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aResearch Center of Nano Science and Tec

200444, P. R. China. E-mail: lnsun@

+86-21-66137153bDepartment of Urology, Renji Hospital, S

University, Shanghai 200127, P. R. China. EcLaboratory of Molecular Neural Biology, Sch

Shanghai 200444, P. R. China

† Electronic supplementary informatiluminescence spectra of UCNC-ErUCNC-Tm-FA. Confocal luminescence imthe Z optical axis. See DOI: 10.1039/c4nr0

Cite this: Nanoscale, 2014, 6, 8878

Received 29th April 2014Accepted 29th May 2014

DOI: 10.1039/c4nr02312a

www.rsc.org/nanoscale

8878 | Nanoscale, 2014, 6, 8878–8883

Folic acid-functionalized up-conversionnanoparticles: toxicity studies in vivo and in vitroand targeted imaging applications†

Lining Sun,*a Zuwu Wei,a Haige Chen,*b Jinliang Liu,a Jianjian Guo,c Ming Cao,b

Tieqiao Wenc and Liyi Shi*a

Folate receptors (FRs) are overexpressed on a variety of human cancer cells and tissues, including cancers of

the breast, ovaries, endometrium, and brain. This over-expression of FRs can be used to target folate-linked

imaging specifically to FR-expressing tumors. Fluorescence is emerging as a powerful new modality for

molecular imaging in both the diagnosis and treatment of disease. Combining innovative molecular

biology and chemistry, we prepared three kinds of folate-targeted up-conversion nanoparticles as

imaging agents (UCNC-FA: UCNC-Er-FA, UCNC-Tm-FA, and UCNC-Er,Tm-FA). In vivo and in vitro

toxicity studies showed that these nanoparticles have both good biocompatibility and low toxicity.

Moreover, the up-conversion luminescence imaging indicated that they have good targeting to HeLa

cells and can therefore serve as potential fluorescent contrast agents.

1. Introduction

Cancer is a major public health problem and greatly endangershuman health; it is becoming the primary cause of death inhumans.1 It is particularly important to detect cancer at an earlystage of the disease. Optical techniques are emerging aspowerful methods for molecular imaging in both detecting andtreating cancer.2,3 Imaging plays a key part in helping cliniciansto detect solid tumors, detect the recurrence of disease, and toassess the success of treatment regimens. The synthesis ofuorescent materials is a critical part of optical imaging tech-niques. There are three general classes of imaging materialsused to aid visualization: organic dyes,4–9 inorganic semicon-ducting quantum dots,10–17 and up-conversion luminescentnanoparticles (UCNs).18–25 Compared with organic dyes andquantum dots, UCNs doped with rare-earth elements are rela-tively new uorescent materials that have received increasingattention in recent years. They have many advantages, such as aweak autouorescence background, low toxicity, and excellentchemical stability.26–28

hnology, Shanghai University, Shanghai

shu.edu.cn; shiliyi@shu.edu.cn; Tel:

chool of Medicine, Shanghai Jiao Tong

-mail: kirbyhaige@aliyun.com

ool of Life Sciences, Shanghai University,

on (ESI) available: Up-conversionand UCNC-Er-FA, UCNC-Tm andaging data collected as a series along2312a

Recent efforts to develop tumor-specic imaging agents havefocused on the use of targeting ligands that deliver radio-emitters or contrast agents to receptors overexpressed on cancercells.29 The folate receptor (FR) is a highly selective tumormarker overexpressed in >90% of ovarian carcinomas as well asnumerous other cancers, including endometrial, kidney, lung,mesothelioma, breast, and brain cancer, and also in myeloidleukemia.30 Because many cancers overexpress FRs while mostnormal tissues express low to negligible levels, FRs could proveto be of clinical signicance, particularly with regard to thecurrent development of folate-targeted imaging and therapeuticagents.30,31 To date, various folate–drug conjugates havedemonstrated possible therapeutic value in animals. Folate-targeted imaging agents are currently being evaluated.31–34

In the work reported here, three kinds of folate-targeted UCNimaging agents (UCNC-FA: UCNC-Er-FA, UCNC-Tm-FA, andUCNC-Er,Tm-FA) were designed and prepared. These nano-particles show good biocompatibility, up-conversion lumines-cence (UCL) emission in water, and low cytotoxicity in vivo andin vitro. Furthermore, these nanoparticles have been success-fully applied to the targeted imaging of cells.

2. Experimental methods2.1 Materials and chemicals

YCl3$6H2O (99.99%), YbCl3$6H2O (99.99%), ErCl3$6H2O(99.99%), TmCl3$6H2O (99.99%), 1-ethyl-[3-(3-dimethylamino)-propyl] carbodiimide hydrochloride, and N-hydroxysuccinimidewere bought from Sigma-Aldrich Co. Ltd. The UCNs weresynthesized by a solvo-thermal method.35 Cysteine, 3-chlor-operoxybenzoic acid, acetone, and cyclohexane were obtained

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from Sinopharm Chemical Reagent Co. Ltd. 1-Octadecene,methanol, and folic acid (FA) were purchased from AladinCompany. Oleic acid was purchased from Alfa Aesar. All thesematerials were used as received without further purication.Deionized water was used for all the experiments.

2.2 Synthesis of cysteine-modied UCNs (named as UCNC:UCNC-Er, UCNC-Tm, and UCNC-Er,Tm)

Cysteine-modied UCNs were prepared based on previouslyreported methods.20 Briey, 2 mL of as-prepared UCN[NaYF4:Yb (20%), Er (2%); NaYF4:Yb (20%), Tm (1%); NaYF4:Yb(20%), Er (1.6%), Tm (0.4%)], 2 mL of cyclohexane, and 2 mL ofCH2Cl2 were mixed with 5 mg of 3-chloroperoxybenzoic acid.The resulting mixture was heated to 40 �C with constant stir-ring. Aer 3 h the mixture was cooled down to room tempera-ture, cysteine (0.02 g) was added, and the mixture was reacted at25 �C for 5 h. The resultant product was then separated bycentrifugation and washed several times with deionized waterand ethanol. The three samples of cysteine-modied hydro-philic UCNs, named as UCNC-Er, UCNC-Tm, and UCNC-Er,Tm,were re-dispersed in 20 mL of H2O.

2.3 Synthesis of FA-conjugated UCNC (named as UCNC-FA:UCNC-Er-FA, UCNC-Tm-FA, and UCNC-Er,Tm-FA)

The modication of UCNC with FA was based on a previouslyreported method.36 To conjugate FA with UCNC, FA (15 mg) wasdissolved in anhydrous dimethyl sulfoxide (DMSO, 20 mL) andactivated by 1-ethyl-[3-(3-dimethylamino)propyl] carbodiimidehydrochloride and sulfo-N-hydroxysuccinimide at room temper-ature under nitrogen for 2 h. The UCNC sample (30 mg)dispersed in DMSO (10 mL) was then added to this solution.37 FAconjugation was performed under nitrogen at room temperaturefor 12 h. The mixture was centrifuged at 12 000 rev min�1 for20 min and subsequently washed several times with water. Thethree samples were then re-dispersed in 10 mL of ethanol.

2.4 Characterization

Transmission electron microscopy (TEM) images were recordedon a JEM-200CX transmission electron microscope with anaccelerating voltage of 120 kV. Zeta potential experiments werecarried out using a Zeta-Sizer 3000HS instrument. Up-conver-sion luminescence spectra were acquired in an Edinburgh FLS-920 uorescence spectrometer with an external 2 W semi-conductor laser (980 nm, Beijing Hi-tech Optoelectronic Co.,China) as the excitation source, instead of the xenon source inthe spectrophotometer. Laser-scanning up-conversion lumi-nescence imaging of HeLa cells and MCF-7 was executed usinglaser-scanning up-conversion luminescence microscopy(LSUCLM) under the excitation of a CW 980 nm laser. Thehistological sections were observed under a uorescencemicroscope.

2.5 Cell culture

HeLa cells were supplied by the Shanghai Institutes for Bio-logical Sciences, Chinese Academy of Sciences. The HeLa cells

This journal is © The Royal Society of Chemistry 2014

were cultivated in Roswell Park Memorial Institute's medium(RPMI) 1640 supplemented with 10% fetal bovine serum at 37�C and 5% CO2.38–40

2.6 Laser-scanning up-conversion luminescence imaging

HeLa cells and MCF-7 were plated on 14 mm glass cover slipsand allowed to adhere for 24 h. Aer washing with PBS the cellswere incubated in a serum-free medium containing 300 mgmL�1 UCNC-Er for 3 h at 37 �C under 5% CO2 and thenwashed with sufficient PBS to remove any excess nanoparticles.Laser-scanning up-conversion luminescence imaging ofHeLa cells and MCF-7 were executed on a laser-scanning up-conversion luminescence microscope using excitation with aCW 980 nm laser.41

2.7 In vivo toxicity studies

To investigate whether UCNC-FA caused any detrimentaleffects, we carried out hematoxylin and eosin (H&E) stainingand blood chemistry analysis on healthy mice.42 Four-week-oldKunming mice were provided by the Shanghai Slack LaboratoryAnimal Co. Ltd. The animal procedures conformed to theguidelines of the Institutional Animal Care and Use Committee.UCNC-Er,Tm-FA at a total dose of 0.3 mg (0.15 mL of 2 mgmL�1

UCNC-Er,Tm-FA in normal saline per mouse) were injected intoKunming mice (n ¼ 2, weight ca. 20 g) via the tail vein; the twomice constituted the test group. Kunming mice (n ¼ 2) injectedwith normal saline were selected as the control group. Forhistological and hematological studies, blood samples andtissues were harvested frommice injected with UCNC-Er,Tm-FA(15 mg kg�1) 7 days post-injection and from the mice whichreceived an injection of normal saline. Blood was collected fromthe hearts of the mice quickly aer they were anaesthetized.Seven important hepatic indicators (alanine aminotransferase,aspartate aminotransferase, alkaline phosphatase, globulin,total bilirubin, albumin, and total protein) and three indicatorsof kidney function (urea nitrogen, creatinine, and uric acid)were measured. Upon completion of the blood collection, themice were killed. The liver, spleen, heart, lungs, and kidneyswere removed and para-xed in formalin, embedded inparaffin, sectioned, and stained with H&E.42 The histologicalsections were observed under a uorescence microscope.

2.8 Cytotoxicity assay

The in vitro cytotoxicity was studied by the methyl thiazolyltetrazolium (MTT) reduction assay.43HeLa cells were cultivated ina 96-well cell-culture plate at a density of 96 cells per well for 12 h.Subsequently, the medium was substituted for fresh medium atdifferent concentrations of UCNC-Er-FA.44 Aer 24 h, themediumcontaining the nanoparticles was removed and 100 mL of MTTsolution (dissolved in RPMI 1640) were added for another 4 h,followed by the addition of DMSO to dissolve the formazannanoparticles.45 The absorbance was measured by a Bio-RadModel-680 microplate reader at a wavelength of 490 nm. Thefollowing formula was applied to calculate the viability of cellgrowth: cell viability (%) ¼ (mean absorbance value of treatmentgroup/mean absorbance value of control) � 100%.46,47

Nanoscale, 2014, 6, 8878–8883 | 8879

Fig. 2 Up-conversion luminescence spectra of UCNC-Er,Tm (black,in water) and UCNC-Er,Tm-FA (red, in water) with excitation at980 nm.

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3. Results and discussion3.1 Synthesis and characterization of FA-conjugated UCNC(UCNC-FA)

The analysis of size and morphology was performed by TEM.As shown in Fig. 1, the TEM images of UCNC are very similar toeach other, as shown by the representative pattern of theUCNC-Er,Tm-FA. From Fig. 1a it can be observed that thesynthesized UCNC-Er,Tm exhibits a very uniform particle sizedistribution of about 30–40 nm, with a regular morphologyand perfect monodispersity. The TEM image of UCNC-Er,Tm-FA (Fig. 1b), as the representative image of UCNC-FA,is very similar to that of UCNC-Er,Tm (Fig. 1a), which showsthat the morphology, size, and dispersity of the UCNC-FA arewell retained aer the conjugation of FA on the surface ofUCNC. The zeta potential experiments were carried out onUCNC and UCNC-FA, and the zeta potential results forUCNC-Er and UCNC-Er-FA were about 41.2 and 24.1 mV,respectively. From the change in the zeta potential data, itcan be deduced that FA was successfully loaded onto thesurface of the UCNCs.

The UCL spectra of UCNC-Er,Tm and UCNC-Er,Tm-FA areshown in Fig. 2. Under excitation of the CW laser at 980 nm,the UCNC-Er,Tm showed distinct emission bands of bothTm3+ and Er3+ ions. The UCL bands at 475, 695, and 800 nmoriginate from the 1G4 / 3H6,

3F3 / 3H6, and3H4 / 3H6

transitions of Tm3+, respectively, and the UCL bands at 521,542, and 654 nm are assigned to the 4H11/2 / 4I15/2,

4S3/2 /4I15/2, and 4F9/2 / 4I15/2 transitions of Er3+, respectively.Compared with the UCL spectrum of UCNC-Er,Tm, the UCLspectrum of UCNC-Er,Tm-FA in H2O (Fig. 2, red line) shows noobvious difference, except for a decrease in the luminescenceintensity. The UCL spectra of UCNC-Er and UCNC-Er-FA,UCNC-Tm and UCNC-Tm-FA are shown in Fig. S1 and S2,†respectively. For UCNC-Er-FA, the luminescence spectrumdisplays the well-known emission peaks at 521, 539, and651 nm, ascribed to the transitions from the 4H11/2,

4S3/2, and4F9/2 energy levels to the ground state 4I15/2 of the Er3+ ion,respectively, similar to that of UCNC-Er. In Fig. S2,† theemission features of UCNC-Tm and UCNC-Tm-FA are similarand they both show emission bands at 475, 695, and 800 nm,assigned to the transitions 1G4 /

3H6,3F3 /

3H6, and3H4 /

3H6 of the Tm3+ ion, respectively.

Fig. 1 TEM images of (a) UCNC-Er,Tm and (b) UCNC-Er,Tm-FA.

8880 | Nanoscale, 2014, 6, 8878–8883

3.2 Cytotoxicity assay

To address the toxicity of UCNC-FA, the metabolic activities ofHeLa cells were measured using an MTT cytotoxicity assay(Fig. 3). The detailed procedure for theMTT assay was presentedin the experimental section. Aer incubation with UCNC-Tm-FAover a range of dosages from 0 to 800 mg mL�1, even at thehighest concentration of 800 mg mL�1 the HeLa cells still showmore than 90% cell viability. On the basis of the MTT assayresults, it can be deduced that UCNC-FAs can be applied in thebiomedical eld with good biocompatibility and can serve assafe luminescent bioprobes.

3.3 Histology and hematology results

For an assessment of in vivo toxicity, the UCNC-FA were injectedinto Kunmingmice via the tail vein. Tissues for histology analysiswere acquired from the harvested organs (heart, lung, liver,spleen, and kidneys). Fig. 4 shows theH&E-stained tissue sectionsfrom mice injected with UCNC-Er,Tm-FA 7 days post-injection(test group) and frommice receiving an injection of normal saline(control group). It was observed that the structures of the organsfrom the test group were normal without any signicant differ-ence from those of the control group; they exhibited no tissuedamage, inammation, or lesions from toxic exposure.

Fig. 3 In vitro cell viability of HeLa cells incubated with UCNC-Tm-FAat different concentrations for 24 h.

This journal is © The Royal Society of Chemistry 2014

Fig. 4 H&E-stained tissue sections from mice injected with UCNC-Er,Tm-FA (dose 15 mg kg�1) 7 days post-injection (test) and micereceiving an injection of normal saline (control). Tissues were har-vested from the heart, liver, spleen, lung, and kidneys.

Fig. 5 Serum biochemistry results obtained from mice injected withUCNC-Er,Tm-FA 7 days post-injection (n ¼ 2, dose 15 mg kg�1, test)and mice receiving an injection of normal saline (n¼ 2, control). Thesefindings do not indicate a trend of toxicity. ALT ¼ alanine amino-transferase, AST ¼ aspartate aminotransferase, ALP ¼ alkaline phos-phatase, GLOB¼ globulin, T-BIL¼ total bilirubin, ALB¼ albumin, TP ¼total protein, BUN ¼ urea nitrogen, CRE ¼ creatinine, and UA ¼ uricacid.

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Established serum biochemistry assays are useful to evaluatethe inuence of UCNC-FA on the exposed mice more quantita-tively, especially for potential injuries to the liver and spleen,and effects on kidney function. Fig. 5 shows the serumbiochemistry results obtained from mice injected with UCNC-Er,Tm-FA 7 days post-injection and mice receiving an injectionof normal saline. In Fig. 5, seven hepatic function markers(alanine aminotransferase, aspartate aminotransferase, alka-line phosphatase, globulin, total bilirubin, albumin, and totalprotein) and three indicators for kidney function (urea nitrogen,creatinine, and uric acid) were measured. All the factors arewithin the normal range and are at similar levels for the testmice and the control mice, suggesting that there was noapparent toxicity of UCNC-Er,Tm-FA in mice at exposure levelsbeyond those commonly used in luminescent in vivo imagingand at an exposure time of up to 7 days. Therefore, these in vivotoxicity assays indicate that the UCNC-FA has no evident toxiceffects and can serve as potential uorescent contrast agents.

3.4 Targeting ability of UCNC-FA

To demonstrate the FA-mediated targeted delivery, we investi-gated the FA-positive [FR(+)] HeLa cell lines and FR-negative[FR(�)] MCF-7 cell lines. The experimental results were

This journal is © The Royal Society of Chemistry 2014

evaluated by LSUCLM under CW excitation at 980 nm (Fig. 6).Aer 1 h of incubation with UCNC-Er-FA at 37 �C, the HeLa cellsshowed an extensive and bright luminescence signal (Fig. 6a–d),whereas the MCF-7 cells displayed a much weaker lumines-cence (Fig. 6e–h). In addition, from overlays of the confocalluminescence and bright-eld images (Fig. 6d and h), it can beobserved that the luminescence was obvious on the cytosol ofthe HeLa cells. These results suggest that the HeLa cells show ahigher-level FR expression than the MCF-7 cells and that theUCNC-FA nanocomposites selectively accumulated on thecytosol of the [FR(+)] HeLa cells.

To further conrm the receptor-mediated endocytosis of theUCNC-FA nanocomposites, we also conducted inhibitionexperiments under the same experimental conditions. For acompetitive reaction, [FR(+)] HeLa cells were simultaneouslyincubated with free FA at 37 �C for 1 h and then with UCNC-Er-FA nanocomposites at 37 �C for 1 h. As shown in Fig. 6i–l, onlyvery weak luminescence signals were observed, suggesting thatthe FR-mediated targeted delivery was effective. Furthermore,the confocal luminescence imaging data collected as a seriesalong the Z optical axis (Z-stack) (Fig. S3†) conrmed that theluminescence signals of the UCNC-Er-FA were indeed localizedwithin the HeLa cells. Therefore this result shows that UCNC-FAcan target tumor cells with large numbers of FRs.

4. Conclusion

In summary, we have developed three kinds of novel folate-targeted up-conversion nanoparticle imaging agents (UCNC-Er-FA, UCNC-Tm-FA, UCNC-Er,Tm-FA). The obtained UCNC-FAimaging agents remain water-dispersible. The MTT assaysuggested that the UCNC-FA displayed good in vitro

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Fig. 6 Confocal luminescence images of [FR(+)] HeLa cells (a–d) and [FR(�)] MCF-7 cells (e–h) stained with UCNC-Er-FA nanocomposites (300mg mL�1) for 1 h at 37 �C. For the competition experiments, [FR(+)] HeLa cells were first incubated with excess free FA and then UCNC-Er-FAnanocomposites under the same conditions (i–l). Fluorescent images were collected at the green (520–560 nm) and red (630–670 nm)channels.

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biocompatibility. The results of histological and hematologicalstudies illustrated that the UCNC-FA show no apparent toxicityto living systems (Kunming mice). Moreover, up-conversionluminescence imaging and the competition experiment showedthat the UCNC-FA have good targeting ability towards HeLacells. The experimental results show that the as-preparedUCNC-FA exhibit excellent properties, including low toxicity,good biocompatibility, and cell-specic targeting, making thempromising potential uorescent contrast agents.

Acknowledgements

We are grateful for nancial support from the National NaturalScience Foundation of China (Grant nos 21001072, 21231004,and 21201117), the Innovation Program of the ShanghaiMunicipal Education Commission (13ZZ073), the Science andTechnology Commission of Shanghai Municipality(13NM1401100, 13NM1401101), the Shanghai Rising-StarProgram (14QA1401800), and the project from State Key Labo-ratory of Rare Earth Resource Utilization (RERU2014012). Weare also grateful to the Instrumental Analysis & Research Centerof Shanghai University.

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