Registered charity number: 207890

Showcasing work from School of Medical Imaging,

Tianjin Medical University, Tianjin, China.

Green and facile synthesis of a theranostic nanoprobe with

intrinsic biosafety and targeting abilities

The simple mixing of transferrin and indocyanine green without

any toxic reagents and tough synthesis conditions enables

self-assembly in aqueous solution to form nanoparticles with

excellent water solubility and colloidal stability, neglectable

cytotoxicity and in vivo toxicity, remarkable active targeting

imaging ability and impressive photothermal therapy of tumors

in vitro and in vivo . This work presents the simplest method for

the fabrication of active targeting theranostic agents, and also the

fi rst example of active targeting theranostic agents synthesized

using green reagents and procedures.

www.rsc.org/nanoscale

Nanoscalewww.rsc.org/nanoscale

ISSN 2040-3364

PAPER G. Kioseoglou, E. Stratakis et al. Spatial non-uniformity in exfoliated WS

2 single layers

Volume 8 Number 36 28 September 2016 Pages 16075–16530

As featured in:

See Shao-Kai Sun et al. , Nanoscale , 2016, 8 , 16204.

Nanoscale

PAPER

Cite this: Nanoscale, 2016, 8, 16204

Received 3rd March 2016,Accepted 31st July 2016

DOI: 10.1039/c6nr01845a

www.rsc.org/nanoscale

Green and facile synthesis of a theranosticnanoprobe with intrinsic biosafety andtargeting abilities†

Cai Zhang,‡b Li Zhou,‡a Jing Zhang,a Yan-Yan Fu,a Xuejun Zhang,a Chunshui Yu,a,b

Shao-Kai Sun*a and Xiu-Ping Yanc

Traditional targeting nanoprobes suffer from the risks of partial loss of targeting activity and nanoparticle

aggregation induced by post-synthetic modifications, ambiguous toxicity, tedious synthesis procedures

and environmentally hazardous processes. Herein, we report a green and facile strategy to fabricate trans-

ferrin–indocyanine green nanoparticles as a smart theranostic agent with intrinsic biosafety and active

targeting abilities for near-infrared fluorescent imaging and photothermal therapy of tumors. Simple

mixing of transferrin and indocyanine green enables their self-assembly in aqueous solution to form

nanoparticles with excellent water solubility, colloidal stability, favorable biocompatibility and impressive

active targeting theranostic effects in vitro and in vivo. The transferrin–indocyanine green nanoparticles

show great potential in theranostic applications of tumors in clinical therapy.

1. Introduction

Targeting theranostic nanoprobes play a vital role in bio-imaging and therapy in vivo, and give profound significancefor the development of precision medicines with minimizedsystematic effects.1–6 Generally, for the purpose of active tar-geted imaging and therapy, the nanoprobes must be modifiedwith targeting ligands such as antibodies, aptamers, peptidesand small molecules.7–10 Unfortunately, the post-syntheticmodifications, based on strong covalent or coordination inter-actions between the ligands and nanoparticles, often suffersfrom the risks of partial loss of the targeting ability of theligands and the aggregation of nanoparticles, leading to lowsensitivity and specificity and poor therapeutic effects.7–10

Therefore, it is a great challenge to develop active targetingtheranostic agents capable of maintaining the activity of thetargeting ligands to the greatest extent.

The biosafety of theranostic nanoprobes also attractsincreasing attention.11–14 Although plenty of nanoprobes withexcellent imaging abilities, effective therapeutic effects andremarkable targeting abilities are available, the ambiguoustoxicity of non-biodegradable components has highly hinderedtheir application in clinical therapy.15–21 In addition, tedioussynthetic procedures, the necessity for toxic reagents, harshreaction conditions and large energy consumption in most ofthe traditional methods have also limited the large-scale andenvironmentally-friendly production of the nanoprobes withhigh reproducibility.3–6 As a result, only a few imaging or thera-peutic nanoparticles have been applied in the clinic, whichaccounts for a negligible proportion of thousands of availabletheranostic nanoprobes.15–18 Recently, albumin-based imagingand therapeutic nanoprobes, on the basis of the hydrophobicinteraction between protein and organic molecules, have pro-vided a versatile way for the fabrication of biocompatible nano-materials, but lack the active targeting ability.22–28 So, thedesign a smart strategy for the fabrication of theranostic nano-probes with excellent active targeting ability and impressivebiocompatibility in a facile and green way is highly desired.

Herein, we report a green and facile strategy to synthesizean active targeting theranostic nanoprobe with intrinsic bio-safety and active targeting ability for near-infrared (NIR)fluorescent imaging and photothermal therapy. The nano-probe was fabricated by simply mixing transferrin (Tf) andindocyanine green (ICG) in aqueous solution under stirring atroom temperature (Scheme 1). Tf, as a human-inherent tumor-targeting ligand, was employed as a smart scaffold to load

†Electronic supplementary information (ESI) available: Additional figures. SeeDOI: 10.1039/c6nr01845a‡These authors contributed equally to this work.

aSchool of Medical Imaging, Tianjin Medical University, Tianjin 300203, China.

E-mail: shaokaisun@163.combDepartment of Radiology, Tianjin Key Laboratory of Functional Imaging,

Tianjin Medical University General Hospital, Tianjin 300052, ChinacCollege of Chemistry, Research Center for Analytical Sciences, State Key Laboratory

of Medicinal Chemical Biology (Nankai University), Tianjin Key Laboratory of

Molecular Recognition and Biosensing, and Collaborative Innovation Center of

Chemical Science and Engineering (Tianjin), Nankai University, 94 Weijin Road,

Tianjin 300071, China

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ICG,29–32 and FDA approved ICG was used as a theranosticagent.19,21,26,33–35 The application of free ICG suffers fromnon-specific binding to proteins, self-aggregation in physio-logical solutions and rapid elimination from the body.26,33–35

The hydrophobic interaction between the hydrophobicbinding pockets of Tf and amphiphilic ICG enables their self-assembly to produce Tf–ICG nanoparticles with excellent watersolubility and colloidal stability, remarkable actively targetingimaging ability and impressive photothermal therapy perform-ance. Additionally, the nanoassembly shows high reproducibil-ity due to the ultra-simple synthesis procedure. To ourknowledge, this work presents the simplest method for thefabrication of active targeting theranostic agents; and alsothe first example of active targeting theranostic agents syn-thesized using green reagents and procedures.

2. Experimental section2.1 Materials

All chemicals were at least of analytical grade and usedwithout further purification. Ultrapure water (HangzhouWahaha Group Co. Ltd, Hangzhou, China) was used inall experiments. Transferrin was purchased from SigmaAldrich (St Louis, MO, USA). Indocyanine green, Na2HPO4·12H2O, NaH2PO4·2H2O, FITC, NaCl and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wereobtained from Aladdin Reagent Co. Ltd (Shanghai, China).DMSO was bought from Concord Technology (Tianjin, China).Bovine serum albumin (BSA) was obtained from BeijingDingguo Biotechnology Co., Ltd (Beijing, China).

2.2 Characterization

The size of Tf–ICG nanoparticles was determined on a PhilipsTecnai G2 F20 (Philips, Holland) field emission high-resolu-tion transmission electron microscope (HRTEM). The Fourier

transform infrared (FT-IR) spectra (750–4000 cm−1) weremeasured on a Nicolet IS10 spectrometer (Nicolet, USA)with pure KBr as the background. The absorption spectrawere obtained on a UV-3600 UV-vis-NIR spectrophotometer(Shimadzu, Japan). The content of ICG in Tf–ICG nano-particles was determined based on the absorption spectra. Itshould be noted the standard curve of ICG was drawn basedthe absorbance of various concentrations of ICG in the pres-ence of Tf. The hydrodynamic size and zeta potential weredetermined on a Malvern Zetasizer (Nano series ZS, UK). Allcircular dichroism (CD) spectra were recorded on a ModelJasco J-715 CD spectrometer at ambient temperature.

2.3 Synthesis of Tf–ICG and FITC-labelled Tf–ICGnanoparticles

The Tf–ICG nanoparticles were prepared using a facile andgreen method. Briefly, 49.7 mg Tf (0.65 μmol) and 10 mg ICG(13 μmol) were dissolved in 5 mL water and reacted under vig-orous stirring at room temperature for 12 h. Then, the mixturewas dialyzed (membrane cut off Mw: 8 kDa) with water at roomtemperature for 24 h to remove unreacted ICG. Finally, theprepared Tf–ICG nanoparticles were freeze-dried and stored at4 °C. Tf was labelled with FITC according to previousreports.36,37 FITC-labelled Tf–ICG nanoparticles were syn-thesized using the same procedure for the preparation ofTf–ICG nanoparticles. All the experiments were carried out inthe dark.

2.4 Measurement of photothermal performance

In order to assess the photothermal efficacy of Tf–ICG nano-particles, water and different concentrations of Tf, ICG and Tf–ICG nanoparticles were irradiated with an 808 nm NIR laser ata density of 2 W cm−2 for 5 min. At the same time, a thermo-couple probe was inserted into the solution to measure thetemperature change of the Tf–ICG nanoparticles solution, andthe temperature was recorded every 30 s.

2.5 Cell cultures

Hela cells (human cervical carcinoma cell) and 3T3 cells werecultured in RPMI-1640 culture medium supplemented with1% penicillin–streptomycin and 10% fetal bovine serum at37 °C under a humidified atmosphere with 5% CO2.

2.6 Tf receptor expression levels of 3T3 and Hela cells

3T3 and Hela cells were seeded in a 96-well plate with 104 cellsper well and cultured in RPMI-1640 medium for 24 h. Then,the cells were incubated in culture medium containing FITC-labelled Tf for another 24 h. After that, the cell samples werewashed with PBS and the luminescence images were acquiredon an inverted luminescence microscope.

2.7 Cytotoxicity of Tf–ICG nanoparticles

Hela cells were seeded in a 96-well plate with 104 cells per welland cultured in RPMI-1640 medium for 24 h. Then, the cellswere incubated in culture medium containing different con-centrations of Tf–ICG nanoparticles or PBS for another 24 h.

Scheme 1 Schematic illustration of synthesis of theranostic transferrin–ICG nanoparticles with intrinsic biocompatibility and targeting abilities viaa green and facile strategy for targeted NIR imaging and photothermaltherapy.

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After that, the cell samples were washed with PBS and treatedwith MTT for 4 h. Finally, DMSO was added into the cells todissolve the formazan crystals, and a muti-mode microplatereader was applied to measure the absorbance of the solutionin each well. Six replicates were carried out for each treatmentgroup.

2.8 Cellular uptake assay

Hela cells and 3T3 cells were plated into 96-well plates with adensity of 104 cells per well and cultured in complete mediumfor 24 h. Then, the medium was replaced by fresh mediumcontaining Tf–ICG nanoparticles with different concentrations,and the cells were further incubated at 37 °C and 5% CO2 for4 h. After that, the cells were washed with PBS 2 times, and thefluorescence imaging of the cell samples was performed on asmall animal imaging system (excitation wavelength: 745 nm,emission wavelength: 820 nm).

2.9 Tf receptor-targeting ability of Tf before and after loadingof ICG

Hela cells were seeded in a 96-well plate with 104 cells per welland cultured in RPMI-1640 medium for 24 h. Then, the cellswere incubated in culture medium containing FITC-labelled Tfor FITC-labelled Tf–ICG for another 24 h. After that, the cellsamples were washed with PBS and the luminescence imageswere acquired on an inverted luminescence microscope.

2.10 In vitro photothermal therapy of Tf–ICG nanoparticles

Hela cells were seeded in a 96-well culture plate with 104 cellsper well in complete medium at 37 °C under 5% CO2 for 24 h.After the medium was refreshed, the cells were treated withTf–ICG nanoparticles or PBS for 0.5 h or 1.5 h and were orwere not irradiated by an 808 nm laser with power densities of0.78 or 2.28 W cm−2 for 10 min. Then, cell viability was evalu-ated by the MTT assay. PI (propidium iodide) and Calcein AM(calcein acetoxymethyl ester) were used to stain dead and livecells, respectively. The luminescence images were acquired onan inverted luminescence microscope.

2.11 In vivo toxicity of Tf–ICG nanoparticles

The in vivo toxicity of Tf–ICG nanoparticles was evaluatedusing Kunming mice (29–31 g, Beijing HFK Bioscience Co.Ltd, Beijing, China) (n = 3 in one group). Comprehensive bio-chemical analysis of blood, especially the biomarkers associ-ated with the functions of liver and kidney, was performed onmice at 1 d, 7 d and 30 d after intravenous injection of 200 μLTf–ICG nanoparticles (7 mg mL−1).

2.12 In vivo imaging

All the animal experiments were approved by the TianjinMedical University Animal Care and Use Committee. Balb/cnude mice (22–24 g, Beijing HFK Bioscience Co. Ltd, Beijing,China) with subcutaneous tumors in the inguinal region wereused as the animal model. The imaging studies were carriedout when the tumor size grew to 6–8 mm. The Tf–ICG nano-particles (7 mg mL−1) or pure ICG (1 mg mL−1) were intra-

venously injected into mice (n = 3 in one group), and thenfluorescence images at different time points were acquired onan in vivo fluorescence imaging system (IVIS Spectrum,PerkinElmer).

2.13 In vivo photothermal therapy of Tf–ICG nanoparticles

200 μL of Tf–ICG nanoparticles solution (7 mg mL−1), PBS orpure ICG (1 mg mL−1) were intravenously injected into thenude mice with subcutaneous tumors (n = 5 in one group). At8 h after injection, the tumors were irradiated with a NIR laserat a density of 1 W cm−2 for 10 min. The surface temperaturechange of the tumor region in nude mice was monitored withan infrared (IR) thermal camera during laser irradiation. Thetumor photos at different time points before and after theinjection of Tf–ICG nanoparticles were recorded to monitorthe volume changes of the tumors. The tumor sizes weremeasured by a caliper at different time points and calculatedas the volume = (tumor length) × (tumor width)2/2.

3. Results and discussion3.1 Synthesis and characterization of Tf–ICG nanoparticles

The Tf–ICG nanoparticles were prepared using a facile andgreen method. Briefly, Tf and ICG were dissolved in 5 mLwater and reacted under stirring for 12 h at room temperature.Tf was chosen as the model template due to its tumor target-ing ability and hydrophobic region for the loading of amphi-philic molecules. On the other hand, ICG is approved by theFDA for clinical use and has been widely employed in NIR fluo-rescence imaging and photothermal therapy. The strongaffinity between Tf and ICG was confirmed by the fact that thefiltrate of the Tf–ICG nanoassembly was nearly colorless evenafter a long period of dialysis. Previous studies show that theinteraction of protein and various small molecules are mainlybased on electrostatic adsorption and/or hydrophobic inter-actions, as proteins contain abundant accessible functionalgroups composed of hydrophilic surfaces and hydrophobicbinding pockets.38–40 Herein, the electrostatic adsorptionbetween Tf and ICG is weak under near neutral pH conditionas both Tf (isoelectric point = 5.6) and ICG are negativelycharged.34,41 Thus, the hydrophobic interaction between thehydrophobic region of Tf and amphiphilic ICG undoubtedlymakes a predominant contribution to the high encapsulationefficiency of ICG, and positively charged regions in Tf may playa supporting role in the formation of Tf–ICG nanoparticlesbased on electrostatic adsorption. The obtained nanoparticleswere easily dispersed in various media including water, saline,PBS and fetal calf serum without visible precipitation over4 months. The possible dissociation of ICG from the nano-particles dispersed in various media was also investigated atdifferent incubation times (1–14 d) via ultrafiltration centrifu-gation. No isolated ICG was present in all studied filtrate,which indicated the high purity of Tf–ICG nanoparticles andthe absence of ICG self-assembly in the purified nanoparticles.Besides that, the structural stability of Tf–ICG nanoparticles in

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the presence of BSA was investigated. The results indicated theTf–ICG nanoparticles possessed favorable structure stability inthe presence of BSA, and ICG did not dissociate from thenanoparticles and bind to competitive BSA. The above experi-mental results demonstrated the proposed Tf–ICG nano-particles not only possessed excellent colloidal stability, butalso owned favorable structural stability. In contrast, ICG alonewas water-soluble, but aggregated in water, NaCl solution andPBS after a long time (Fig. S1†). The excellent water solubilityand enhanced colloidal stability of Tf–ICG nanoparticleshighly benefit their biological application.

The Tf–ICG nanoparticles showed a sphere-like geometrywith a diameter of 15 nm (Fig. 1A). The hydrodynamic dia-meter of Tf nanoparticles was 25 nm, which is an appropriatesize for application in vivo. The zeta potential of the nano-particles was −26 mV due to the many carboxyl groups in Tfthat facilitate the excellent water solubility and colloidal stabi-lity. The presence of Tf in the prepared Tf–ICG nanoprobeswas verified by the characteristic bands for amide I at1644 cm−1 and amide II bands at 1532 cm−1 (Fig. 1B).42,43 TheCD spectrum of Tf–ICG nanoparticles was similar to that ofpure Tf, which revealed the secondary structure of Tf was notsignificantly changed after the loading of ICG (Fig. 1C). Themild and stable hydrophobic interaction between ICG and Tfensured the negligible change of the molecular structure of Tf,including typical α helix, β sheet and random coil, and main-tained the targeting activity of Tf in Tf–ICG nanoparticlesefficiently.25,44 The as-prepared nanoprobes emitted strongNIR fluorescence at 820 nm and showed a strong absorptionin the NIR region, facilitating deep tissue fluorescenceimaging in vivo and ensuring the favorable photothermaltherapy ability (Fig. 1D). The content of ICG in Tf–ICG nano-particles was about 14.29%. It was found that low molar ratios

of ICG to Tf will lead to the fluorescence quenching of ICG(Fig. S2†); however, high molar ratios of ICG to Tf will result inthe presence of many free ICG particles due to the limitedloading ability of Tf. Thus, a moderate molar ratio of ICGto Tf was used for the fabrication of the nanoprobes. Theinteraction between Tf and ICG was studied based on thefluorescence quenching of Tf induced by ICG (Fig. S3†).The dynamic process of fluorescence quenching was analyzedby the Stern–Volmer equation. The binding constant of Tfand ICG was determined to be 3.2 × 104 M−1 at 25 °C,which was big enough to ensure the formation of a stable self-assembly.

FITC-labelled Tf was synthesized according to the pre-vious reports.36,37 The successful conjugation of FITC ontoTf was proved by the characteristic fluorescence peak at522 nm of FITC from FITC-labelled Tf (Fig. S4†). FITC-labelled Tf–ICG nanoparticles were constructed using thesame procedure for the fabrication of Tf–ICG nanoparticles.After loading of ICG, the fluorescence intensity at 522 nm ofFITC-labelled Tf–ICG was much lower than that of FITC-labelled Tf due to the fluorescence quenching induced byICG (Fig. S4†).

3.2 Measurements of photothermal performance

To assess the photothermal performance, water and differentconcentrations of Tf, ICG and Tf–ICG nanoparticles were ir-radiated with an 808 nm laser at a power density of 2 W cm−2

for 5 min. The temperature of the Tf–ICG solution was raisedby 11.6 to 30.4 °C in 5 min, depending on its concentration(Fig. 2A). In contrast, the temperature of pure water was only

Fig. 1 (A) HRTEM image of the Tf–ICG nanoparticles. (B) FT-IR spectraof Tf and Tf–ICG nanoparticles. (C) The absorption and fluorescencespectra of the Tf–ICG nanoparticles. (D) CD spectra of Tf–ICG nano-particles and pure Tf.

Fig. 2 (A) Photothermal heating curves of pure water and Tf–ICGnanoparticles with different concentrations, which were irradiated by a808 nm laser (2 W cm−2). (B) Relative viability of Hela cells treated withdifferent concentrations of Tf–ICG nanoparticles. (C) Fluorescenceimages of Hela cells and 3T3 cells treated with different concentrationsof Tf–ICG nanoparticles (0, 62.5, 125, 250 mg L−1 (35.7 mg L−1 ICG)). (D)The cellular viability of Hela cells after treatment with various combi-nations of Tf–ICG nanoparticles and laser irradiation (808 nm, 0.78 or2.28 W cm−2).

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increased by 4.3 °C under the same conditions (Fig. 2A).Expectably, the temperature of different concentrations of Tfalso showed negligible increases (Fig. S5†). Although pure ICGshowed an acceptable temperature increase under the laserirradiation, the photothermal performance of pure ICG wasworse than that of Tf–ICG nanoparticles with the samecontent of ICG (Fig. S5†). The above results indicated the pres-ence of Tf could improve the chemical stability of ICG due toself-assembly. Thus, Tf–ICG nanoparticles with strong NIRabsorption can rapidly induce heating efficiently under808 nm laser irradiation, and could serve as an effective photo-thermal agent to ablate tumors.

3.3 Tf receptor expression levels of Hela and 3T3 cells

Hela cells (high expression level of Tf receptor) and 3T3 cells(low expression level of Tf receptor) are two classic cell modelsfor the investigation of tumor-targeted imaging and therapy ofTf-based nanoprobes.31,45 To give rigorous evidence of theamounts of Tf receptors in these two cells, FITC-labelled Tfwas employed for cellular fluorescence imaging. Fig. S6† indi-cated that the Hela cells emitted much stronger fluorescencethan 3T3 cells after incubation with FITC-labelled Tf, whichclearly demonstrated Hela cells expressed much more Tf recep-tors than 3T3 cells.

3.4 Cytotoxicity of Tf–ICG nanoparticles

We then evaluated the cytotoxicity of Tf–ICG nanoparticles.Hela cells were incubated with different concentrations of Tf–ICG nanoparticles or PBS for 24 h and then the MTT assay wascarried out to measure the cell viability. High cell viability(86%) was observed with the concentration as high as 200mg L−1 and the cell viability was still over 70% even whenthe concentration reached up to 500 mg L−1 (Fig. 2B). Theabove results clearly show the low cytotoxicity of Tf–ICGnanoparticles.

3.5 Cellular uptake assay

To investigate the ability of specific recognition and enhancedcellular uptake, Hela cells and 3T3 cells were incubated withdifferent concentrations of Tf–ICG nanoparticles for 4 h andwashed with PBS two times to remove free nanoparticles.31,45

Then, the fluorescence of Tf–ICG nanoparticles wasmeasured on an in vivo fluorescence imaging system to evalu-ate cellular uptake of the nanoprobes (Fig. 2C). The fluo-rescence signal of Hela cells treated with PBS was negligible,showing no inherent fluorescence of Hela cells at 820 nm. Thefluorescence intensity of Hela cells treated with Tf–ICG nano-particles obviously increased with the concentration of thenanoprobe. However, the fluorescence intensity of 3T3 cellswas much lower under the same conditions as the lowexpressed Tf receptors on 3T3 led to less specific uptake of Tf–ICG nanoparticles. These results clearly proved that the Tf–ICGnanoparticles could specifically recognize the cancer cellshighly expressed Tf receptor, thus enabling enhanced uptakeby cancer cells.

3.6 Receptor-binding ability of Tf before and after theloading of ICG

To evaluate the receptor-binding ability of Tf before and afterthe loading of ICG, FITC-labelled Tf and FITC-labelled Tf–ICGnanoparticles were employed for cellular fluorescenceimaging. The amounts of Hela cells with green fluorescenceafter the incubation with FITC-labelled Tf or FITC-labelled Tf–ICG nanoparticles were similar (Fig. S7†), which demonstratedthe receptor-binding ability of Tf after loading of ICG was notchanged obviously. It should be noted that the fluorescenceintensity of Hela cells treated with FITC-labelled Tf–ICG nano-particles was a little weaker than those treated with FITC-labelled Tf, which was due to the fluorescence quenching ofFITC induced by Tf–ICG nanoparticles. The above results indi-cated that the proposed non-covalently assembled nanoparticlescould retain the receptor binding ability of Tf effectively.

3.7 In vitro photothermal therapy of Tf–ICG nanoparticles

To evaluate the photothermal effect of the Tf–ICG nano-particles in vitro, Hela cells were incubated with Tf–ICG nano-particles and then irradiated by an 808 nm laser at differentpower densities. The MTT assay revealed the cells treated withTf–ICG nanoparticles or laser irradiation alone had no appar-ent change in cell viability compared to the negative controlwithout nanoparticles and laser irradiation (Fig. 2D). However,the viability of the Hela cells treated with the combination ofTf–ICG nanoparticles and laser ablation significantlydecreased, and only 6.67% of the Hela cells survived when thecells were exposed to a 2.28 W cm−2 NIR laser for 10 min(Fig. 2D). Calcein-AM/PI dual staining was performed as wellto differentiate the live and dead cells (Fig. 3A–D). Hela cellstreated with Tf–ICG nanoparticles plus laser irradiation experi-enced substantial cellular death, while all other treatments ledto negligible cellular destruction. All the results demonstratedthat the Tf–ICG nanoparticles had significant photothermaltherapy efficacy in vitro.

3.8 In vivo toxicity

Prior to the in vivo application, the in vivo toxicity of Tf–ICGnanoparticles was assessed. Blood biochemical analysis ofKunming mice after intravenous injection with 200 μL of Tf–ICG nanoparticles was performed, and then several indicatorswhich reflected the hepatic and kidney function were analyzed.The liver function makers, including alanine aminotransferase(ALT), aspartate aminotransferase (AST), total protein (TP),serum albumin (ALB), globulin (GLB) and gamma glutamyltransferase (GGT), and the kidney function markers, includingcreatinine (Crea) and urea (Urea), were all measured atdifferent time points after injection of Tf–ICG nanoparticles.All the parameters tested in the experimental group had noobvious difference compared to those of the control group(Fig. 3E), indicating that the Tf–ICG nanoparticles did notcause obvious hepatic damage and kidney disorder. Therefore,the Tf–ICG nanoparticles with low in vivo toxicity could beused for further in vivo fluorescence imaging and photo-thermal therapy.

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3.9 In vivo imaging

We then applied Tf–ICG nanoparticles to in vivo fluorescenceimaging. Nude mice bearing Hela tumors were used in thisexperiment when the diameter of the tumors reached 6–8 mm.The fluorescence imaging of mice before and after the intra-venous injection of Tf–ICG nanoparticles were performed on asmall animal imaging system (Fig. 3F). At the beginning, theTf–ICG nanoparticles were widely spread all over the body ofmice, and tumor contrast was clearly observed at 0.5 h postinjection of the nanoprobes. The most obvious contrastbetween the tumor and the surrounding tissues appeared after24 h. The mice injected with Tf–ICG nanoparticles were sacri-ficed at 48 h post injection, and then the major organs andtumors were collected for ex vivo imaging. The Tf–ICG nano-particles mainly accumulated in the tumor region where thefluorescent intensity was much higher than any other organsincluding mononuclear phagocyte systems such as the liverand spleen (Fig. 3G). In contrast, the fluorescence signal–noiseratio in the tumor regions of mice treated with pure ICG waslower than those treated with Tf–ICG nanoparticles due to thelack of active tumor targeting abilities by the pure ICG(Fig. S8†). These results showed that Tf–ICG nanoparticlescould contribute to distinguishing tumor from surroundingtissues effectively.

3.10 In vivo photothermal therapy of Tf–ICG nanoparticles

The significant photothermal therapy effect in vitro, low tox-icity and good tumor targeting abilities encouraged us toutilize Tf–ICG nanoparticles for the photothermal therapy oftumors in vivo. Mice bearing Hela tumors were intravenously

injected with Tf–ICG nanoparticles or PBS, and the tumorswere exposed to laser irradiation with the power density of1 W cm−2 after 8 h. The surface temperature change of the micewas monitored with an IR thermal camera during laserirradiation. Fig. 4 shows that the tumor surface temperatureof the mice injected with Tf–ICG nanoparticles increased to55 °C rapidly under laser irradiation in a short period of time.However, for the mice intravenously injected with PBS, thesurface temperature of the tumors had little change under thesame conditions. Photos were also taken to monitor thevolume change of the tumors (Fig. 5). The tumors of the micetreated with the Tf–ICG nanoparticles plus NIR laserirradiation were inhibited significantly and finally dispelledcompletely after two weeks. In contrast, the tumors of the mice

Fig. 3 (A)–(D) Hela cells were stained with calcein AM and PI after different treatments, and the live and dead cells were stained to be greenand red respectively. (A) Hela cells treated without Tf–ICG nanoparticles and laser irradiation. (B) Hela cells treated with Tf–ICG nanoparticles(250 mg L−1) alone. (C) Hela cells treated with 808 nm laser irradiation (0.78 W cm−2) for 10 min. (D) Hela cells treated with Tf–ICG nanoparticles(250 mg L−1) for 1.5 h and then irradiated with laser (808 nm, 0.78 W cm−2) for 10 min. (E) The blood levels of ALB, ALT, AST, Crea, TP, UA and Ureafrom the mice treated with or without Tf–ICG nanoparticles at different time points (200 μL, 7 mg mL−1). (F) In vivo fluorescence images of micebearing Hela tumors taken before and after intravenous injection of Tf–ICG nanoparticles (200 μL, 7 mg mL−1). (G) Ex vivo fluorescence images ofmajor organs and tumors dissected from the mice at 48 h after the injection of Tf–ICG nanoparticles (200 μL, 7 mg mL−1).

Fig. 4 IR thermal images of mice bearing Hela tumors before and afterthe treatment with PBS or Tf–ICG nanoparticles and laser irradiation(808 nm, 1 W cm−2).

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treated with PBS or ICG kept growing (Fig. S9†). The cure ratecurves indicated only the Tf–ICG nanoparticles could providean impressive therapeutic effect towards tumors (Fig. S10†).These results clearly demonstrate that the Tf–ICG nano-particles could be used not only as a NIR fluorescence imagingnanoprobe, but also as an effective photothermal therapyagent with superior tumor targeting abilities and favorable bio-compatibility in vivo.

4. Conclusions

In summary, we have shown the fabrication of an efficienttheranostic nanoprobe with intrinsic biocompatibilities andtargeting abilities for NIR fluorescence imaging and photo-thermal therapy of tumors. A green and facile strategy forloading ICG on Tf was proposed based on the hydrophobicinteraction-mediated self-assembly of Tf and ICG by simplemixing in aqueous solution without the need of any toxicreagents or tough synthetic conditions. The novel theranosticnanoprobe offered uniform size, excellent water solubility andcolloidal stability, favorable biocompatibility and impressiveactive targeting theranostic effects in vitro and in vivo. The pro-posed method opens a new way for the fabrication of bio-compatible and targeting nanoprobes using a green and facilestrategy, and could be easily extended to construct other nano-probes for the diagnosis and treatment of malignant tumorsusing other functional proteins and FDA approved smallorganic molecules.

Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (Grants 21435001, 21405112, 21505099)

and the China Postdoctoral Science Foundation (Grants2014M550146, 2015M571270).

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