Low-Density

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Biological and Medical Applications of Materials and Interfaces

Polyelectrolyte-Mediated Nontoxic AgGaxIn1-xS2 QDs/ Low-Density Lipoprotein Nanoprobe for Selective 3D Fluorescence Imaging of Cancer Stem Cells Jiangluqi Song, Yan Zhang, Yiwen Dai, Jinhang Hu, Lixin Zhu, XiaoLiang Xu, Yue Yu, Huan Li, Bo Yao, and Huixin Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00121 • Publication Date (Web): 19 Feb 2019 Downloaded from http://pubs.acs.org on February 26, 2019

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ACS Applied Materials & Interfaces

Polyelectrolyte-Mediated Nontoxic AgGaxIn1-xS2 QDs/Low-Density Lipoprotein Nanoprobe for Selective 3D Fluorescence Imaging of Cancer Stem Cells Jiangluqi Song,†,* Yan Zhang,§ Yiwen Dai,ǁ Jinhang Hu,┴ Lixin Zhu,§,* Xiaoliang Xu,# Yue Yu,† Huan Li,† Bo Yao,† Huixin Zhou†,* †School

of Physics and Optoelectronic Engineering, Xidian University, Xi’an 710071, China

§Central

Laboratory, First Affiliated Hospital of Anhui Medical University, Hefei 230022, China

ǁDepartment

of General Surgery, The Second Hospital of Anhui Medical University, Hefei

230601, China ┴Shaanxi

Collaborative Innovation Center of Chinese Medicinal Resources Industrialization,

Shaanxi University of Chinese Medicine, Xianyang 712046, China #Key

Laboratory of Strongly-Coupled Quantum Matter Physics, Chinese Academy of Sciences,

and Department of Physics, University of Science and Technology of China, Hefei, Anhui 230026, China KEYWORDS: cation exchange, quantum dots, low-density lipoprotein, colon cancer stem cells, selective three-dimensional imaging

ACS Paragon Plus Environment

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ABSTRACT: Cancer stem cells, which are a population of cancer cells sharing common properties as normal stem cells, have strong self-renewal ability and multi-lineage differentiation potential to trigger tumor proliferation, metastases and recurrence. From this, targeted therapy for cancer stem cells may be one of the most promising strategies for comprehensive treatment of tumors in the future. We design a facile approach to establish the colon cancer stem cells-selective fluorescent probe based on the low-density lipoprotein (LDL) and the novel AgGaxIn(1-x)S2 quantum dots (AGIS QDs). AGIS QDs with a high crystallinity are obtained for the first time via cation exchange protocol of Ga3+ to In3+ starting from parent AgInS2 QDs. Photoluminescence emission wavelength of AGIS QDs can be turned from 502 to 719 nm by regulating the reaction conditions, with the highest quantum yield up to 37%. Subsequently, AGIS QDs-conjugated LDL nanocomposites (NCs) are fabricated, in which a cationic polyelectrolyte were used as a coupling reagent to guarantee the electrostatic self-assembly. The structural integrity and physicochemical properties of the LDL-QDs NCs are found to be maintained in vitro, and the NCs exhibit remarkable biocompatibility. The LDL-QDs can be selectively delivered into cancer stem cells that overexpress LDL receptor, and three-dimensional imaging of cancer stem cells is realized. The results of this study not only demonstrate the versatility of nature-derived lipoprotein nanoparticles, but also confirm the feasibility of electrostatic conjugation using cationic polyelectrolyte, allowing reseachers to design nanoarchitectures for targeted diagnosis and treatment of cancer.

Introduction


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As one of the great dangers facing humanity, cancer is a global public health issue with high mortality. In the research of cancer, the concept and the confirmation of cancer stem cells is considered to be the most significant development during the past few decades.1,2 Cancer stem cells, according to the current study, are known as the seeds of tumorigenesis as well as the driving force of metastases and relapses, which maintains the vitality of tumor cell populations through their self-renewal and infinite proliferation.3-5 As such, targeted treatment against cancer stem cells may be the radical cure for cancer. To this end, accurate location and tracking of cancer stem cells should be adopted, which is the prerequisite for precise treatment. However, due to the low content of the cancer stem cells in tumor tissues and blood,3 it is very difficult to detect them in the present situation. Over the past few years, researchers have been searching for an ideal carrier that can specifically transport contrast agent or medicament to the lesion. A variety of nanomaterials with unique physicochemical properties and loading capacity have been inspired, such as carbon materials,6 metal-organic frameworks (MOFs),7 porous silica,8 shaped metal particles,9 dendrimers10 and polymeric micelles11. Despite the in-depth developments of these formulations, the carrier systems still face a lot of burning problems, including cytotoxicity, immunological rejection, drug release control and intratumoral diffusion.12 To this end, biological fabrication-based designs, especially those materials directly or indirectly derived from nature,13 have been intensively explored to build novel delivery vehicles. Among these nanovehicles, low-density lipoprotein (LDL) is of particular interest for its natural biocompatibility and small size, as well as excellent loading capacity.14,15 Typically, LDL is ~30 nm in diameter and consists of a monolayer of phospholipids encapsulating cholesterol esters and triglycerides with apolipoprotein embedded thereon. The surface apolipoprotein ApoB-100 in LDL is rich in functional groups, and can be recognized by the LDL


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receptors on the surface of the cell membrane.16 Additionally, LDL has an appropriate size of bellow 40 nm, which in favors of its diffusion among the interfibrillar openings in solid tumors.17 Thus, LDL is frequently chosen as a carrier for drug delivery and theranostic applications. Since hydrophobic molecules were successfully loaded into the core of LDL by Krieger,18 three major strategies, including apolipoprotein conjugation, hydrophobic core substitution and phospholipid molecule interpolation, have been developed to reconstruct LDL up to now. 17 Despite the different applicability of these protocols, they still suffer from the disadvantage of being complicated to operate, and facile methods of loading functional nanoparticles have not been reported. In order to achieve accurate diagnosis and localization of tumors, a mass of nanotechnologyinspired tracers have been explored to exactly evaluate therapeutic effects at the cellular level.19,20 Among various imaging materials, fluorescent quantum dots (QDs) exhibit a number of appealing features, including large Stokes shifts, tunable emission, robust chemical stability and vigorous photobleaching resistance.21 Although QDs with diverse types and multiple structures have been fabricated, such as C,22 CdTe,23 Mn-doped ZnSe,24 CdSe/ZnSe/ZnS,25 they are failed to meet the demand of biocompatibility and nontoxicity, due to the incorporated Class A and Class B elements (Pb, Cd, As and Se) and organic ligands. In recent years, there have been substantial explorations of ternary I-III-VI semiconductors, such as AgInS2 (AIS) and CuInS2, because of their significant defect tolerance, component-dependent emission and extremely low cytotoxicity.19,26 Both simulations and experiments, however, indicated that it is difficult to tailor the bandgap (Eg) as well as the emission wavelength by adjusting the size of I-III-VI QDs,27,28 which is quite different from those II-VI semiconductors. Although doping with transition metal ions can broaden their bandgap to some extent, 29,30 for example, the Eg of AIS QDs could be tuned from ~1.9 eV to ~2.4 eV of Zn-doped AgInS2,29 improvements in crystalline quality and photoluminescence (PL)


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quantum yields continue to be the major roadblocks due to the radius difference between the introduced ions and native ions. Considering their component-dependent emission, a common strategy has been proposed to alloy the semicondutors with other elements in the congener group. Among I-III-VI semiconductors, the only member that has a large bandgap and a similar crystal structure is AgGaS2 (Eg=~2.7 eV).31,32 Unfortunately, due to differences in reactivity between Ag+ and Ga3+, biological application-oriented AgGaS2 or AgGaxIn(1-x)S2 alloy QDs with water dispersity have never been studied. Hence, innovative strategies for synthesizing these novel QDs are still expected. In this study, we report a facile method to conjugate native LDL of human with fluorescent QDs via electrostatic interaction using polyelectrolyte. Nontoxic AgGaxIn(1-x)S2 (AGIS) QDs with tunable photoluminescence wavelengths and prominent biocompatibility were first synthesized through cation exchange protocol, in which AgInS2 was employed as the pristine template. The elemental composition-dependent optical properties and the evolution of crystals structure were studied in detail. By varying the incorporation of Ga3+ as well as the extraction of In3+, AGIS QDs with a broad color window and excellent quantum yield (QY) were obtained. Subsequently, fluorescent LDL-QDs nanocomposites (NCs) were fabricated by modification of cationic polyelectrolyte and adsorption of negatively charged AGIS QDs. We would demonstrate that LDL-QDs NCs could be used as the specific-marker-free fluorescent probe with selectivity for three-dimensional visualization of colon cancer stem cells (CCSCs). Additionally, the results also showed that polyelectrolyte was a versatile cross-linker to establish a bridge between biologically inspired carriers and inorganic materials. Experimental Section


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Materials. All the chemicals were used without further purification. Silver nitrate (AgNO3, 99%),

gallium

nitrate

(Ga(NO3)3·xH2O,

99.9%),

sodium

citrate

tribasic

dehydrate

(Na3C6H5O7·2H2O, 99.0%), N-acetyl-L-cysteine (NAC, 99%), L-glutathione reduced (GSH, 98%), Poly(diallyldimethylammonium chloride) solution (PDDA, 20 wt % in H2O), sodium chloride (NaCl, 99.5%) were purchased from Sigma-Aldrich Chemical Reagent Co. Ltd. (Shanghai, China). Thioacetamide (TAA, 99%), sodium hydroxide (NaOH, 96%), and absolute ethanol were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Indium acetate (In(OAc)3, 99.99%) was purchased from Alfa Aesar Chemical Reagent Co. Ltd. (Shanghai, China). All the water used in experiments was deoxidized deionized water (18.25 MΩ·cm). Native LDL was isolated from human plasma according to the previous report.33 Human colon cancer cell line (SW480), colonic epithelial cell line (NCM-460) and colon cancer stem cells was provided by Anhui Medical University. CCSCs were sorted from SW-480 cells using CD133 (Prominin-1) monoclonal antibody as a marker. High glucose Dulbecco’s modified Eagle medium (DMEM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), penicillin−streptomycin, fetal bovine serum (FBS), phosphate buffer solution (PBS) and streptomycin were purchased from commercial sources. Preparation of water-dispersible AgInS2 as a pristine template. Ternary AgInS2 QDs were synthesized using a hydrothermal protocol as described in our previous research with large modifications.38 Briefly, Ag-NAC and In-NAC precursors were prepared by mixing AgNO3 (5 mL, 0.01 M), In(OAc)3 (7.5 mL, 0.05 M) and NAC solution (7.5 mL, 0.3 mM) under stirring, followed by adjusting the pH to 8.5 using NaOH solution (1.5 M). Subsequently, 12.5 mL of freshly prepared TAA solution (0.05 M) was injected into the above solution, and sealed in an exclusive Teflon-lined stainless steel autoclave. The autoclave was heated to 120 °C, and


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maintained for 3 h. After the reaction, excess amount of ethanol was added, and the liquid was centrifuged to remove the remnant reactant. The precipitation was re-dispersed in water for further use, noting as S1. CE for synthesis of AGIS QDs. 10 mL of purified AIS QDs (0.1 M) was added in a three-neck flask with a condenser and heated to 100 °C. Then, a precursor containing a desire amount of Ga(NO3)3 (0.02 M) and sodium citrate (0.01 M) was injected swiftly under stirring, and the mixture was refluxed for 1 h with condenser attached. Aliquots of the solution were taken out at different time intervals to monitor the reaction. The purification of the samples were the same as the AIS QDs. We set the molar ratio of Ga3+ (in gallium nitrate precursor) to In3+ (in the AIS QDs) as 0.85, 1.56, 2.88 and 3.73, noting as S2-S5. Electropositive modification of LDLs. The native LDLs have negative surface charges because of the abundant anionic phosphate groups in the phospholipid monolayer. The electronegative LDLs were modified with cationic polyelectrolyte (PDDA). The procedure is schematically illustrated in Figure 3a. PDDA was diluted to 1 g L-1 in NaCl solution (0.8 M), and the pH was adjusted to 9.5 using hydrochloric acid and sodium hydroxide. Then, 2 mg of LDLs was dispersed in 10 mL of PBS solution, and added to the PDDA solution using a peristaltic pump under moderate stirring. The obtained modified LDLs were collected by centrifugation at 15 000 rpm for 30 min, and were redispersed in PBS solution for further use. Conjugation of QDs and LDLs. The QDs-LDL NCs were fabricated via electrostatic interaction. 1 mL of QDs dispersion (0.06 M) was added to the modified LDL solution (pH=9.5) using a peristaltic pump, followed by stirring for 12 h at 5 °C. The NCs dispersion was filtered


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through 0.8/0.2 μM pore size syringe filters in order to sterilize the solution, and centrifuged (18 000 rpm for 30 min) and redispersed in sterile PBS solution. Characterization. The morphologies and the element analyses of nanoparticles were determined on a transmission electron microscope (TEM, model JEM-1400) equipped with an energy dispersive X-ray spectrometer (EDS) attachment. EDS results were normalized by the S content. The crystallographic structures of QDs were performed on a high-resolution transmission electron microscope (HRTEM, model JEM-2100F). Photoluminescence (PL) spectra were obtained on an RF-5301 fluorescence spectrophotometer. UV-vis absorbance analyses were carried out by using a UV-3600 spectrophotometer. X-ray diffraction (XRD) measurements were conducted on a Philips X'Pert Pro X-ray powder diffractometer with Cu Kα (0.154 nm) serving as the incident radiation. Elemental composition was analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES) using a Plasma Quad 3 spectrometer. X-ray photoelectron spectroscopy (XPS) was determined using an ESCLAB 250 spectrometer, and the spectra were corrected by the C 1s peak (@284.8 eV). Quantum yield was determined using Rhodamine 6G (QY = 95%) as a reference.34 Fourier transform infrared (FTIR) spectra were recorded using the KBr pellet technique on a Nicolet 6700 spectrometer. UV circular dichroism (CD) measurements were carried out by a Jasc0 J-815 CD Spectrometer in the wavelength range of 200–260 nm. Particle size, polydispersity and zeta potential were measured with a Malvern Zetasizer Ultra.

Results and discussion Synthesis and characterization of fluorescent AgGaxIn1-xS2 QDs. According to the hard and soft acids and bases (HSAB) theory, Ag+ is a soft Lewis acid, which can readily react with soft


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Lewis base, i.e. S2-, to form a stable product. However, Ga3+ is a hard Lewis acid. As a consequence, it is difficult to directly synthesize AgGaS2 QDs with high crystallinity and uniformity because of the different reactivity between the two ions. In the realm of materials synthesis, cation change (CE) is a versatile and repeatable method for fabricating bulk materials as well as their nanocrystals that are difficult to obtain.35 CE is a kind of manageable reaction involves cation replacement between guest cations (in the reaction solution) and host cations (in the initial material) in the surface area, followed by cation diffusion within the material, where the anionic sublattice remains intact. We have demonstrated in our previous work that QDs with high quality and excellent optical properties can be prepared by the CE method through regulating the thermodynamic and kinetic parameters.36 In this study, AIS QDs were prepared as a pristine template using a “green” hydrothermal route. N-acetyl-L-cysteine (NAC) was used as a surfactant to form a cationic precursor, for the reason that NAC is a soft Lewis base ligand, in which the thiol group tends to coordinate with soft Lewis acid, i.e. Ag+. While, In3+ is a harder Lewis acid for its larger charge density and smaller ionic radius compared with Ag+.37,38 By this, a 7.5-fold excess of In3+-precursor was prepared to ensure the reaction. Transmission electron microscopy (TEM) image (Figure 1a) displayed that the sphere-shaped AIS QDs (denoted as sample S1) have an average size of 4.2 nm with excellent monodispersity and narrow size distribution (Figure S1, Supporting Information). The high resolution TEM (HRTEM) image exhibited the (202) lattice plane of the orthorhombic AIS, with a measured dspacing of 2.42 Å (Figure 1b). Energy dispersive X-ray spectrometer (EDS) spectrum (Figure 1c) presented the characteristic peaks of Ag, In and S with an atom ratio of [Ag]:[In]:[S]=1.03:0.98:2, which is in accordance with the ICP result (Table S1, Supporting Information) and the stoichiometry of AgInS2. Meanwhile, X-ray photoelectron spectra (XPS) confirmed the valence


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states of Ag+, In3+ and S2-, respectively (Figure S2, Supporting Information). In stead of a definite excitonic peak, the absorption spectrum showed a broad shoulder with an absorption onset, which is different with those of II-VI QDs. In addition, PL emission of AIS QDs demonstrated a broad profile with a full width at half maximum (FWHM) larger than 100 nm. These features, which are the characteristics of ternary I-III-VI semiconductors, may be the result of donor−acceptor (D-A) transition and trap states-related recombination.39,40 Moreover, the double-peak profile that was frequently seen in the previous studies was resulted from the surface states and intrinsic states transition respectively.39 X-ray diffraction (XRD) pattern illustrated an atacamite-like AgInS2 with an orthorhombic phase (Figure 1e), which matched well with the HRTEM data. Thus, the above results exhibited the synthesis of AIS QDs as the parent nanotemplate. To obtain AGIS QDs, various amounts of Ga3+ were introduced into AIS QDs through CE reaction (samples with increased Ga contents were denoted as S2~S5, details were shown in Experimental Section). Our previous studies illustrated that the key to a successful CE reaction is to balance the surface replacement and the internal diffusion of cations.36 In this reaction, glutathione (GSH) was employed as a Lewis base ligand, for that Ga3+ (hard Lewis acid) can easily coordinate with the amino (–NH2) in GSH.41,42 As revealed by TEM analysis (Figure 2a), the obtained AGIS QDs retained the original spherical shape and the monodispersity (Figure S3, Supporting Information). HRTEM image depicted continuous lattice fringes (Figure 2b), indicating the single crystalline nature of the AGIS QDs. The appearance of Ga peak in the EDS spectrum confirmed the incorporation of Ga3+ and the formation of AGIS after CE reaction (Figure 2c), which was also verified by the XPS observation (Figure S4, Supporting Information). With the addition of Ga3+ in precursors, increased amount of In3+ was replaced. It has been extensively proved that reduced reaction barrier on the nanoscale makes it possible to control the direction and


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progress of CE.35,43 During the reaction, both –SH and –NH2 in GSH could coordinate with In3+ that increased its dissociation from QDs and dissolution in the aqueous system, as confirmed by further FTIR determination (Figure S5, Supporting Information). In addition, high curvature surfaces of QDs, coupled with the difference in solubility of Ga3+ and In3+ which resulted from the participation of NAC and GSH ligands, enabled a dramatically expedite replacement in the surface area. Furthermore, reduced lattice energy before and after the reaction and the similar ionic valence of Ga3+ and In3+ in favored of the cationic diffusion progress inside the nanocrystals.37 As a consequence, CE could be manipulated through the adjustment of the reaction conditions. Specifically, we note that the Ag content remained invariant during the CE process (Figure S6, Supporting Information), which means that Ag+ did not exchanged by Ga3+. This may be the result of the larger ionic radius of Ag+ (1.15 Å) with respect to that of Ga3+ (0.62 Å)

37

and the strong

bond strength of Ag-S, suggesting that the replacement of Ga3+ with In3+ is more favorable than Ag+. At an initial ratio of [Ga]/[In]=3.73, In3+ ions were entirely replaced by Ga3+ ions, giving rise to the formation of AgGaS2 with a stoichiometry of [Ag]:[Ga]:[S]=1:0.98:2.11 confirmed by ICP (Table S1, Supporting Information) and XPS measurement (Figure S7, Supporting Information). TEM picture (Figure 2d) of AGS QDs exhibited nice monodispersity and narrow size distribution (Figure S8, Supporting Information) similar to the as-prepared AIS and AGIS QDs. HRTEM observation declared the high crystallinity of AGS QDs, with clear lattice fringes of (202) facet of the tetragonal phase with a d-spacing of 2.51 Å (Figure 2e). The characteristic peaks of Ag, Ga and S depicted by the EDS spectrum showed an atom ratio of [Ag]:[Ga]:[S]=0.96:0.92:2 (Figure 2f), suggesting the formation of AgGaS2 QDs.


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Analysis of absorption and PL spectra of the exchanged AGIS and AGS QDs yielded a detailed view on the evolution of the bandgap. As seen in Figure 2g, these samples displayed evident component-dependent absorption and PL emission. Similar to the AIS QDs, the absorption spectra of the AGIS QDs didn’t show a definite excitonic peak, and a large FWHM of each sample was also observed. With the incorporation of Ga3+, the absorption onset of each sample gradually shifted from ~595 nm of AIS to ~450 nm of AGS. Meanwhile, the PL wavelength shifted from 719 nm for AIS to 548 nm for AGIS (Table S2, Supporting Information), implying the broadening of the bandgaps. It has been reported that the conduction band of bulk AgInS2 semiconductor is formed by hybrid orbitals of In 5s5p and S 3p, while the conduction band of AgGaS2 is made up of Ga 4s4p hybridized with S 3p, where both of the valence bands of them is composed of Ag 4d and S 3p.44,45 Because of the similar electronic configuration of AIS and AGS, the position of the conduction band in AGIS can be elevated by decreasing the number of In 5s5p through the exchange of In to Ga. As a result, the bandgap was broadened with the continuous incorporation of Ga and the extraction of In, and thus AGS QDs with a emission wavelength of 502 nm and a QY up to 32% were achieved (Table S2, Supporting Information). Furthermore, it should be mentioned that the FWHM of each sample was gradually reduced, presumably may be the result of the reduction in structural defects during the CE reaction. Interestingly, XRD measurements demonstrated a successive evolution of the crystalline structure (Figure 2h). With the incorporation of Ga3+ and the extraction of In3+ (sample S2~S4), the diffraction patterns changed and some of the peaks vanished. After the complete replacement of In3+ by Ga3+, the XRD pattern of AgGaS2 (sample S5) could be indexed as the thermodynamically stable tetragonal phase with a chalcopyrite feature rather than the metastable orthorhombic phase. It is known that CE reaction is essentially a thermodynamically activated


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isotropic process which involves the dissociation of surface host cations (i.e. In3+ in this case) and the introduction of guest cations (i.e. Ga3+), where the anionic sublattice remains unchanged. For the previously reported exchange of Cu2S to ZnS,46 the initial hexagonal crystal structure was maintained due to the very similar ionic radius of Cu+ and Zn2+. The crystalline phase transformation in this reaction illustrates that low symmetry orthorhombic AIS underwent a rearrangement to the AGIS solid solution and finally to the tetragonal AGS with a higher symmetry (Figure 2i), owing to the comprehensive interaction of lattice strain, crystal energy and ionic diffusion rate.47 We further studied the evolution of XPS composition of the sample during the CE reaction (Table S3, Supporting Information). The determination provided a dramatic result that the [Ga]:[In] ratio in the obtained QDs (sample S3) increased sharply in the early stage of the reaction process (0~20 min), then gradually decreased (20~50 min) and remained unchanged eventually (>50 min), as shown in Figure S9. Since the XPS technique is known to be sensitive to the surface elements, this result manifests that cation exchange occurs mainly in the surface area of the particles, resulting in an inhomogeneous structure of AGIS. With prolonging the reaction time, Ga3+ ions gradually diffused inward along with the In3+ diffused outward, and a uniform alloy was achieved in the end. By tuning the amount of precursor, thus, AGIS and AGS QDs with a QY higher than 30% (Table S2) can be obtained. These results inspire us that the structure of the materials can be facilely tailored by controlling the thermodynamic and kinetic parameters during the CE reaction. This may be the first report of aqueous synthesis of the AGIS and AGS QDs. Polyelectrolyte modification of LDL and conjugation of nanoprobe. To facilitate targeted imaging using the overexpression of LDL receptors in tumor cells, LDL-based nanoprobe was fabricated by the cationic polyelectrolyte (PDDA) modification and subsequent electrostatic


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coupling with negatively charged QDs (Figure 3a). In order to achieve a better imaging result, QDs with the highest QY (sample S3) were chosen as the luminophore. TEM image of the isolated LDL (Figure 3b) showed an average size of 32.3 nm with a determined PDI of 0.163 and a zeta potential of −33.5 mV (Table S4, Supporting Information), suggesting that LDL was negatively charged with a good monodispersity. After coated with PDDA, the zeta potential switched to a positive value of +31.8 mV, which confirmed that LDL was modified by the cationic polyelectrolyte. Further adsorption of QDs again indicated a negative zeta potential of −13.1 mV with a PDI of 0.211, manifesting that the negatively charged QDs were successfully conjugated to LDL/PDDA via electrostatic attraction, as proved by the FTIR analysis (Figure S10, Supporting Information). TEM images of LDL/PDDA and LDL-QDs NCs also demonstrated good monodispersity with the maintained morphology (Figure 3c and 3d). Additionally, the result of ELISA assay (Figure 3e) revealed that no distinct difference in oxidation among LDL, LDLPDDA and LDL-QDs, illustrating that the polyelectrolyte modification and electrostatic grafting had no prominent effect on the morphology of LDLs. It should be mentioned that the adsorption of PDDA-decorated LDL and negatively charged QDs exhibited notable sensitivity and relevance to the concentration of salt in the dispersion (Figure 3f), with the other conditions being constant. As the NaCl concentration increased, the loaded QDs increased significantly. At the NaCl concentration of 0.6 M, the result showed the highest adsorption content of QDs. Higher concentration of NaCl, conversely, led to a decrease in the loaded QDs. Since metal ions may alter the spatial configuration of polymer chain to a certain extent,48 we speculate that due to the increased ion concentration in the solution, the ion shielding effect shrinked the polyelectrolyte chain, thereby greatly facilitated the assembly of QDs and polyelectrolytes. Further increase in


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NaCl concentration may unduly block the electronegativity of the QDs, resulting in a decrease in adsorption content. As a consequence, the optimized NaCl concentration should be 0.6 M. Structural stability, cytotoxicity and apoptosis assay. The UV/vis absorption of LDL depicted a distinct peak at 280 nm (Figure 4a), which was consistent with the previous paper and was the absorption of ApoB-100.49 The absorption spectrum of LDL-QDs NCs was similar with that of the LDL, except for a long tail marked with green-shaded area, which could be assigned to the absorption of the conjugated QDs. The PL emission of LDL-QDs didn’t show substantial changes compared with QDs. The far UV circular dichroism (CD) spectra were carried out in order to determine the structural stability of LDL before and after QDs conjugation. The negative peak at 220 nm with a broad shoulder around 208 nm (Figure 4b), derived from the absorption of αhelix and β-sheet secondary structure of ApoB-100 in LDL,50 were seen in both samples. These results indicated that the electrostatic adsorption of QDs didn’t alter the structure and properties of LDL. Furthermore, time-dependent hydrodynamic diameter and photostability investigations also revealed the high colloidal stability with vigorous bonding strength of LDL-QDs NCs (Figure S11, Supporting Information). The toxicity of QDs and corresponding nanoprobe is a key factor in determining whether it is feasible to be applied to cellular and clinical purposes. The cytotoxicity induced by AGIS QDs and QDs-LDL NCs was carefully examined by the MTT assay. As displayed in Figure 4c (left), QDs and LDL-QDs NCs didn’t exhibit significant cytotoxicity (P=n.s, t-test was showed in Figure S12, Supporting Information) for NCM-460 cells during the incubation. For comparison, the viability of cells incubated with CdTe QDs decreased rapidly with the increase of the incubating time (Figure 4c, right). Additionally, the cell viability of QDs and LDL-QDs treated cells was higher than 80% at a concentration of 0.75 mg mL-1. Conversely, cells treated with CdTe QDs


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demonstrated significantly lower viability at both concentrations (P

Low-Density

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