triple. 2.6. Characterization. The 1H NMR spectra were measured on a Bruker DMX500 spectrometer operating at 300 MHz using CDCl3 or DMSO-d6 as the solvent. And the NOSEY spectrum was obtained using Bruker DMX500 spectrometer (500 MHz). UV-Vis absorption spectra were recorded with a Lambda 25 UV-Vis spectrophotometer (Perkin Elmer, America). PL spectra were recorded by a LF-1204009 fluorescence spectrophotometer (Thermo Fisher Scientific, South Korea). All optical measurements were performed at room temperature. The size and ζ-potentials measurements of Sb-CD-DOX-ZAISe/ZnS@FA NPDs were characterized by Mastersizer 2000 Laser Particle Size Analyzer (LPSA, Malvern, British). In addition, the TEM images of the samples were taken on a JEOL JEM-2100 transmission electron microscope with an acceleration voltage of 200 kV (Carbon coated copper grid was dipped in sample solution to deposit it on the film). The absorbance for the MTT assay was measured with a Tecan’s Infinite M200 microplate reader at a wavelength of 490 nm. Finally, the fluorescence images of cells were obtained by Olympus Fluoview 300 confocal laser scanning system. 2.7. In Vitro Cell Study. Cell Culture: The cells (U87MG, L02 and H22) 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. Remark: the H22 cells were obtained from the mouse cancerous ascites.


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Cytotoxicity Evaluation: The in vitro cytotoxicity of prodrug nanocomposites was assessed preliminarily by the colorimetric MTT assay using L02, and U87MG cell lines. Typically, the cells were seeded onto 96-well plates (1 × 104 cells per well) and cultured at 37 °C in a humidified incubator with 5% CO2 for 24 h. Then, the cells were incubated with sample solutions at a wide concentration range from 10 µg/mL to 300 µg/mL for another 24 h at 37 °C. The cells were rinsed using fresh DMEM medium, followed by incubation with 20% MTT (10 µL) at 37 °C for 4 h. After the medium was discarded, DMSO (150 µL) was used to lyse the cells. The absorbance at 570 nm was recorded by microplate reader. Every experiment was carried out quintic, and the cell viability was calculated by referring to the control group without the sample treatment. In Vitro Cell Imaging: The cell imaging experiment was carried out using human U87MG cells and mouse H22 cells. In brief, these cells were seeded in laser scanning confocal microscope (LSCM) culture dishes, and incubated at 37 °C in a humidified incubator with 5% CO2 until the whole cells occupied about 70% of the dish bottom space. Subsequently, SbCD-DOX-ZAISe/ZnS@FA DMEM solution (200 µL) was added to cells for co-incubation 2 h to stain. Before imaged by LSCM, the cells were co-incubated further with Hoechst 33342 for another 30 min to stain nuclei. At last, the cells were washed by fresh DMEM for three times, and then measured under laser light (λex = 405, or 488 nm) excitation. 2.8. In Vivo Study. Establishment of H22 tumor model: H22 tumor model was built up by subcutaneous injection of H22 cells (obtained from the mouse cancerous ascites) into the axilla of each ICR mouse (4−6 weeks old, 18−20 g). After growing for 2 weeks, the tumor volume reached approximately 50−70 mm3. Remark: The animal experimental protocols were approved by the Department of Science and Technology of Jiangsu Province and Jiangsu Association for Laboratory Animal. All 9 ACS Paragon Plus Environment


animal experiments were carried out in accordance with the Laboratory Animal Management Rules of the Jiangsu Provincial People’s Government (Document No. 45, 2008). In Vivo Fluorescence Imaging: 5% glucose injection (200 µL) containing prodrug nanocomposites (100 µg) was injected into mice (n ≥ 3) with different volume of tumors in bilateral armpits (tumor in left is larger than the right one, as the mouse model was constructed with different inoculability time of H22 cells) through the vena caudalis. Then, the mice were imaged at predetermined time intervals (0-72 h) post-injection (P.I.) with a homemade in vivo small animal NIR imaging system 31, and the background image was taken before injection. NOTE: The similar procedure was used to trace the distribution of NPD in normal mice. Furthermore, the mouse was sacrificed to measure the enrichment of the NPD in liver using 7700x quadrupole ICP-MS (Agilent Technologies, Tokyo, Japan). In Vivo Antitumor Experiment: When the volume of H22 tumor tissues reached approximately 50−70 mm3, the tumor-bearing mice were randomly divided into six groups (n = 5 for each group) and were treated by intravenous injection with glucose solution, CDZAISe/ZnS, DOX·HCl, CD-DOX-ZAISe/ZnS, Sb-CD-DOX-ZAISe/ZnS, and Sb-CD-DOXZAISe/ZnS@FA respectively. Each formulation was injected via the tail vein at an equivalent dose of 5 mg/kg (DOX/bodyweight) at a 2-day interval. The tumor size and body weight were measured every day during the treatment (Tumor volume (mm3) = width2×length⁄2). After 15 days treatment, the mice were sacrificed to separate the liver, heart, spleen, lung, kidney and tumor tissues. After washed with PBS solution, these isolated organs were fixed in 10% formaldehyde for histological examination. 2.9. Histology Examination. Histology analysis was carried out at the 15th day after the treatment. Typical heart, liver, spleen, lung, kidney and tumor tissues of the mice in the control group and the best treatment 10 ACS Paragon Plus Environment

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group were isolated. Then, the organs were dehydrated using buffered formalin, ethanol with different concentrations, and xylene. After that, they were embedded in liquid paraffin. The sliced organs and tumor tissues (3-5 mm) were stained with hematoxylin and eosin (H&E), and examined by a microscope.

3. Results and discussion This work focuses on tumor theranostic using hierarchical self-assembled QDs-based NPDs. As the depiction in the overall route in Scheme 1, the construction of the dual-sensitive SbCD-DOX-ZAISe/ZnS via Host-Guest chemistry (the 1st SA strategy) was presented in section 3.1, which describes the design, characterizations, and sensitive drug release of the NPD in detail. Subsequently, the obtained NPDs were used to image tumor (H22) tissues in vivo by optical imaging techniques (section 3.2). In this section, the EPR effects of the tumors having different sizes were also investigated. Finally, the Sb-CD-DOX-ZAISe/ZnS was further modified with anionic FA ligand via electrostatic interaction (the 2nd SA strategy) to construct Sb-CD-DOX-ZAISe/ZnS@FA NPD with pH-controlled surface charge reversal ability, and the antitumor effect was studied in section 3.3.

3.1. Fabrication of the pH and H2O2 Dual-Sensitive Host-Guest Nanoparticle System The differences of cell environments between cancer cells and their normal counterparts provide an opportunity to prepare smart drug nanocarriers,32,33 which can control drug release at special time and space on demand, and be achieved with a “zero release” effect in blood circulation to protect healthy tissues from toxic drug. Supramolecular nanotechnology has been recognized as a significant approach in the design of drug delivery systems.19 The efficient assembly via “Host-Guest interaction” is crucial to supramolecular nanotechnology for synthesis of supramolecular system due to the simple synthesis procedure, dynamically 11 ACS Paragon Plus Environment


tunable properties, and even easy implementations of functional groups and new stimulus properties.20 On account of the lower pH value and higher concentration of H2O2 in tumor microenvironment,34,35 a dual-sensitive nanoparticle system was designed via Host-Guest strategy. 3.1.1. Construction of Sensitive Host Parts: α-Cyclodextrin Derivatives. α-Cyclodextrin (α-CD), a low toxicity seminatural compound, possesses a hydrophilic exterior surface and hydrophobic interior cavity on the truncated cone. Owing to its excellent inclusion ability, α-CD is commonly used in the design and construction of supramolecular structures. Modification of α-CD using polymers is popular in drug delivery systems. These polymers were usually grafted to the exterior surface of α-CD to provide a platform with multi-function, for instance enhancing molecular stabilization, inducing sensitive trigger, etc.20, 26 Generally, modification strategies have only minor influence on the cavity, which still has the “Host” characteristics to encompass “Guest” molecular. In this work, an α-CD based “Guest” molecule with long circulation time was obtained by linked with sulfobetaine (Sb) and DOX into CD ring using ester and hydrazide (a sensitive functional group for acidic pH and H2O2) bonds (Figure 1, the corresponding 1H NMR spectra of intermediate molecules were shown in Figure S1). 1H NMR spectrum (Figure 1B) of the as-prepared Sb-CD-DOX showed the appearance of the aromatic signals between 7 and 8 ppm. The integration ratio of the peak “r” and the peaks corresponding to b,c-positioned hydroxyl groups of the α-CD ring (5.32 ppm) were approximately 1:4, suggesting that about three DOXs had been uploaded into one parental α-CD (the drug content of CD-DOX was calculated to be 45.32%; the ratio of DOX:α-CD could be controlled by initial molar ratio of precursors). According to the steric hindrance, the optimal spatial relationship among DOX molecules in α-cyclodextrin should be meta-position. And the integral area of methyl peaks “m” at around 2.9 ppm was about 1.5 folds to b,c-positioned hydroxyl groups, indicating that almost all of the residual three 12 ACS Paragon Plus Environment

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hydroxyls of maternal CD ring had been connected to Sb (the ligand content was about 25.8%). 3.1.2. Water Transfer of Oil-Soluble ZAISe/ZnS QDs via Host-Guest Strategy. Multi-functional nanodrug systems constructed with inorganic nanoparticles and organic molecules have attracted wide attention recently, as they could introduce fluorescence, heat, magnetism, etc. besides delivering drugs.36 Supramolecular nanoparticles constructed using “Host-Guest chemistry” could retain the original surface ligands of inorganic nanoparticles, and thus keep their initial excellent functions. The formation of the Host-Guest complex requires combination of several elemental noncovalent interactions such as hydrophobic interactions and geometric fittings within the interaction structures.20 It has been proven that the cavity structure of α-CD could accommodate the aliphatic chain of oleic acid, resulting in transferring the oleic acid stabilized nanoparticle (Fe3O4) into water.24 Our previous studies have revealed that the surface ligands of ZAISe/ZnS QDs, a versatile probe for multiscale bioimaging, were similar to oleic acid.28 Herein, α-CD was used to water-solubilize hydrophobic ZAISe/ZnS QDs, according to the matching degree between its hydrophobic cavity and the surface alkane chains of QDs.20 Remarkably, the efficiency of water transfer was positive correlation with the initial α-CD concentration. And when the “guest molecule” concentration reached 10 mM/L, the saturated water transfer phenomenon occurred (Figure S2). Interestingly, using ultrasound instead of the published stirring method, 24 the process of phase transfer has been shortened from 10 h to 6 min. (Figure 2C) Due to the concentration dependent phase transfer, the dosages of α-CD based guest molecules used in this paper were equal to 10 mM/L. Figure 2 showed the absorption and photoluminescence (PL) spectra of ZAISe/ZnS QDs before and after water transfer using SbCD-DOX. Compared with initial QDs, after water solubilization, the absorption spectra (Figure 2A) contained wide absorption of ZAISe/ZnS from 400 nm to 785 nm and 13 ACS Paragon Plus Environment


characteristic absorption peaks of DOX (495 nm). However, the PL spectra (Figure 2B) showed that the as-prepared Sb-CD-DOX-ZAISe/ZnS emitted the strong fluorescence mainly inherited from oil-soluble QDs but no characteristic PL emission of Sb-CD-DOX (PL peak = 595 nm). In addition, the intensity of PL emission dropped slightly (~15%) after water transfer, which revealed that the FRET effect is naturally present in this Host-Guest nanoparticle system. FRET phenomenon is an energy transfer process, which is extremely enslaved to the spacing between donor and acceptor.37 For intensively validating FRET effect between DOX and ZAISe/ZnS QD, a contrast solution, containing water-soluble ZAISe/ZnS QDs treated with α-CD and free Sb-CD-DOX molecule, was designed. The absorbance curve (Figure S3A) of the mixed solution was smoother than the Sb-CD-DOX-ZAISe/ZnS one, which is a composite curve including two separate individuals and does not display the characteristic absorption peak (495 nm) of DOX. In addition, the PL spectrum (Figure S3B) was also a simple stack of Sb-CD-DOX and ZAISe/ZnS QDs. Instead, the Sb-CD-DOX-ZAISe/ZnS nanoparticle system exhibited the distinctive fluorescence originated from ZAISe/ZnS QDs. That is, the PL emission of DOX was absorbed by ZAISe/ZnS QDs, resulting in the fluorescence quenching of DOX (Figure S4). TEM measurements (Figure 2D) indicated that the average hydrodynamic diameter of single-dispersed QD-based complex was approximately 10 nm (Figure S5). The 2D 1H NOESY spectrum (Figure S6) provided direct evidence for the formation of a Host-Guest inclusion complex in D2O. The cross-peak signals at 3.66 ppm (1-dodecanethiol, DT) and 3.57–3.77 ppm (Sb-CD-DOX) suggested a close distance between DT and the internal H of the CD cavity, indicating the supramolecular interactions between DT chains and CDs. In addition, the Molecular Operating Environment (MOE) model displayed clearly that the distance between the surface of ZAISe/ZnS QD and DOX was below 10 nm, which serves to explain, at least in part, the red-shift of PL peak in Figure 2B induced by the FRET. 14 ACS Paragon Plus Environment

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3.1.3. In vitro and Intracellular Drug Release of the Host-Guest Nanoparticle System. The “smart” drug releases of the Sb-CD-DOX-ZAISe/ZnS were investigated at different pH values (pH = 5 or 7) without or with H2O2 (Figure 3A). Specifically, the curve of drug release involved two distinctive release behaviors during the test: fast release for the first 4 h, and slow delivery within the rest of time. The release rate of DOX from prodrug system was faster at pH 5.0 (the pH value of tumor microenvironment) than that at pH 7.4. Specifically, after immersed at pH 5.0 for 48 h, ~49% DOX was released from the Sb-CD-DOX-ZAISe/ZnS. By contrast, the drug release rate was dramatically decelerated under simulated body fluid (buffer at pH 7.4), with only 7% released during the same immersing time. That is, under pH 5.0, DOX was cleaved from the Host-Guest nanoparticle system rapidly, leading to the burst drug release. Accordingly, it is reasonable to infer that Sb-CD-DOX-ZAISe/ZnS can effectively release DOX triggered by endosomal pH, after the drug delivery system is internalized into cancer cell. Furthermore, the drug release data were minor increasing after H2O2 addition. Owing to the unique fluorescence of the prodrug nanoparticle system, the cellular uptake and release behavior (Figure 3B) could be easily detected by LSCM using U87 cells, of which the nucleus were dyed blue using Hoechst 33342 (λem = 405 nm) (Figure 3C). To eliminate the interference of DOX’s red light (λem = 595 nm), Sb-CD (precursor of Guest molecule) was used for water transfer of ZAISe/ZnS QDs (the absorbance and PL spectra was shown in Figure S7). As the low cytotoxicity of ZAISe/ZnS QDs-based nanoparticle systems (MTT assay was shown in Figure S8. The nanocomposite showed stronger cell growth inhibition ability for U87MG cell, which may be because the cell uptake of NPD by tumor cell is higher than that of normal cell), U87MG cell line was incubated with Sb-CD-ZAISe/ZnS and SbCD-DOX-ZAISe/ZnS to track DOX release in vitro. During the 4 h co-incubation process, strong red fluorescence from Sb-CD-ZAISe/ZnS was only observed in the cytoplasm, 15 ACS Paragon Plus Environment


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indicating that the nanoparticle could not cross the nucleopore to locate nucleus. However, the cellular distribution of Sb-CD-DOX-ZAISe/ZnS is dynamic. At the initial 1 h of coincubation, the red signal from Sb-CD-DOX-ZAISe/ZnS had appeared on the cytoplasm. With the extension of incubation, entire cell, including cytoplasm and nucleus, was dyed red, which suggested that DOX had been cleaved from the nanoparticle backbone and escaped from the endosome. After 4 h, almost all of the red fluorescence from DOX was accumulated in nucleus area. Meanwhile, the morphology of tumor cells changed observably, caused by the tumor-killing effect of the released free DOX. These results confirmed that Sb-CD-DOXZAISe/ZnS NPD was able to be swallowed by cancer cells, and could release free DOX into nucleus at the intracellular microenvironment. 3.2. Selective Tumor Targeting of the Host-Guest Nanoparticle System Because of the penetrating ability to deep tissue of NIR fluorescence, Sb-CD-DOXZAISe/ZnS prodrug nanoparticles could also be used as a detective probe for tumor-imaging in vivo. EPR effect is a critical reason for tumor accumulation of nanoparticle.38-40 On the one hand, the size of nanomedicine is the decisive factor of EPR effect. And we have proved previously that QDs-based nano-probe (ZAISe/ZnS QDs-clusters) around 20‒100 nm is optimal.28 On the other hand, the size of tumor, an easily ignored condition, also played an important role for the tumor location of nano-drugs.41 To investigate the accumulation capacities of tumors with different sizes, the bilateral tumor models (BTMs), which respectively reflect early, intermediate, advanced cancer, were established, and next imaged by small animal imaging systems using Sb-CD-DOX-ZAISe/ZnS NPDs (Figure 4). The images in Figure 4A visually displayed that early phase (~2.6 mm3) is not easy to image by nanoparticles via EPR effect. Meanwhile, intermediate (~59.2 mm3) and advanced (~400 mm3) tumors could be imaged obviously. And the intensity of fluorescence was increasing with the course of a malignancy

(Figure

4C).

The

results


were

consistent

with

the

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pathophysiological basis of tumor, which was reported that the EPR effect relies heavily on the peripheral blood capillary around malignancy. In brief, at the initial stage of tumor, the EPR effect is weak as the micrangium just formed soon (the number of vessels is low). With the development of cancer course, there are more and more micro-vessels around tumor tissue; and the uptake capacities for nanoparticles become higher. The data also indicated that using nanoparticles to detect early tumors in vivo is enslaved to the EPR effect of tumor. 3.3. Antitumor Activity of the FA Coating Host-Guest Nanoparticle System Although the sulfobetaine-modified Host-Guest nanoparticle could escape the capture of reticulo-endothelial system (RES) (e.g., the liver and spleen) to further prolong the circulation time in vivo, the cationic Sb-CD-DOX-ZAISe/ZnS complex also has nonspecific protein binding in blood.21,22 In order to minimize the drug losses, folic acid (FA), which is an essential vitamin with anionic charges composed of a pteridine ring, paraminobenzoic acid, and glutamic acid, was induced. In addition, FA is often used as a ligand for FA-specific receptor-mediated drug delivery systems to H22 tumor.22 After overcoating with FA, the ζ-potential of the complex was reversed into -21.2 mv at pH 7.4. And the ζ-potential of Sb-CD-DOX-ZAISe/ZnS@FA could be reversed into positive charge upon the pH value of solution below 5, which is a simulated tumor micro-environment. Meanwhile, the ζ-potential of the mixed solution, containing free Sb-CD-DOX-ZAISe/ZnS nanoparticles and FA molecule (pH ≤ 5), is smaller than the complex, indicating that FA was able to modify Sb-CD-DOX-ZAISe/ZnS electrostatically at buffer solution (Figure 5A). In the process of FA coating, the fluorescence of Sb-CD-DOX-ZAISe/ZnS decreased with the increasing of FA concentration (Figure 5B).42 In this paper, the concentration of FA was 0.5 mg/mL, and the PL signals of the obtained nanocomposites were able to be captured by LCSM (visible light region) and CCD system (NIR region).



The cell distribution of Sb-CD-DOX-ZAISe/ZnS@FA NPD was investigated using H22 mice tumor cells (Figure 5C). The drug release of Sb-CD-DOX-ZAISe/ZnS@FA is similar to the release behavior of Sb-CD-DOX-ZAISe/ZnS in U87MG cell. Briefly, the nano-drug has entered into cells after 1 h cultivation; and the DOX will be released and spread into nucleus during the prolonged incubation process. Herein, the endocytic mechanisms of Sb-CD-DOXZAISe/ZnS@FA NPD should be mediated by FA-receptor and charge absorption, concomitantly. The data confirmed that the abilities of endocytosis and drug release of SbCD-DOX-ZAISe/ZnS were slightly affected by FA coating. Encouraged by the favorable tumor-targeting of Sb-CD-DOX-ZAISe/ZnS@FA in vivo (Figure 5D), we further investigated the feasibility and curative effect of Sb-CD-DOXZAISe/ZnS@FA. Herein, H22 tumor bearing mice were randomly divided into six groups (n = 5) receiving specific treatments as follows: (1) sucrose solution alone; (2) CD-ZAISe/ZnS; (3) free DOX·HCl; (4) CD-DOX-ZAISe/ZnS (the synthetic routes were shown in Figure S9 and S10); (5) Sb-CD-DOX-ZAISe/ZnS; and (6) Sb-CD-DOX-ZAISe/ZnS@FA. During the investigation period, the body weights of these groups were almost same (Figure 6A). However, after 14 days treatment, the relative tumor volume of the control groups (sucrose and CD-ZAISe/ZnS groups) reached about 1000 mm3 (Figure 6B), which is a symptom of a late-stage tumor. By contrast, the tumor grew at a comparatively low speed in DOX group, showing the effectiveness of DOX·HCl as an anticancer chemotherapeutic. Compared with the positive DOX, the as-prepared CD-DOX-ZAISe/ZnS showed similar curative effect. What made the more encouraging results were that the tumor growth of the groups treated with SbCD-DOX-ZAISe/ZnS and Sb-CD-DOX-ZAISe/ZnS@FA nanocomposites were effectively suppressed (the tumor inhibitory up to 50%). In addition, the relative volumes of tumors invivo (Figure 6C) and ex-vivo (Figure 6D) in these groups were significantly smaller than that of DOX·HCl treated group, whereas the most remarkable suppression of tumor deterioration 18 ACS Paragon Plus Environment

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was observed in Sb-CD-DOX-ZAISe/ZnS@FA group. Furthermore, histological observation of the excised tumor intuitively revealed the differences in tumor tissues after 14 days postinjection of the above therapeutic agent (Figure 6E). For sucrose and CD-ZAISe/ZnS groups treated mice, purple stained nuclei occupied most of the area. Necrosis area and neoplastic cells were hardly observed in most areas. Oppositely, in tumor tissue treated with DOX·HCl, the necrosis area was obvious. And more serious necrosis and neoplastic cells were found in Sb-CD-DOX-ZAISe/ZnS@FA groups, confirming the excellent anti-tumor efficacy. Meanwhile, Figure 6F displayed the fluorescence intensity of DOX in different cryo sectioned tumors, reflecting the inequable drug accumulation in lesion and giving the most favorable evidence for the discrepant therapeutic effect. The other important reason for developing the targeting drug system is to reduce the side effects of free drug.43 The pathomorphology analysis of the main organ (Figure S11) was investigated. The heart section of the DOX treating mice exhibits significant lesion and lymphocytic invasion, indicating the high cardiotoxicity. By contrast, there is no apparent damage in the detected organs from other groups. However, it still is a challenging work to fully tracing QDs in vivo.44 In this work, the stay time of the Sb-CD-DOX-ZAISe/ZnS@FA in body was investigated further to assess the metabolism preliminarily (Figure S12). The result was consistent with the distribution in tumor-bearing mice (Figure 5D), and showed that i) beside tumor tissues, the fluorescent signal can be also detected clearly in liver (the important detoxifying organ), which is mainly related to the uptake by RES;45,46 ii) the concentration of the NPD in liver decreased with the prolongation of experimental time; iii) most of the NPD could be cleaned up at 10 days P.I. Combining with the data of pathomorphology analysis of liver (Figure S11), it could be concluded that the as-prepared QDs-based nanocomposites have good biocompatibility in vivo. 19 ACS Paragon Plus Environment


4. Conclusion In summary, a pH and H2O2 dual-sensitive NPD was constructed via ingenious hierarchical self-assembly strategies. The “Guest” prodrug was designed in combining Sb-CD with DOX by the dual-sensitive chemical bond. After phase transfer (the 1st SA strategy), the integrated nanocomposite not only possessed highly fluorescence emission, but also inherited pesticide effect for tumor with a series of favorable attributes, such as multicolor bioimaging, prolonged circulation time in blood, effective accumulation at tumor site, enhanced internalization by tumor cells and timely drug release. By multiscale optical imaging techniques, Sb-CD-DOX-ZAISe/ZnS have been confirmed to have the dual-responsive drug releasing property in cell, and to be a potential fluorescent probe for malignant tumors in metaphase and advanced stage. Moreover, after overcoating FA layer (the 2nd SA strategy), the as-prepared Sb-CD-DOX-ZAISe/ZnS@FA NPD was provided with pH-controlled negativeto-positive charge reversal ability further, and showed better curative effect than free DOX. This study is interesting for exploiting a stagey to fabricate biocompatible QD-based NPD to improve the clinical applications of hydrophobic QDs, and also provide an intriguing phase transfer method for the inorganic nanoparticles with similar structure.


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Scheme 1. Overall synthetic scheme for Sb-CD-DOX-ZAISe/ZnS@FA NPD by “HostGuest” assembly and electrostatic interaction strategy, as well as the following bioapplications.



Figure 1. (A) Synthetic scheme and (B) 1H NMR spectrum for “Guest” Sb-CD-DOX molecule (The shifts at 2.5 and 3.3 ppm should be assigned mainly to the solvent of DMSO and residual H2O. The signals of protons “h” and “j” were at 2.62 and 3.36 ppm respectively, which were overlapped by these strong solvent peaks, although the obvious peaks could be seen in Figure S1).


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A1.8 1 3

0.9 0.6

PL Intensity (a.u.)

1.2

B800

Initial oil-soluble ZAISe/ZnS QDs Sb-CD-DOX Sb-CD-DOX-ZAISe/ZnS NPDs

1.5

Absorbance

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


2

0.3 0.0 400

600

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1000

1 3 2

600 400 200 0 500

600

700

800

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900

Figure 2. (A) Absorption, (B) PL spectra (λex = 450 nm) and (C) schematic illustration of ZAISe/ZnS QDs before and after water transfer by Sb-CD-DOX molecule. The insets were the true-color photographs of samples captured under room light and UV light (λmax = 365 nm) excitation, respectively. (D) The TEM image and cartoon model (the inset) of resultant aqueous Sb-CD-DOX-ZAISe/ZnS NPD.



A 0.7

Sb-CD-DOX-ZAISe/ZnS at pH 5.0 without H2O2

0.6

Sb-CD-DOX-ZAISe/ZnS at pH 7.4 without H2O2

Accumulative Weight (%)

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

Sb-CD-DOX-ZAISe/ZnS at pH 5.0 with H2O2 Sb-CD-DOX-ZAISe/ZnS at pH 7.4 with H2O2

0.5 0.4

*

0.3 0.2 0.1 0.0 0

1

2

3

4

5

6

7

Time (h)

8 12 24 48

Figure 3. (A) Drug release rate curves of Sb-CD-DOX-ZAISe/ZnS NPD in the simulated physiological and tumor micro-environment. Data were shown as mean ± S.D (n = 3). Significance is defined as *P < 0.05. (B) Schematic illustration of the of drug release process of the Sb-CD-DOX-ZAISe/ZnS NPD. (C) LSCM images of U87MG cells incubated with SbCD-ZAISe/ZnS for 4 h, and Sb-CD-DOX-ZAISe/ZnS for 1 h, 2 h, 4 h (λex = 405 nm for hoechst, and λex = 488 nm for QDs). 24 ACS Paragon Plus Environment

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C4

*

Tumormax/Normal Ratio

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*

*

3

2

1

0 Early Stage

Middle Stage Advanced Stage

Figure 4. (A), (B) Dynamic distributions, and (C) corresponding Tmax/N (muscle) fluorescence intensity ratios of Sb-CD-DOX-ZAISe/ZnS nanocomposites in bilateral H22 tumor models (The left lower extremity was chosen as reference at 10 h, respectively). (*P < 0.05, n = 5 mice in each group).


A60

B 900 PL Intensity (a.u.)

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

Zeta Potential (mV)


30

0

FA Coating QDs

FA Separated

FA Mixing

0 mg/mL 0.1mg/mL 0.5mg/mL 1mg/mL 3mg/mL

600

300

0 450

-30

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600

750

Wavelength (nm)

900

Figure 5. (A) ζ-potential variation trend of Sb-CD-DOX-ZAISe/ZnS during the coating of FA molecule (0.5 mg/mL), and (B) PL spectra of Sb-CD-DOX-ZAISe/ZnS coated with different concentration of FA molecule. The inset is the pseudocolored photograph taken under NIR laser light (λex = 660 nm). Dynamic distributions of Sb-CD-DOX-ZAISe/ZnS@FA NPD in H22 (C) tumor cells (λex = 405 nm for hoechst, and λex = 488 nm for QDs) and (D) tumorbearing model (λex = 660 nm). 26 ACS Paragon Plus Environment

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A40

B1200

Glucose CD-ZAISe/ZnS DOX CD-DOX-ZAISe/ZnS Sb-CD-DOX-ZAISe/ZnS Sb-CD-DOX-ZAISe/ZnS@FA

30 20 10 0 0

2

4

6

8

10

Time (d)

12

Glucose CD-ZAISe/ZnS DOX CD-DOX-ZAISe/ZnS Sb-CD-DOX-ZAISe/ZnS Sb-CD-DOX-ZAISe/ZnS@FA

1000

Tumor Volume(mm3)

Body Weight (g)

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


14

16

800 600

*

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*

200

*

0 0

2

4

6

8

10

Time (d)

12

14

16

Figure 6. (A) Body weight curves, (B) tumor growth curves and (C) photographs of H22 tumor-bearing mice in the six groups (sucrose, CD-ZAISe/ZnS, DOX·HCl, CD-DOXZAISe/ZnS, Sb-CD-DOX-ZAISe/ZnS, Sb-CD-DOX-ZAISe/ZnS@FA) on different days of treatments (1, 3, 5, 7, 9 and 14 d). (D) The digital photo, (E) H&E stained images and (F) LSCM images of the H22 tumors collected from different treatment groups (14 d). (*P < 0.05, n = 5 mice in each group). 27 ACS Paragon Plus Environment


ASSOCIATED CONTENT Supporting Information: Detailed experimental procedures and additional data (1H NMR spectra of “Guest” molecules; conditional optimization test of the water transfer; absorption and PL spectra of CD-ZAISe/ZnS, Sb-CD-ZAISe/ZnS, and Sb-CD-DOX-ZAISe/ZnS; schematic presentation for the FRET in Sb-CD-DOX-ZAISe/ZnS; MTT assay of Sb-CDDOX-ZAISe/ZnS; H&E stained images of normal organs; and the stay time of NPD in body, 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] (Z. Gao) ORCID：Dawei Deng: 0000-0002-1391-1845

Author contributions 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-120974) in University of the Ministry of Education of China, and the Graduate Innovation Foundation of Huahai Pharmaceutical (1010110002).


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REFERENCES (1) D'Mello, S. R.; Cruz, C. N.; Chen, M. L.; Kapoor, M.; Lee, S. L.; Tyner, K. M. The Evolving Landscape of Drug Products Containing Nanomaterials in the United States. Nat. Nanotechnol. 2017, 12, 523-529. (2) Bobo, D.; Robinson, K. J.; Islam, J.; Thurecht, K. J.; Corrie, S. R. Nanoparticle-Based Medicines: A Review of FDA-Approved Materials and Clinical Trials to Date. Pharm. Res. 2016, 33, 2373-2387. (3) Cormode, D. P.; Jarzyna, P. A.; Mulder, W. J.; Fayad, Z. A. Modified Natural Nanoparticles as Contrast Agents for Medical Imaging. Adv. Drug Delivery Rev. 2010, 62, 329-338. (4) Diaz, S. A.; Giordano, L.; Azcarate, J. C.; Jovin, T. M.; Jares-Erijman, E. A. Quantum Dots as Templates for Self-Assembly of Photoswitchable Polymers: Small, Dual-Color Nanoparticles Capable of Facile Photomodulation. J. Am. Chem. Soc. 2013, 135, 3208-3217. (5) Deng, D. W.; Hao, C. L.; Sen, S.; Xu, C. L.; Král, P.; Kotov, N. A. Template-Free Hierarchical Self-Assembly of Iron Diselenide Nanoparticles into Mesoscale Hedgehogs. J. Am. Chem. Soc. 2017, 139, 16630–16639. (6) Chinen, A. B.; Guan, C. M.; Ferrer, J. R.; Barnaby, S. N.; Merkel, T. J.; Mirkin, C. A. Nanoparticle Probes for the Detection of Cancer Biomarkers, Cells, and Tissues by Fluorescence. Chem. Rev. 2015, 115, 10530-10574. (7) Garcia-Orue, I.; Pedraz, J. L.; Hernandez, R. M.; Igartua, M. Nanotechnology-Based Delivery Systems to Release Growth Factors and Other Endogenous Molecules for Chronic Wound Healing. J. Drug Delivery Sci. Technol. 2017, 42, 2-17.



(8) Hartshorn, C. M.; Bradbury, M. S.; Lanza, G. M.; Nel, A. E.; Rao, J. H.; Wang, A. Z.; Wiesner, U. B.; Yang, L.; Grodzinski, P. Nanotechnology Strategies to Advance Outcomes in Clinical Cancer Care. ACS Nano 2018, 12, 24-43. (9) Probst, C. E.; Zarazhevskly, P.; Bagalkot, V.; Gao, X. Quantum Dots as a Platform for Nanoparticle Drug Delivery Vehicle Design. Adv. Drug Delivery Rev. 2013, 65, 703-718. (10) Zorn, G.; Dave, S. R.; Weidner, T.; Gao, X.; Castner, D. G. Direct Characterization of Polymer Encapsulated CdSe/CdS/ZnS Quantum Dots. Surf. Sci. 2016, 648, 339-344. (11) de Lima França, C. C.; da Silva Terto, E. G.; Dias-Vermelho, M. V.; Silva, A. C. A.; Dantas, N. O.; de Abreu, F. C. The Electrochemical Behavior of Core-Shell CdSe/CdS Magic-Sized Quantum Dots Linked to Cyclodextrin for Studies of the Encapsulation of Bioactive Compounds. J. Solid State Electrochem. 2016, 20, 2533-2540. (12) Ma, L.; Tu, C.; Le, P.; Chitoor, S.; Lim, S. J.; Zahid, M. U.; Teng, K. W.; Ge, P.; Selvin, P. R.; Smith, A. M. Multidentate Polymer Coatings for Compact and Homogeneous Quantum Dots with Efficient Bioconjugation. J. Am. Chem. Soc. 2016, 10, 3382-3394. (13) Fedosyuk, A.; Radchanka, A.; Antanovich, A.; Prudnikau, A.; Kvach, M. V.; Shmanai, V.; Artemyev, M. Determination of Concentration of Amphiphilic Polymer Molecules on the Surface of Encapsulated Semiconductor Nanocrystals. Langmuir 2016, 8, 1955-1961. (14) Petryayeva, E.; Krull, U. J. Quantum Dot and Gold Nanoparticle Immobilization for Biosensing Applications Using Multidentate Imidazole Surface Ligands. Langmuir 2012, 28, 13943-13951. (15) Reisch, A.; Klymchenko, A. S. Fluorescent Polymer Nanoparticles Based on Dyes: Seeking Brighter Tools for Bioimaging. Small 2016, 12, 1968-1992. 30 ACS Paragon Plus Environment

Page 30 of 36

Page 31 of 36 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


(16) Ye, D. X.; Ma, Y. Y.; Zhao, W.; Cao, H. M.; Kong, J. L.; Xiong, H. M.; Mohwald, H. ZnO-Based Nanoplatforms for Labeling and Treatment of Mouse Tumors without Detectable Toxic Side Effects. ACS Nano 2016, 10, 4294-4300. (17) Evans, C. M.; Love, A. M.; Weiss, E. A. Surfactant-Controlled Polymerization of Semiconductor Clusters to Quantum Dots Through Competing Step-Growth and Living Chain-Growth Mechanisms. J. Am. Chem. Soc. 2012, 134, 17298-17305. (18) Zhao, M. X.; Zhu, B. J. The Research and Applications of Quantum Dots as NanoCarriers for Targeted Drug Delivery and Cancer Therapy. Nanoscale Res. Lett. 2016, 11, 207. (19) Busseron, E.; Ruff, Y.; Moulin, E.; Giuseppone, N. Supramolecular Self-Assemblies as Functional Nanomaterials. Nanoscale 2013, 5, 7098-7140. (20) Hu, Q. D.; Tang, G. P.; Chu, P. K. Cyclodextrin-Based Host-Guest Supramolecular Nanoparticles for Delivery: from Design to Applications. Acc. Chem. Res. 2014, 47, 20172025. (21) Zhang, Q.; Shen, C.; Zhao, N.; Xu, F.-J. Redox-Responsive and Drug-Embedded Silica Nanoparticles with Unique Self-Destruction Features for Efficient Gene/Drug Codelivery. Adv. Funct. Mater. 2017, 27, 1606229. (22) Kurosaki, T.; Morishita, T.; Kodama, Y.; Sato, K.; Nakagawa, H.; Higuchi, N.; Nakamura, T.; Hamamoto, T.; Sasaki, H.; Kitahara, T. Nanoparticles Electrostatically Coated with Folic Acid for Effective Gene Therapy. Mol. Pharmaceutics 2011, 8, 913-919. (23) Hu, X.-Y.; Liu, X.; Zhang, W.; Qin, S.; Yao, C.; Li, Y.; Cao, D.; Peng, L.; Wang, L. Controllable Construction of Biocompatible Supramolecular Micelles and Vesicles by Water-



Soluble Phosphate Pillar [5, 6] arenes for Selective Anti-Cancer Drug Delivery. Chem. Mater. 2016, 28 (11), 3778-3788. (24) Wang, Y.; Wong, J. F.; Teng, X.; Lin, X. Z.; Yang, H. “Pulling” Nanoparticles into Water: Phase Transfer of Oleic Acid Stabilized Monodisperse Nanoparticles into Aqueous Solutions of r-Cyclodextrin. Nano lett. 2003, 3, 1555-1559. (25) Liu, Y.; Perez, L.; Mettry, M.; Easley, C. J.; Hooley, R. J.; Zhong, W. SelfAggregating Deep Cavitand Acts as a Fluorescence Displacement Sensor for Lysine Methylation. J. Am. Chem. Soc. 2016, 138, 10746-10749. (26) Wang, Y.; Wang, H.; Chen, Y.; Liu, X.; Jin, Q.; Ji, J. pH and Hydrogen Peroxide Dual Responsive Supramolecular Prodrug System for Controlled Release of Bioactive Molecules. Colloids Surf., B 2014, 121, 189-195. (27) Yum, K.; Wang, N.; Yu, M. Electrochemically Controlled Deconjugation and Delivery of Single Quantum Dots into the Nucleus of Living Cells. Small 2010, 6, 2109-2113. (28) Deng, T; Peng, Y.; Zhang, R.; Wang J.; Zhang J.; Gu Y.; Huang, D.; Deng, D. WaterSolubilizing Hydrophobic ZnAgInSe/ZnS QDs with Tumor-Targeted cRGD-SulfobetainePIMA-Histamine Ligands via a Self-Assembly Strategy for Bioimaging. ACS Appl. Mater. Interfaces 2017, 9, 11405-11414. (29) Deng, D.; Chen, Y.; Cao, J.; Tian, J.; Qian, Z.; Achilefu, S.; Gu, Y. High-Quality CuInS2/ZnS Quantum Dots for In vitro and In vivo Bioimaging. Chemistry of Materials 2012, 24, 3029-3037.


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(30) Deng, D.; Qu, L.; Zhang, J.; Ma, Y.; Gu, Y. Quaternary Zn-Ag-In-Se quantum dots for biomedical optical imaging of RGD-modified micelles. ACS Appl. Mater. Interfaces 2013, 5, 10858-65. (31) Du C.; Deng D.; Shan L.; Wan S.; Cao J.; Tian J.; Achilefu S.; Gu., Y. A pH-sensitive doxorubicin prodrug based on folate-conjugated BSA for tumor-targeted drug delivery. Biomaterials 2013, 34, 3087-3097. (32) Bauleth-Ramos, T.; Shahbazi, M.-A.; Liu, D.; Fontana, F.; Correia, A.; Figueiredo, P.; Zhang, H.; Martins, J. P.; Hirvonen, J. T.; Granja, P.; Sarmento, B.; Santos, H. A. Nutlin-3a and Cytokine Co-loaded Spermine-Modified Acetalated Dextran Nanoparticles for Cancer Chemo-Immunotherapy. Adv. Funct. Mater. 2017, 42, 1703303. (33) Choi, H. W.; Kim, J.; Kim, J.; Kim, Y.; Song, H. B.; Kim, J. H.; Kim, K.; Kim, W. J. Light-Induced Acid Generation on a Gatekeeper for Smart Nitric Oxide Delivery. ACS Nano 2016, 10, 4199-4208. (34) Khawar, I. A.; Kim, J. H.; Kuh, H. J. Improving Drug Delivery to Solid Tumors: Priming the Tumor Microenvironment. J. Controlled Release 2015, 201, 78-89. (35) Melamed, J. R.; Riley, R. S.; Valcourt, D. M.; Day, E. S. Using Gold Nanoparticles To Disrupt the Tumor Microenvironment: An Emerging Therapeutic Strategy. ACS Nano 2016, 10, 10631-10635. (36) Onoshima, D.; Yukawa, H.; Baba, Y. Multifunctional Quantum Dots-Based Cancer Diagnostics and Stem Cell Therapeutics for Regenerative Medicine. Adv. Drug Delivery Rev. 2015, 95, 2-14.



(37) Li, S.-Y.; Liu, L.-H.; Rong, L.; Qiu, W.-X.; Jia, H.-Z.; Li, B.; Li, F.; Zhang, X.-Z. A Dual-FRET-Based Versatile Prodrug for Real-Time Drug Release Monitoring and In Situ Therapeutic Efficacy Evaluation. Adv. Funct. Mater. 2015, 25, 7317-7326. (38) Fang, J.; Nakamura, H.; Maeda, H. The EPR effect: Unique Features of Tumor Blood Vessels for Drug Delivery, Factors Involved, and Limitations and Augmentation of the Effect. Adv. Drug Delivery Rev. 2011, 63, 136-151. (39) Maeda, H. Toward a Full Understanding of the EPR Effect in Primary and Metastatic Tumors As Well As Issues Related to Its Heterogeneity. Adv. Drug Delivery Rev. 2015, 91, 36. (40) Iyer, A. K.; Khaled, G.; Fang, J.; Maeda, H. Exploiting the Enhanced Permeability and Retention Effect for Tumor Targeting. Drug Discovery Today 2006, 11, 812-818. (41) Maeda, H.; Fang, J.; Inutsuka, T.; Kitamoto, Y. Vascular Permeability Enhancement in Solid Tumor: Various Factors, Mechanisms Involved and Its Implications. Int. Immunopharmacol. 2003, 3, 319-328. (42) Geszke-Moritz, M.; Clavier, G.; Lulek, J.; Schneider, R. Copper- or Manganese-Doped ZnS Quantum Dots As Fluorescent Probes for Detecting Folic Acid in Aqueous Media. J. Lumin. 2012, 132, 987-991. (43) Li, Z.; Tan, S.; Li, S.; Shen, Q.; Wang, K. Cancer Drug Delivery in the Nano Era: An Overview and Perspectives. Oncol. Rep. 2017, 38, 611-624. (44) Eunkeu, O.; Liu, R.; Nel, A.; Gemill, K.; Bilal, M.; Cohen, Y.; Medintz, I. L. Metaanalysis of cellular toxicity for cadmiumcontaining quantum dots. Nat. Nanotechnol. 2016, 11, 479-463. 34 ACS Paragon Plus Environment

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(45) Schädlich, A.; Caysa, H.; Mueller, T.; Tenambergen, F.; Rose, C.; Göpferich, A.; Kuntsche, J.; Mäder, K. Tumor Accumulation of NIR Fluorescent PEG–PLA Nanoparticles: Impact of Particle Size and Human Xenograft Tumor Model. ACS Nano 2011, 5, 8710-8720. (46) Nissinen, T.; Näkki S.; Laakso H.; Kučiauskas D.; Kaupinis A.; I. Kettunen, M.; Liimatainen, T.; Hyvönen, M.; Valius, M.; Gröhn, O.; Lehto, V.-P. Tailored Dual PEGylation of Inorganic Porous Nanocarriers for Extremely Long Blood Circulation in Vivo. ACS Appl. Mater. Interfaces 2016, 8, 32723–32731.



Table of content graphic:

Fabrication and bio-application of multi-functional self-assembled quantum dot-based nanoprodrug (Sb-CD-DOX-ZAISe/ZnS@FA).


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Quantum Dots-Based Multifunctional Nano-Prodrug Fabricated by

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