Folic Acid-conjugated AgInS2 Quantum Dots for in vitro Cancer Cell Imaging

BU Cheng-fei,LIU Li-wei*,WANG Qian,ZHANG Bu-tian, HU Si-yi,REN Yu,ZHU Ling-xi

(1.International Joint Research Center for Nanophotonics and Biophotonics,School of Science, Changchun University of Science and Technology,Changchun 130022,China; 2.School of Electrical and Electronic Engineering,Nanyang Technological University,Singapore) *Corresponding Author,E-mail:liulw@cust.edu.cn

Folic Acid-conjugated AgInS2Quantum Dots for in vitro Cancer Cell Imaging

BU Cheng-fei1,LIU Li-wei1*,WANG Qian1,ZHANG Bu-tian2, HU Si-yi1,REN Yu1,ZHU Ling-xi1

(1.International Joint Research Center for Nanophotonics and Biophotonics,School of Science, Changchun University of Science and Technology,Changchun 130022,China; 2.School of Electrical and Electronic Engineering,Nanyang Technological University,Singapore) *Corresponding Author,E-mail:liulw@cust.edu.cn

AgInS2quantum dots(QDs)were synthesized using hot colloidalmethod and transferred intowater via a ligand exchange route.To enhance the stability of these QDs,dBSA was coated on the aqueous QDs as a ligand shell.After dBSA modification,a size increase was observed from the transmission electron microscopy(TEM)results.The prepared dBSA-MPA QDs exhibited good monodispersity,enhanced stability(stable for 4 weeks)and bright photoluminescence.Then the QDswere functionalized with folic acid(FA)and the successful conjugation was confirmed by the Fourier transform infrared(FT-IR)spectroscopy.The resulting FA-dBSA-MPAQDs nanocomposites were tested in breast cancer cells(MCF-7 cells)with elevated folate receptor expression.Compared to the unconjugated dBSA-MPA QDs,the presence of FA on the surface of QDs significantly improved the uptake rate of the nanoparticles by the cancer cells.

AgInS2;quantum dots;fluorescence;in vitro;cell imaging

1 Introduction

Nanotechnology has received a great deal of attention in the fields ranging from biology to medicine[1-6].Quantum dots(QDs),the semiconductor nanocrystals,are known to be a potential candidate as optical probes for long term imaging in vitro and in vivo due to their unique optical properties,including high quantum yield,excellent photostability, broad absorption spectra with narrow photoluminescence(PL)spectra and long fluorescence lifetime[7-10].Nowadays,themost frequently used QDs are cadmium-based,which raise people's concern on the toxicity issue of these QDs,especially considering that they may degrade and release the Cd2+in the biological fluids.Cadmium-free QDs,as promising candidates for optical probes,are attracting increasing attention because of their lower intrinsic toxicity.However,it is still a challenge tomake the cadmium-free QDs with comparable optical propertieswith the cadmium-based QDs.The AgInS2QDs, containing low toxic components and as a type of ternary QDs,could tune the absorption and emission wavelength by adjusting the composition ratio.For instance,Xie et al.[11]developed a“greener”method for synthesizing nearlymonodispersed AgInS2QDs using indium stearate and silver nitrate in a non-coordinating organic solvent.In general,the fabrication of AgInS2QDs is commonly carried out in organic solvents using hydrophobic ligands to produce high-quality nanocrystals.These QDs with hydrophobic surfactants are not water dispersible.In order to employ QDs for biosensing and biomedical imaging applications,they need to be transferred into aqueous phase to acquire compatiblity with the biological environment[12-14].However,there remains a significant challenge to preserve the optical property of water-dispersible QD formulations with long shelf lives.Currently,many of the QD formulations are known to have short shelf lives from few hours to few days[3,15].Furthermore,some of these methods have led to the broadening of emission spectra and severe quenching of the emission intensity[16].

After phase transfer,the water-dispersible QDs are required to be functionalized with biomolecules for intended biological applications.For example, folic acid(FA)is often conjugated on QDs surface for specific uptake by cancer cells.FA is generally taken up into the cells by the folate receptor(FR) by initiating the receptor-mediated endocytosis[17]. FR is overexpressed in many human cancer tumors. For example,it is reported to be overexpressed in human nasopharyngeal carcinoma cells,and several other established cancer cell lines such as cancer cells originating from ovary,cervix,pancreas, breast,and myeloid leukemia[18].Our group has previously demonstrated the use of FA conjugated QDs as highly biocompatible probes for two-photon imaging of cancer cells[19].

In this study,we report the preparation of the high-quality luminescent ternary AgInS2QDs.These QDs were transferred into aqueous phase using MPA,stabilized by dBSA,and finally functionalized with FA.The resulting nanocomposites were tested in MCF-7 cell cultures with overexpression of folate receptors.By fluorescent imaging study,the presence of FA on the surface of QDs significantly improved their uptake rate by cancer cells.The excellent optical properties,high stability and high targeting ability suggest that the FA-dBSA-MPA QDs are potential candidates for cancer diagnosis.

2 Experiments

2.1 M aterials

Silver nitrate(AgNO3,99.9%),indium acetate(In(Ac)3,99.99%),sulfur(99.5%),1-dodecanethiol(98%)were purchased from Alfa Aesar.Stearic acid(98.5%),oleylamine(98%), oleic Acid(70%),1-octadecene(ODE,90%), 3-mercaptopropionic acid(MPA,99%),folic acid (FA,＞98%),N-hydroxysuccinimide(NHS), ammonium hydroxide solution(NH4OH,28.0%-30.0%NH3basis),and N-ethyl-N-(3-dimethylaminopropyl)carbodiimide(EDC)were purchased from Sigma-Aldrich.All organic solvents were purchased from EM Sciences.All chemicals were used directly without further purification.

2.2 Synthesis of AgInS2QDs

In a typical synthetic reaction,0.1 mmol Ag-NO3,0.1 mmol In(AC)3,0.3 mmol stearic acid, 0.6 mmol oleic acid,3 mmol 1-dodecylthiol and ODE(10 mL)in a 50 mL three-neck flask were heated to 80℃under flowing argon with magnetic stirring for 30 min.Afterwards,the temperature of the mixture was raised to 120℃.Subsequently, 0.3 mmol of oleylamine(4 mL)was injected as fast as possible.The reaction solution wasmaintained at 120℃.The obtained AgInS2nanocrystals were washed with ethanol by centrifugation at7 800 r/min for 5 min to remove the unreacted precursors,and the washing process was repeated three times.The purified nanoparticleswere then dispersed in chloroform for storage.

2.3 Preparing of dBSA-MPA QDs

For acquiring water dispersiblity of QDs,a short thiol ligand,MPA,was used to transfer the hydrophobic QDs to aqueous phase.Typically,MPA (4μL)dissolved in 1 mL of chloroform wasmixed with 1 mg of AgInS2QDs dispersed in 5mL of chloroform and themixture was stirred for 5 min.After 5 min,1mL of NH4OH solution(3%NH3basis)was added into the reaction mixture to from a biphasic solution system.After stirring for 5 h,majority of QDs were transferred to aqueous phase.The QDs were then collected and washed by centrifugation with ethanol,and subsequently redispersed and stocked in 1 mL of deionized water.

To obtain the dBSA for coating the QDs surface,BSA was firstly denatured.300mg of BSA was dissolved in 10 mL DIwater,then 7 mg of NaBH4was added into the BSA solution and the solution was stirred at30℃for 1 h until no hydrogen is generated.The concentration of the obtained dBSA solution was 100 mg/mL.40 mg/mL of dBSA was injected into 1 mg/mL of MPA-QDs solution and themixture was stirred for 30 min at the temperature of 80℃. The obtained dBSA-MPA QDs were purified by centrifugation.

2.4 Preparation of FA-conjugated QDs

In order to applying the dBSA-MPA QDs for applications of immuno-labeling and fluorescent imaging of cancer cells,these nanoparticleswere conjugated with FA.The conjugation of FA with dBSAMPA QDs was completed through the reaction between amino and carboxyl groups under activation of NHS and EDC.Typically,2 mg of dBSA-MPA QDs were dissolved in 2 mL of dimethylsulphoxide(DMSO),mixed with 2mg of FA,15mg of NHS,and 6 mg of EDC with magnetic stirring and kept for 4 h. The nanoparticleswere alternately rinsed and washed with ethanol for four times,and dispersed in 2mL of deionized water for further use.

2.5 Characterization

Fourier transform infrared(FT-IR)spectra weremeasured by a Thermo Scientific Nicolet iS50 FT-IR spectrometer.Transmission electronmicroscopy(TEM)imageswere obtained using a Tecnai G2 F20 microscope at an acceleration voltage of 200 kV.TEM samples were prepared by dropping a dilute solution of QDs on carbon-coated copper grids (Formvar/Carbon 300 Mesh)and by slowly evaporating the solvent in air at room temperature.The measurements of PL spectra were carried out by using a Agilent Cary Eclipse spectrofluorometer equipped with a 150 W xenon lamp.Cell imaging was performed on a Leica DMI3000B inverted confocalmicroscope(Leica Microsystems).We used a Zetasizer Nano ZS90(Malvern Instruments)tomeasure the Zeta potential of dBSA-MPA QDs and FA-conjugated QDs dispersed in 1mL of ultrapure water.

3 Results and Discussion

Energy dispersive X-ray spectroscopy(EDS) shown in Fig.1(a)was employed to determine the elemental ratios of the AgInS2QDs.From the EDS spectra,the weight ratio of Ag:In:S is 36.66:34.9: 28.44 and the corresponding elemental ratio was calculated to be 1:0.9:2.6.The strong signals for Cu come from the copper grid on which the AgInS2QDswere supported.The deviation of themeasured S emental ratio from its precursor ratio suggests an excess S content in the obtained QDs andmay be attributed to the capping of n-dodecylthiol[20].The normalized PL emission spectra of the AgInS2QDs, dBSA-MPA QDs,and FA-conjugated QDs are pres-ented and compared in Fig.1(b).The emission peaks of AgInS2QDs in chloroform solution,dBSAMPA QDs and FA-conjugated QDs in the aqueous phase locate at631,650,665 nm,respectively.It isworthmentioning that there is a 19 nm red shift of emission peak after ligand exchange using MPA and another 15 nm red shift of emission peak after FA conjugation.A similar red-shift trend can be observed in the absorption spectra of the modified QDs.The phase transfer of the QDs using thiol ligand(MPA)was accompanied by a red shift in PL spectra,which may be a consequence of the newformed oxide layer or defects during the ligand exchange process[14,16,21].The quantum yield(QY)of the AgInS2QDs,dBSA-MPA QDs and FA-conjugated QDs dispersed in buffer was estimated to be about 16%,14%and 13%,respectively.After modification,we observe the QDs dispersion stable for four weeks.The Zeta potential values of dBSAMPA QDs and FA-conjugated QDs dispersed in ultrapure water were-28.4 mV and-23.6 mV, respectively.After conjugated with FA,the Zeta potential of QDs is almost unchanged,indicating the repulsive electrostatic force between the nanoparticles for keeping their stability.

Folate targeting is an emerging approach for cancer theranostics[22-23].Folate receptor(FR)has been extensively considered as a tumor maker because of the selectively high expression of FR on the surface ofmany human cancer cells[24](e.g.ovarian, lung,breast,kidney,brain and pancreatic cancer cells).FA exhibits high affinity for FR,which could be captured by FR from the extracellular environment and traffic inside the cell within the recycling endosomal compartments[25].Therefore,we functionalized the fluorescent dBSA-MPA QDs with FA to target the MCF-7 cells based on FA-FR-mediated endocytosis.Carbodiimide coupling reaction was used to conjugate the carboxyl group of the MPA/dBSA molecules and amino group of FA[26-27]. The conjugation was examined by comparing the FTIR spectra of dBSA-MPA QDs and that of the FA-dBSA-MPA QDs(Fig.2).The successful conjugation was confirmed by the appearance of the FA characteristic peaks(1 695,1 606,1 485 cm-1)in the spectra of FA-dBSA-MPA QDs.

Fig.1 (a)EDS spectrum and calculated weight ratio(inset)of AgInS2 QDs.(b)Absorption and normalized PL spectra of the hydrophobic AgInS2 QDs(in chloroform),dBSA-MPA QDs and FA-dBSA-MPA QDs (in water).

Fig.2 FTIR spectra of FA,dBSA-MPA QDs,and FA-dBSA-MPA QDs,respectively.

Fig.3(a)shows the TEM image of the organically dispersible AgInS2QDs,The high resolution image shows that the QDs are highly crystalline and have an average diameter of4.7 nm.From the TEM images,the average size of dBSA-MPA QDs and FA-dBSA-MPA QDswas estimated to be 9.6 nm and 14.8 nm,respectively(Fig.3(b)and 3(c)). Therefore,a size increase was observed when coating BSA ligand shell and conjugating FA molecules.

Fig.3 TEM images of the synthesized AgInS2 QDs(a),dBSA-MPA QDs(b),FA-dBSA-MPA QDs(c),and their corresponding digital photographs(inset).

The potential clinical applications of the resultant luminescent FA-conjugated QDs in the diagnosis and treatment of cancers or other diseases depend on their cytotoxicity.In this case,the MTT assay was used to evaluate the cytotoxicity of FA-conjugated QDs and the viability of cells.As shown in Fig.4, the viability of the MCF-7 cells still remained above 75%after incubation with QDs even at the concentration of 300μg·mL-1for 24 h and 48 h.The results confirm that our FA-conjugated QDs show an excellent biocompatibility and have no adverse effect to MCF-7 cells at the concentration we used for cell imaging.

Fig.4 Relative cell viability ofMCF-7 cells treated with FA-conjugated QDs formulation for 24 and 48 h post-treatment

Fig.5 Fluorescent images of MCF-7 cells treated with FA-conjugated QDs and unconjugated QDs

In Fig.5,the selective uptake of different QDs by MCF-7 cells was monitored by fluorescence microscopy.The MCF-7 cells were separately treated with 50μg/mL MPA-QDs,dBSA-MPA QDs or FA-dBSA-MPA QDs for 4 h before they were examined under the microscope.In Fig.5(a),the red fluorescent signals demonstrated a strong uptake of FA-dBSA-MPA QDs by MCF-7 cells.As a comparison, weak fluorescence was observed for MCF-7 cells treated with dBSA-MPA QDs(Fig.5(d)).In order to reduce FR on the surface of the MCF-7 cell,we added folic acid solution to cells first.Then we incubated the FR-negative MCF-7 cells with FA-conjugated QDs of 50μg/mL for 4 h and then washed by PBS for two times.As shown in the Fig.6,the fluorescent signal displayed in the MCF-7 cells is much weaker than that in the cells treated with the FA-conjugated QDs only.These cell fluorescent imaging results suggest that the conjugated FA efficiently increased the uptake of dBSA-MPA QDs by MCF-7 cells.

Fig.6 Overlay(a),transmission(b),and fluorescent(c)images of MCF-7 cells treated with excess folic acid and FA-conjugated QDs of 50μg/mL,respectively.

4 Conclusion

In summary,we report a successful preparation of the high-quality luminescent ternary AgInS2QDs. The AgInS2QDswere synthesized using a hot colloidal method and exhibit strong fluorescence.After phase transfer by MPA and coating by BSA,the resulted QDs remain their fluorescent properties and acquire good dispersibility and stability in water. Later,the dBSA-MPA QDswere conjugated with FA for specific uptake by cancer cells.The resulting nanocomposites were tested using MCF-7 cell line with overexpressed folate receptors.The presence of FA on the surface of QDs significantly improved the uptake of these nanocomposites by targeted cells. Overall,the excellent optical properties,good stability and enhanced cancer-targeting efficiency suggest that FA-dBSA-MPA QDs are potential candidates for cancer diagnosis.

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卜承飛(1990-),男,江蘇揚州人,碩士研究生,2013年于長春理工大學獲得學士學位,主要從事納米光子學與生物光子學方面的研究。

E-mail：buchengfei1990@163.com

劉麗煒(1979-),女,吉林長春人,博士生導師,2013年于長春理工大學獲得博士學位,主要從事納米光子學與生物光子學方面的研究。

E-mail：llw_cust@163.com

葉酸修飾的AgInS2量子點在體外癌細胞成像上的應用

卜承飛1,劉麗煒1*,王 倩1,張卜天2,胡思怡1,任 玉1,朱泠西1

(1.長春理工大學理學院國際納米光子學與生物光子學聯合研究中心,吉林長春 130022; 2.南洋理工大學電氣與電子工程學院,新加坡)

成功制備出高品質的三元AgInS2量子點。通過配體交換法將油溶性AgInS2量子點轉為水溶性量子點,通過dBSA修飾水溶性量子點形成配位體殼,使量子點具有更好的穩定性(4周)。從透射電子顯微鏡(TEM)觀察到dBSA修飾后的量子點的粒徑增加,分散性較好,并且在可見光區域有明顯的光致發光。用葉酸對dBSA-MPA量子點進行修飾,并通過傅立葉變換紅外光譜進行了驗證。將得到的FA-dBSA-MPA納米復合材料應用于能與葉酸受體特異性結合的乳腺癌細胞中,并在熒光倒置顯微鏡中檢測到量子點成功對乳腺癌細胞進行了標記。與dBSA-MPA量子點相比,表面被葉酸修飾后的量子點與癌細胞的結合效率顯著提高。

AgInS2;量子點;熒光;體外;細胞成像

2015-06-14;

2015-07-24

國家自然科學基金(11204020);吉林省國際納米光子學與生物光子學重點實驗室支撐項目(20140622009JC);長春市科技計劃(14GH005)資助項目

O482.31 Document code：A

10.3788/fgxb20153609.0989

1000-7032(2015)09-0989-07