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Cytotoxicity and3+

aNanoBioMedical Centre, Adam Mickiewi

Poznan, Poland. E-mail: wozniaka@amu.edbDepartment of Experimental Physics, W

Wyspianskiego 27, 50-370 Wroclaw, PolandcFaculty of Physics, Adam Mickiewicz Univ

Poland

Cite this: RSC Adv., 2016, 6, 95633

Received 12th August 2016Accepted 1st October 2016

DOI: 10.1039/c6ra20415e

www.rsc.org/advances

This journal is © The Royal Society of C

imaging studies ofb-NaGdF4:Yb Er3+@PEG-Mo nanorods

Anna Wozniak,*a Agnieszka Noculak,b Jacek Gapinski,ac Daria Kociolek,b

Agnieszka Bos-Liedke,ac Tomasz Zalewski,a Bartosz F. Grzeskowiak,a

Anna Kołodziejczak,a Stefan Jurga,a Mateusz Banski,b Jan Misiewiczb

and Artur Podhorodeckib

Multimodal imaging based on nanostructures has become a subject of interest for numerous biomedical

laboratories. The main focus was placed on applying nanocrystals for the purpose of two types of clinical

imaging (contrast and fluorescent agents) due to their excellent luminescence and/or paramagnetic

properties. Such systems should also be characterized by low toxicity and high cellular uptake efficiency.

Since bare rare earth fluoride nanocrystals influence the cell membrane integrity, it is expected that their

coatings will improve biocompatibility profile, as well as increase hydrophilicity, dispersion and chemical

stability. Hence, by synthesis of b-NaGdF4:Yb3+Er3+ nanorods (NRs) coated with noncovalently bounded

polyethylene glycol monooleate (PEG-Mo), it should be possible to obtain multimodal imaging

biomarkers meeting established criteria. Synthesis of b-NaGdF4:Yb3+Er3+@PEG-Mo NRs was performed

by the co-precipitation method. These nanostructures were characterized in terms of their size,

morphology, zeta potential, magnetic and optical properties as well as their cytotoxicity profile and

cellular internalization was evaluated. It was shown that the shape and size of nanocrystals, namely 20

nm nanorods, present generally accepted parameters for biomedical purpose. Ligand attraction of PEG-

Mo 860 resulted in the encapsulation of oleic acid coated NRs and formation of hydrophilic bilayer.

Superparamagnetic and luminescence properties were highly efficient. Cytotoxic profiles of normal and

cancer cell lines were low and determined by dose and time. Cellular uptake was confirmed by the

presence of upconversion luminescence in cell interior. These findings are showing multimodal imaging

properties of rod shaped b-NaGdF4:Yb3+Er3+@PEG-Mo NRs which may be useful in some biomedical

applications.

Introduction

Multimodal imaging (MI) combines two or more imagingmethods for acquiring different images of the same objectsimultaneously or sequentially. The signicant advantage of MIlies in the possible overlapping of two or more images with thehighest precision, thus MI offers complementary informationabout the organ or tissue in relatively short period of time.1 Forsome recent years, the biggest interest has lied in the combi-nations of expensive nuclear methods like positron emissiontomography (PET) which requires an injection of radioactivetracer for imaging of physiological processes, with radiologicalmethods, e.g. computed tomography (CT) or magnetic reso-nance imaging (MRI). Although CT is a perfect tool for

cz University, Umultowska 85, 61-614

u.pl

roclaw University of Technology, Wyb.

ersity, Umultowska 85, 61-614 Poznan,

hemistry 2016

radiotherapy planning, MRI is considered to possess a clearadvantage in the process of acquiring high resolution anatom-ical and physiological images. In addition to the techniquesmentioned above, optical imaging (OI) techniques e.g. uores-cence and bioluminescence imaging, show a clear trend fortranslation into clinic due to the high imaging sensitivity andthe low cost of imaging facilities.

Despite the advantages offered by all the above-mentionedmethods, it is necessary for the majority of these cases to usea contrast agent in order to acquire a high-contrast image thatcan further represent the physiological parameters. Hence,parallel to multimodal imaging, the idea of multimodal nano-particles (MNs) has been developed as the method which allowsthe process of imaging by two different imaging techniques atthe same time.

Due to the physical phenomenon on which OI techniques isbased, contrast agents which are to be used for in vitro and invivo examination must meet some special requirements. First ofall, such markers should be photostable, display possibly longbody persistence time and reveal emission in the “optical

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transmission window” of tissue.2 Furthermore, they should bealso biologically compatible and non-toxic. Unfortunately, themajority of organic uorescent dyes do not meet all of theserequirements what makes most of them useless in clinical OI.3

Therefore, nding a novel marker for OI complying all theserequirements became a challenging topic of the last decade.

Recently, magnetic and optical properties of rare-earthdoped uoride nanocrystals (RENCs), aroused an interest ofscientists, and became a potential candidate as multimodalcontrast agents. For example, the Gd arising paramagneticproperties of NaGdF4 NCs host matrix are further developed bydoping with light-emitting rare earth (RE) ions, e.g. Eu+3, Tm+3,Er+3.4,5 RE ions pair like Yb+3/Er+3, which have some specialoptical properties associated with f-electron, i.e. relatively highYb3+ absorption cross-section, give rise to the unique upcon-version uorescent process where near-infrared (NIR) light isabsorbed and converted into multiplex emission of Er3+ span-ning over wide range of light from UV to NIR.3–7 This process ismuch more promising from the biological point of view,because excitation at 980 nm does not cause any optical pho-todamage of biological tissue and strongly reduces the uo-rescence background noise. Furthermore, it was shown thatNaGdF4 NCs due to the presence of the paramagnetic Gd3+ ionsin the host matrix can be used as MR contrast agent as well.4,8,9

Their main advantage over the standard Gd-based chelates isthe higher density of the Gd3+ ions per unit volume of contrastagent that leads to higher signal detection sensitivities neces-sary for molecular and cell tracking.10 Moreover, Gd-containingRENCs are efficient contrast agents due to the easy surfaceconjugation and long blood retention time.11,12 Combination ofthe upconversion uorescent and magnetic properties intoa single nanosystem allows to obtain a new nanoplatform formultimodal imaging, which can be applied in MRI and OItechniques.

The main factor which determines the applicability ofRENCs in biomedicine is their biocompatibility. One of themost important trait of the nanoparticles determining theircytotoxic prole is their shape. It has been shown already thatthe shape of nanocrystals entering the cell structure plays animportant role, and the endocytosis process can be different forisotropic and anisotropic (i.e. nanorods) forms.13 The majorityof the already mentioned examples of using NaGdF4:Yb, Ernanocrystals referred to their cubic or hexagonal shape, whichare both very symmetric.7,14–24 The published data have provedthat as synthesized oleic acid capped RENCs are hydrophobicand more cytotoxic than those coated with various polymers orligands (i.e. PEG, PAA, SiO2, SiO–NH2).18,25 It was shown thatcationic polymers increase their hydrophilicity, dispersion, andalso improve cellular uptake processes due to electrostaticinteractions with negatively charged cell membrane.21 Further-more, such cubic and hexagonal nanocrystals, which have beendescribed so far, meet some moot issues concerning mainlybiological aspects of cytotoxicity and cellular uptake dependingon size, surface chemistry and electrokinetic potential.21,24,25

There exists a signicant need to develop new RENCs based onnanosystems which will be characterized by a stable

95634 | RSC Adv., 2016, 6, 95633–95643

composition, a low cytotoxicity prole, a proper cellular inter-nalization capacity, luminescence efficiency and magneticproperties.

Consequently, for the purpose of this study, NaGdF4 nano-rods co-doped with Er3+ and co-doped with Yb3+, and non-covalently coated with polyethylene glycol monooleate 860, werechosen due to their anisotropic shape and high upconversionefficiency and good biocompatible prole.24,26 Among surfacemodication methods, noncovalent ligand attraction viahydrophobic van der Waals interactions was chosen in order toachieve encapsulation of RENCs without surface changes.24,27,28

The main objective of this study was to determine potentialof b-NaGdF4:Yb

3+Er3+ rod-shape nanocrystals coated with poly-mer polyethylene glycol monooleate 860 in biological applica-tions. For this purpose, their physicochemical, magnetic,optical and biological properties have been investigated.

ExperimentalSynthesis of nanocrystals

Reagents. Gadolinium(III) acetate (99.9%), ytterbium(III)acetate (99.9%), erbium(III) acetate (99.9%), sodium hydroxide(98%), ammonium uoride (98%), polyethylene glycol mono-oleate 860, 1-octadecene (90%), oleic acid (90%), as well as allthe solvents, were purchased from Sigma-Aldrich. All chemicalswere used as received without further purication.

General procedure for the synthesis of nanocrystals

The synthesis method of b-NaGdF4:Yb3+Er3+ NRs preparation

was similar to the co-precipitation method presented by Wanget al.19 In a typical procedure of NaGdF4:Yb

3+, Er3+ NRssynthesis, 4 ml of water solution of Ln(CH3CO2)3 (Ln ¼ Gd, Yb(0.2 M), and Er (0.02 M)) was added to a 50 ml ask containing3 ml of oleic acid. The mixture was heated at 150 �C for 30 minto remove water from the solution. 1-Octadecene (7 ml) wasthen quickly added to the ask and the resulting mixture washeated at 150 �C for another 30 min and then cooled down to50 �C. Shortly thereaer, 5.4 ml of freshly prepared methanolsolution containing NH4F (1.36 mM) and NaOH (1 mM) wasadded and the resulting solution was stirred for 2 hours. Whenmethanol evaporated, the solution was heated to 290 �C undernitrogen for 1 h and then cooled down to room temperature.The resulting NCs were precipitated by addition of ethanol,collected by centrifugation at 10 000 rpm for 4 min, washedwith ethanol several times, and re-dispersed in 4 ml ofcyclohexane.

Functionalisation of the obtained nanocrystals withpolyethylene glycol

The functionalisation protocol was based on the methoddeveloped by Prakash et al. and Das et al.24,27 2 ml of NRssolution was added to 10 ml of cyclohexane and stirred for20minutes. Then, 250 ml of PEG-Mo 860 was added and solutionwas stirred overnight at room temperature. Aer this time,cyclohexane was removed slowly under a rotary evaporator at30 �C until waxy liquid was obtained at the bottom of the ask.

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10 ml of distilled water was added to the waxy liquid andsonicated for 20 minutes. In order to remove residues ofcyclohexane, the ask was put under a rotary evaporator forabout 1 hour at 30 �C. The obtained solution was puried by0.22 mm PTFE mesh lter.

Morphology of the nanocrystals

The morphology and size characterization of b-NaGdF4:-Yb3+Er3+@PEG-Mo NRs were carried out using transmissionelectron microscopy (TEM). For the TEM experiments, hydro-phobic samples were prepared by evaporating a diluted cyclo-hexane solution of the nanoparticles onto carbon coated coppergrids and a FEI Tecnai G2 20 X-TWIN microscope, equippedwith an energy-dispersive X-ray microanalyser, was used toobtain the TEM images and EDXS spectra of the nanoparticles.

Zeta potential measurements

The zeta (z) potential of the NRs suspension in water wasdetermined by electrophoretic light scattering using a MalvernZetasizer Nano ZS (UK).

Magnetic resonance imaging

T2-Weighted magnetic resonance images were obtained using9.4 tesla MRI horizontal scanner (Agilent) with volume RFMillipede coil (40 mm). The resolution was 0.112 � 0.112 �2 mm3. Prior to imaging, the experiment samples with differentconcentration of nanoparticles were placed in a designedholder. The reference specimen with solvent (water) was placedin the central part of the holder. The measurements were per-formed at 16 �C. For the purpose of this experiment, spin echoCPMG sequence was used, with echo time TE ¼ 100–3200 msand repetition time TR ¼ 15 000 ms, the number of echoes wasset to 32.

The optical methods used for sample characterization

The size distribution of b-NaGdF4:Yb3+Er3+@PEG-Mo NRs in

diluted suspension was investigated by means of photoncorrelation spectroscopy (PCS). PCS measures the distributionof diffusion coefficients which can be further converted intosize distribution using the Stokes–Einstein formula. In the PCSexperiments, a 532 nm laser operating at the power of 100 mW,an ALV goniometer and an ALV7000 correlator were used (ALV-GmbH, Germany). CONTIN algorithm incorporated into theALV soware (ver. 3.0) was used to convert the PCS correlationfunctions into the size distributions. The temperature of thesample was maintained at 20 �C.

For the steady state photoluminescence (PL) as an excitationsource, a 980 nm laser (Shanghai Dream Lasers technology SDL-980-LM-1000T) was used. A HR4000 spectrometer (OceanOptics, Dunedin, FL, USA) and InGaAs linear CCD detector(Symphony® I line, Horiba Jobin-Yvon) were used as detectionsystems for measurements in the vis and NIR spectral range,respectively. The PL decays were measured using pulse laser(Opolette™, Opotek Inc., Carlsbad, CA, USA) coupled to a gateddetection system.

This journal is © The Royal Society of Chemistry 2016

Cell culture

The human colorectal cancer HT-29 cell line was obtained fromthe Sigma-Aldrich, human cervical cancer HeLa cell line,human bone osteosarcoma U-2 OS cell line and normal HumanEmbryonic Kidney HEK293T cell line were obtained from theAmerican Type Tissue Collection (ATCC). HT-29 cell line wasmaintained in McCoy's 5A (Sigma-Aldrich), U-2 OS andHEK293T cell lines were maintained in DMEMmedium (Sigma-Aldrich). The media were supplemented with 10% foetal bovineserum (FBS, Sigma-Aldrich), penicillin (100 IU ml�1, Sigma-Aldrich), and streptomycin (100 mg ml�1, Sigma-Aldrich). Thecells were incubated at 37 �C, 95% humidity, and 5% CO2. Thegrowth of the cells, as well as their morphology were evaluatedin an inverted microscope Leica DMIL LED. When the culturesreached 80% conuence, the cells were trypsinized (0.25%trypsin, 0.02% EDTA, Sigma-Aldrich) and seeded on cultureplates in an appropriate concentration.

WST-1 assay

WST-1 assay (Water-Soluble Tetrazolium salt, the PremixedWST-1 Cell Proliferation Reagent, Clontech) was used to assessproliferation of the analysed cells. The cells were trypsinizedand seeded at the densities of 105 per ml in complete mediumin standard 96-wells plate. Aer 24 h, the medium was removedand then b-NaGdF4:Yb

3+Er3+@PEG-Mo NRs were added. Thestock solution of NCs were prepared (1 mg ml�1) before exper-iment and diluted with the complete medium at the followingconcentrations: 1, 8, 16, 32, 64, 100, 300 mg ml�1. The samplesmeasurements were repeated 2–4 times with the positivecontrol (cells w/o nanoparticles) and the negative control (10%DMSO). The cell proliferation was measured aer 24, 48, and72 h. 10 ml of WST-1 reagent were added into the individual welland proceeded according manufacturer's protocol. The opticaldensity was measured by a plate reader (Zenyth) at the wavelength of l � 450 nm (l � 620 nm as reference).

Apoptosis assay

Muse® Annexin V & Dead Cell Assay Kit (Merck Millipore) wasused to determine apoptosis of the analysed cells. The cells weretrypsinized and seeded at the densities of 2 � 105 per ml incomplete medium in a standard 6-wells plate. Aer 24 h, themedium was removed and then b-NaGdF4:Yb

3+Er3+@PEG-MoNRs were added at the following concentrations: 1, 8, 16, 32,64, 100, 300 mg ml�1. Aer 24, 48 or 72 h of incubation, cellswere trypsinized and added to a tube with annexin V & dead cellreagent and proceeded according manufacturer's protocol. Themeasurements were performed using Muse® Cell Analyzer(Merck Millipore).

Confocal imaging

The HT-29 and HeLa cells were seeded at the density of 105 perml in complete medium in the Lab-Tek™ 8 Chamber Slide witha removable, polystyrene media chamber attached to a standardglass microscope slide (25 � 75 mm). Aer 24 h, the mediumwas removed and b-NaGdF4:Yb

3+Er3+@PEG-Mo NRs in the

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concentration of 300 mg ml�1 were added. Aer 24 h of incu-bation, the NRs were washed, the cells were xed (4% para-formaldehyde in PBS at 37 �C for 15 min, 0.2% Triton X-100 for5 min) and stained with concanavalin A, Alexa Fluor 647Conjugate (Con A, Molecular Probes, the working concentra-tion: 100 mg ml�1) and Fluoroshield™ with DAPI (Sigma-Aldrich). Confocal imaging was carried out using the ZeissLSM 780 system equipped with a tunable infrared femtosecondlaser (Chameleon, Coherent). Due to long luminescence life-times, the scans speed was reduced so that the pixel dwelltimes exceeded 25 ms. The spectral detector of LSM 780 was usedto measure the emission spectrum of luminescence underinfrared excitation to make sure that it originates from theupconversion process in the NRs and not from two-photonexcitation of DAPI or from the residual amounts of other dyes.

Results and discussionMorphology analysis

Fig. 1 shows TEM images obtained for b-NaGdF4:-Yb3+Er3+@PEG-Mo NRs recorded at different magnications. Inaddition, Fig. 1c shows two histograms (the number ofmeasured NRs was 100), where two characteristic sizes (diam-eter – a and length – b) of obtained NRs are equal to 15 and 22nm, respectively. The HRTEM image of one nanorod (Fig. 1d)shows interplanar spacing of 0.52 nm corresponding to the (10–

Fig. 1 TEM (a and b) and high resolution TEM images (d) of b-NaGdF4:magnification. Histograms (c) calculated for 100 NCs for a and b axis of

95636 | RSC Adv., 2016, 6, 95633–95643

10) plane of hexagonal NaYF4, conrming that the nanorodsgrow along the c-axis, namely, the [0001] direction.29

The data described so far in literature concerned mostlyRENCs with a spherical and/or hexagonal shape.14,21,22,24,30 Thus,our results on nanorods providing physicochemical and bio-logical analyses resulted in a new insight into the concept ofutilizing anisotropic RENCs in biomedical application.

Functionalisation of the obtained nanocrystals withpolyethylene glycol was chosen due to the fact, that withouta polymer shell, NCs will rapidly aggregate through interactionsbetween themselves or with biological molecules and precipi-tate out of the solution. The coating layer regulates the inter-action between the NC and the environment, e.g., it determinesthe solubility of NCs in a given solvent. The coating keepsthe NCs from coming into contact and aggregatingbecause surfactant molecules on different NCs have repulsivesteric and/or electrostatic interactions. Proper surfactant designcan provide good NC stability and presentation of functionalgroups in a qualitatively and quantitatively dened manner.31,32

We selected specic approach of noncovalent boundingbecause this method does not introduce any additional defectsto the NCs surface making NCs same bright as before thetransfer to water. The other approaches like ligands exchangegave maybe more stable coatings but introduce oen number ofdefects to the NCs surface making them well dispersed andstable in water but useless in practical use because they char-acterize with very low emission intensity. On the other approach

Yb3+Er3+@PEG-Mo NRs dispersed in water and measured at differentnanorods separately.

This journal is © The Royal Society of Chemistry 2016

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used in this work is relatively simple and cheap thus gave a highpotential from the practical point of view.

Zeta potential measurements

Zeta potential of b-NaGdF4:Yb3+Er3+@PEG-Mo NRs suspension

in water was (�17.7 � 0.98) mV.

Magnetic resonance imaging

T2-Weighted images were obtained for 2 samples of b-NaGdF4:Yb

3+Er3+@PEG-Mo NRs dispersed in water (highconcentration, low concentration) and for sample with water asreference, using spin echo experiment for different values ofecho time (TE). Although from images in Fig. 2 it is clearly seenthat signal decay is faster for the sample with higher concen-tration of NRs, the attenuation of MRI signal is rather slow.Apparently, at the applied (strong) magnetic eld the exchangesof energy between b-NaGdF4:Yb

3+Er3+@PEG-Mo NRs and watersprotons are rather weak, so that the examined NRs have lowinuence on proton relaxation T2 time. This mechanism isinuenced by the distance between ions and protons which isincreased by PEG molecules and by the strength of magnetic

Fig. 2 Cross MRI profile of samples in different concentration of b-NaGdF4:Yb

3+Er3+@PEG-Mo NRs as a function of TE. 1 – sample filledwith pure water, 2 – concentration of 300 mg ml�1, 3 – concentrationof 100 mg ml�1.

Fig. 3 Two emission spectrameasured in the water suspension of b-NaGmicroscopic mode and with CW excitation at 980 nm in the macroscopithe 978 nm excitation pulse profile (b).

This journal is © The Royal Society of Chemistry 2016

eld. Authors suppose that due to presence of rare earth atomsin the structure of NRs, stronger relaxivity properties will beobserved at lower (clinical) magnetic elds.

Optical study

Prior to each confocal imaging experiment, the emission spec-trum was measured to ensure that the luminescence signal isproduced by the upconverting particles, and not by the two-photon excitation of other dyes present in the sample. Anexample of the upconversion spectrum measured at femto-second pulse excitation (red line) is shown in Fig. 3a togetherwith the spectrum recorded at continuous wave (CW) excitationat 980 nm wavelength (green line). In addition, Fig. 3b showsemission decay measured for b-NaGdF4:Yb

3+Er3+@PEG-MoNRs. Based on obtained results effective emission decay timehas been estimated as 50 ms.

To get information on hydrodynamic radius of investigatedNRs the PCS technique has been used. The measured PCScorrelation function was analyzed in terms of the correlationtime distribution, which was then converted to the hydrody-namic radius distribution (Fig. 4). As in light scattering exper-iments, the signal is proportional to the particle volume, thesize distribution obtained directly from a PCS measurementdrastically overexposes the contribution of larger particles,compared to their relative weight concentration.

The correlator soware (ALV ver. 3.0) allowed for conversionof the scattered light intensity size distribution to the NCsweight concentration distribution, however assuming the exis-tence of a certain simple model relating the hydrodynamic sizeto the mass of the aggregates. Such obtained weight concen-tration distribution (dashed line in Fig. 4) unequivocally indi-cates that the sample contains primarily small particles of 4–20 nm in diameter. The larger component of diameter close to150 nm – scatters a comparable amount of light; however, such

dF4:Yb3+Er3+@PEG-MoNRsmeasured: with the excitation at 980 nm in

c mode (a). Emission intensity decay recorded at 545 nm together with

RSC Adv., 2016, 6, 95633–95643 | 95637

Fig. 4 Scattered light intensity (solid line) and weight concentration(dashed line) distribution of hydrodynamic size obtained from PCSmeasurement of dilute NRs suspension.

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a strong signal results only from the large volume and hence thescattering strength of such particles. The size distributionshows also the third component of sub-micron size particleswhich is most likely the result of their residual aggregability.

Several factors do not allow for any precise characterizationof the NRs size distribution in water suspension by means ofPCS: (i) non-spherical shape; (ii) polydispersity of the NRs; (iii)presence of smaller and larger aggregates; and (iv) possibleeffect of electrostatic interactions leading to an apparentreduction of hydrodynamic size at nite concentrations.

Nevertheless, the main goal has been achieved: the PCSresults clearly show that the NRs are well dispersible in waterand that a vast majority (probably over 99% weight concentra-tion) of the sample remains in the form of single NRs with onlya residual amount of aggregates.

Biological study

Nanotoxicology concerns the response of cells or whole organ-isms to the contact with nanoparticles. Some negative interac-tion may cause biochemical, enzymatic or genetic changes inhomeostasis, which usually lead to the death of cells. It iscrucial to establish the toxicity of synthetized nanosystems,especially if they are intended for some biomedical applica-tions. Provided proliferation and apoptosis analysis revealedcytotoxicity prole of b-NaGdF4:Yb

3+Er3+@PEG-Mo NRs. Therst screening test was a colorimetric assay dedicated to assessthe viability of examined cells –WST-1, which was performed onthe cancer (HT-29, HeLa, U-2 OS) and normal (HEK293T) cells.The tested NRs concentrations ranged from 1 to 300 mg ml�1.The WST-1 assay shows the ability of the living cells to theenzymatic conversion of the tetrazolium salt WST-1 into a water-soluble formazan dye, which can be quantied by the absor-bance at 420–480 nm. The optical density values correspond toa percentage of the cell viability in relation to the control.

95638 | RSC Adv., 2016, 6, 95633–95643

The HT-29 cells average viability aer 24 h of incubation withb-NaGdF4:Yb

3+Er3+@PEG-Mo NRs was 97.5% (concentrations of1–100 mg ml�1) and was slightly lower at 300 mg ml�1 – 82.8%.Aer 48 h of incubation, cells presented very high proliferationpotential above 100% and decreased at the two highestconcentrations to 85.5% and 76.6%. On the third day, thepotential for the WST-1 salt reduction was also very high, withthe exception of the concentration level at 100 and 300 mg ml�1

(77.5% and 86.6%, respectively) (Fig. 5a).NaGdF4:Yb

3+Er3+@PEG-Mo NRs, aer 24, 48, and 72 h ofincubation, did not decreased the viability prole of U-2 OS cellscomparing to the control (average 97.1%, 91.8%, 87.5%,respectively). Only at the concentration of 300 mg ml�1, theviability was very low and placed at 17.9% (aer 24 h), 8.6%(aer 48 h) and 5.1% (aer 72 h) (Fig. 5b).

Aer 24 and 48 h of incubations with analyzed nanocrystals,the HeLa cells showed the survival rate approximately at 100%(ranging from 85.5% to 107.7% at all the tested concentrations).Cytotoxicity increased only at the highest concentrations of theNaGdF4:Yb

3+Er3+@PEG-Mo NRs (100 and 300 mg ml�1), aer 48and 72 h (Fig. 5c).

HEK293T was the most sensitive cells used in the experi-ment. Aer 24 h of HEK293T cells incubation with b-NaGdF4:-Yb3+Er3+@PEG-Mo NRs, the cytotoxic effect was visible only atthe concentration of 300 mg ml�1. The very similar situation wasobserved aer 48 h of incubation. A systematic decline in thevitality and the lowest viability aer 72 h of exposition at thehighest nanoparticles' concentration comparing to the control(100%) was observed. The viability of HEK293T cells decreasedgradually starting from the concentration of 64 mg ml�1 andshowing the viability of 36.8%, 23.3% and 12.2%, respectively(Fig. 5d).

In vitro viability study has revealed the cytotoxic prole ofanalyzed b-NaGdF4:Yb

3+Er3+@PEG-Mo NRs in cancer (U-2 OS,HeLa, HT-29) and normal (HEK293T) cells. The inuence ofnanoparticles on the cellular toxicity was further examined witha wide spectrum of particle concentrations, as well as at thethree time intervals. The results showed that nanoparticles aretoxic in the dose dependant manner for the cancer cells U-2 OS.The signicant reduction of the WST-1 salt conversion only atthe highest concentration was observed. The HeLa cellsprovided both, dose and time dependency of cytotoxicity. Themost biocompatible prole was detected for the cancer HT-29cells. The normal cells HEK293T has shown the lowestviability at the highest nanoparticles' concentrations, especiallyon the second and third day of incubation. It is worth notinghere that different types of cells may vary in response to thepresence of nanoparticles.33–35 Our observations are in line withthe fact that cells in the logarithmic growth phase are moresensitive than those in the stationary phase.33

According to the suggestions presented by other authors, itis important to examine the cytotoxicity prole of nano-composites using more than one method (enzymatic activitytests and cell labelling) to verify and evaluate the results.36 TheHT-29 cells, which presented the highest prole of viability,were additionally investigated in more detail, using notonly WST-1 assay, but also apoptosis analyses. The performed

This journal is © The Royal Society of Chemistry 2016

Fig. 5 Viability of HT-29 (a), U-2 OS (b), HeLa (c), HEK392T (d) cells after 24, 48 and 72 h of incubation with b-NaGdF4:Yb3+Er3+@PEG-Mo NRs.

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apoptosis microcapillary ow cytometer test included tworeagents, where each of them indicates different types of cellsconditions. The rst one – uorescent active annexin V –

specically binds to phosphatidylserine, an important interiorphospholipid cell membrane component which moves outsidethe cell membrane in the early stage of apoptosis. The secondone, propidium iodide PI, passes the damaged cell membraneand binds to the DNA of dead or late apoptotic cells. Thus, thistest allows for the distinction of four types of analysed cells:living (annexin V�, PI�), early apoptotic (annexin V+, PI�), lateapoptotic (annexin V+, PI+), and dead (annexin V�, PI+). TheHT-29 cells represented the growing percentage of lateapoptotic cells corresponding to the decreasing number ofliving cells. This growth was occurred at the highest nano-particles' concentrations. During exposition process, cellsundergo apoptosis but in analysed range of time intervals thedeath of cells was not reported (Fig. 6a–c).

The investigation showed that both methods allow to specifythe cytotoxic prole of nanoparticles. Although the viability andapoptosis tests provided information about different indicators ofviability, the conclusions from both experiments are consistent.37

Our results indicate that the analysed b-NaGdF4:Y-b3+Er3+@PEG-Mo NRs are biocompatible in a particular rangeof concentrations and above the concentration threshold cancause the loss in the viability of cells in a time and dosedependent manner.

This journal is © The Royal Society of Chemistry 2016

Evaluation of the cytotoxicity of the upconversion nano-particles is a key factor for their further use in in vivo studiesand in clinical trials. The indexes of cytotoxicity are series ofphysiological changes leading to damage and usually, to celldeath. The most common parameters that may be changed asreaction on upconversion nanoparticles are: failure of cell sig-nalling, the activity of enzymes, the ability to proliferation andreplication, conformation and location of proteins or othersurface structures, the permeability of cellular bilayer, and cells'morphology. Those abnormalities oen leading to decreasedviability, apoptosis, oxidative stress and DNA damage.38 In thediscussion concerning the toxicity of rare earth-doped uoridenanocrystals, we should consider the central dogma of nano-toxicology, which includes three major factors inuencing thecytotoxic prole of nanoparticles, namely the shape, size, andsurface chemistry/coatings. The RENCs shape analysed in thispaper was determined by TEM as a rod-like structure. Incontrary – as mentioned above – the majority of publicationsdescribed the spherical or hexagonal morphology of RENCs.There is only one publication which has mentioned the egg-likeor cocoon-like amine-functionalized RENCs (diameters of 20nm and lengths of 20–40 nm).39 From the biological point ofview, nanorods undergo a more efficient internalization processwhich is based on endocytosis. Huang et al. described the freeenergy analysis and concluded that spherocylindrical nano-particles undergo a specic internalization pathway that leads

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Fig. 6 The HT-29 cells apoptosis profile after 24 h (a), 48 h (b), 72 h (c).

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to a more efficient cellular uptake comparing to the sphericalone.13 According to our best knowledge, there are no reportsconcerning any complex physicochemical, optical and biologicalstudy of the rod-shaped RENCs (b-NaGdF4:Yb

3+Er3+@PEG-Mo).The size of nanoparticles can determine some of their

physical properties as well as inuence the dispersion andaggregation process.40 Small nanoparticles demonstrate thetendency to aggregate andmay cause the loss of viability in high

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concentrations. In the case of RENCs, the size exerts alsoinuence on the luminescence activity. The upper size limit forthe cytotoxicity of RENCs has not been fully claried yet. Ingeneral, many authors suggest the existence of biocompatiblenanoparticles' diameter within the range of 20–50 nm which islimited by the kinetics of endocytosis and biodistributionprocess.13,41 Provided TEM analysis revealed that the dominantpart of the analysed b-NaGdF4:Yb

3+Er3+@PEG-Mo NRs have thesize of about 20 nm and meet the suggested range criteria. Forcomparison, the TEM data published by other authorsdescribed cubic or hexagonal RENCs in the size ranging from 10to 300 nm.22,25

The surface chemistry is one of the main factors inuencingnanocrystals cytotoxicity. In the analysed b-NaGdF4:-Yb3+Er3+@PEG-Mo NRs (PEG-Mo) their coating strongly inu-ences the cell viability and NRs' internalization. Polyethyleneglycol 860 monooleate, noncovalently adhered to original oleateligands on the surface of b-NaGdF4:Yb

3+Er3+ NRs, is a well-known coating material, which stabilizes nanoparticles,increases their hydrophilicity and dispersion in polar solvents.24

In general, cationic polymers are commonly used as biocom-patible coating, which also increases efficiency of the internal-ization process.21 Some authors declare only positive aspects oncells' viability and the internalization process of RENCs coatedwith polymers. Other in vitro cytotoxicity analyses presented inliterature have shown the low toxicity of upconverting nano-crystals with various ligands determined as functions ofconcentration and the time of incubation.7,15–18,20,22–24,39,42,43

Xiong et al. described egg-like or cocoon-like shaped amine-functionalized RENCs (diameters of 20 nm and lengths of 20–40 nm) with folic acid as low cytotoxic (MTT assay, HeLa cells,concentrations of 50, 100, 200, 400 mg ml�1, 24 h of incuba-tion).39 Jin et al. reported the cytotoxicity of cubic NaGdF4:-Yr3+Er3+ NCs (50 nm in diameter) coated with differentpolymers: poly(acrylic acid) (PAA), polyethylenimine (PEI), orpolyvinylpyrrolidone (PVP) (MTT assay, HeLa, U87MG,concentrations 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 mg ml�1,24 h of incubation). The PAA coated RENCs did not reduce theviability of the examined cells, but the PEI and PVP coated onesdecreased the viability by approximately 20–30%, however, thislevel was still described as tolerable. Their paper emphasisesthe role of polymer surface coatings as an important factordetermining the toxicity actions towards cells and the efficiencyof cellular uptake.21 A close study of literature also revealed thedata describing the negative or moot inuence of differentcoatings. It shows a wide range of issues which determine thecytotoxicity and cellular uptake efficiency. There are reportsconcerning the high cytotoxicity prole of RENCs coated withpolymers (i.e. PEG).24,25Das et al. described specic properties ofcubic PEG-Mo coated RENCs of 36–40 nm diameter that causedincreased toxicity even aer 24 h of incubation with the HAECcell line (MTT and calcein–propidium iodide live/dead cellassays, the concentration range of 5–75 mg ml�1). Namely, thepH and ionic dependant destabilization of PEG-Mo RENCs mayoccur during incubation, which could result in micellesformation and cells death.24 Generally, pH of the mediumduring the cultivation time is stabilised by CO2 concentration

This journal is © The Royal Society of Chemistry 2016

Fig. 7 Confocal microscope image of the HeLa cells after 24 h of incubation with b-NaGdF4:Yb3+Er3+@PEG-Mo NRs (a and b) and emission

spectra (c). Confocal image shown in (a) was taken in the channel mode (exc. 405, 633, 980 nm) with the power density of �1 MW cm�2 in theinfrared line and�1 kW cm�2 in the other two lines. The image shown in (b) was taken in the lambdamode using only the 980 nm excitation withthe power density�1 MW cm�2. The spectra shown in (c) are the spectra measured in the points indicated in (b) by crosses in respective colours.Cell membrane – concanavalin 647 nm (red colour (a), violet colour (b)), b-NaGdF4:Yb

3+Er3+@PEG Mo NCs – NIR luminescence (green colour),cell nuclei – DAPI (blue colour).

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and should remain stable aer long term culture. However, theyproposed that the RENCs transit dissociation was a result ofcellular or proteins action. There may be several reasons whyresults described in our paper are different from resultsdescribed by Das et al., despite of the similarity of NCs' type.First, they investigated a different type of cell line. We shouldtake into account that every cell line aer exposure to thenanocrystals may react in a specic way, depending on thephase of growth, cell cycle, normal or cancer tissue origin,culture conditions, incubation time interval, concentration ofthe RENCs, as well as methods of their synthesis, puricationand modication.33 Secondly, we tested NCs of different shapeand size compared to NCs tested by Das et al. Both parametersare important in cellular uptake and may directly inuence thismechanism.24

These discrepancies may result from the nature of analysednanoparticles but also may be caused by the lack of restrictedregulations concerning nanoparticles, including the algorithmsof synthesis' purity, as well as physicochemical and biologicalevaluations. That is why the cytotoxicity study should be per-formed for every type of nanoparticles, especially if producedwith the use of different compositions and under variousconditions.24

The confocal microscopy study of b-NaGdF4:Yb3+Er3+@PEG-

Mo NRs' internalization by the HeLa and HT-29 cells conrmedthe effective cellular uptake by the presence of upconversionuorescence inside the cells (Fig. 7). The analysed emissionspectrum of the b-NaGdF4:Yb

3+Er3+@PEG-Mo NRs in intracel-lular matrix under the excitation at 980 nm correlated with theemission spectrum obtained in the optical study of NRssuspension. The observed upconversion efficiency stays in linewith the data concerning dopants on the NaYF4, which indicatethat Yb3+ and Er3+ are known to be the most efficient NIR tovisible upconverting ions.26 Those results indicate a widepotential of tested NCs in the eld of uorescence imaging bothat in vitro and in vivo level. As mentioned above, the type ofsurface coating strongly inuences the internalization process.In the literature it was shown that RENCs with different coating

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polymers enter into the cells via the clathrin endocytic mecha-nism, but the effectiveness of this process is inuenced by theelectrostatic attraction of the cell membrane.21 The zeta poten-tial (z) indicates the electrokinetic phenomena that also candetermine interaction of the NCs with the cellular membrane orinterior compartments of the cell. It depends on many factors(e.g. the type of solvent, or coating, and pH). The zeta potentialof the b-NaGdF4:Yb

3+Er3+@PEG-Mo NRs suspension in water(pH 7.0) was (�17.7 mV � 0.98) mV which species gooddispersion stability. The literature reports on RENCs coatedwith various types of polymers or silica coatings producing boththe negative and positive zeta potential. Good cellular inter-nalization was found for both of them, with some special focuson the positive one, due to electrostatic interactions with thenegatively charged cell membrane.21 It should be noted here,however, that the zeta potential of RENCs with coatings canacquire different values, depending on the solvent used(complete medium, serum, phosphor buffer, water). Thus, onlythe zeta potential of RENCs measured in the same solvent canbe compared and discussed as a factor determining the cellularinternalization efficiency.

Taking into consideration the stable and efficient cellularuptake and the luminescence of the b-NaGdF4:Yb

3+Er3+@PEG-Mo NRs aer 24 h of incubation with the cells as well as timeand dose dependent cytotoxicity prole, we can conclude thatthe analysed nanorods coated with PEG-Mo remain stable. Ourresults also revealed that noncovalently attached PEG-Mo didnot dissociate form the complexes with NRs nor formed anymicelles. Instead, it stabilised RENCs enabling an efficientcellular uptake aer 24 h of incubation.

Conclusions

This report concerns complex physicochemical, optical andbiological data of the rod-shaped b-NaGdF4:Yb

3+Er3+ nano-crystals. We considered the multimodal in vitro imagingand cytotoxic prole using RENCs with luminescenceand paramagnetic properties. Such systems were based on the

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b-NaGdF4:Yb3+Er3+ nanocrystals coated noncovalently with

PEG-Mo 860. The morphology study revealed nanorods 20 nmin length with hydrophilic bilayer. Respective studies demon-strated strong paramagnetic and efficient luminescence prop-erties. The time and dose dependant cytotoxic proles wereestimated as acceptable. The confocal microscopy studyconrmed the cellular uptake by NIR excitation visible lumi-nescence in the cells. Despite the fact that such RENCs presenta great application potential, the discussion about the inuenceof the coating type on their chemical stability is important. Toour best knowledge, this is the rst cytotoxicity report whichconsiders RENCs of anisotropic rod-like shape. In our research,we have mainly focused on the discussion concerning thecytotoxicity and optical properties of RENCs addressing theissue of their unique shape, thus casting a new light on theprocess of designing biocompatible and efficient nanotools formultimodal imaging.

Contribution of each authors

Anna Wozniak – designed biological studies and analysed thesestudies results, coordinated research and analysed data, draedthe manuscript. Agnieszka Noculak – performed synthesis of thenanorods and measured TEM, EDXS and XRD. Jacek Gapinski –performed optical studies (PCS, PL) of the nanorods, upconver-sion and confocal studies in vitro. Daria Kociołek – performedfunctionalisation of the nanorods with PEG-Mo. Agnieszka Bos-Liedke – performed cell culture for in vitro imaging, draed partof introduction part of the manuscript. Tomasz Zalewski – per-formed MRI study. Bartosz Grzeskowiak – performed zetapotential measurements. Anna Kołodziejczak – performed cellcultures, WST1 and apoptosis assays for of biological studies.Stefan Jurga – coordinated research and analysed data. MateuszBanski – assisted in nanorods functionalization, analysed data.Jan Misiewicz – analysed data. Artur Podhorodecki – coordinatedresearch and analysed data, designed synthesis, performedspectroscopic measurements (PL and PL decay).

Acknowledgements

A. P. would like to express his gratitude for the nancial supportto the National Science Centre, Sonata Bis 3 Project No. UMO-2013/10/E/ST5/00651. A. W. would like to express her gratitudefor the nancial support to the National Centre for Research andDevelopment under the research grant “Nanomaterials and theirapplication to biomedicine”, the contract number PBS1/A9/13/2012. B. F. G. would like to express his gratitude for the Inter-national PhD Projects Programme of Foundation for PolishScience, part of the Innovative Economy Operational Programme(IE OP) 2007–2013 within the European Regional DevelopmentFund and NanoBioMedical Centre, Adam Mickiewicz University.

References

1 S. R. Cherry, Semin. Nucl. Med., 2009, 39, 348–353.2 M. Mitsunaga, M. Ogawa, N. Kosaka, L. T. Rosenblum,P. L. Choyke andH. Kobayashi,Nat. Med., 2011, 17, 1685–1691.

95642 | RSC Adv., 2016, 6, 95633–95643

3 Y. Min, J. Li, F. Liu, P. Padmanabhan, E. K. L. Yeow andB. Xing, Nanomaterials, 2014, 4, 129–154.

4 R. Kumar, M. Nyk, T. Y. Ohulchanskyy, C. A. Flask andP. N. Prasad, Adv. Funct. Mater., 2009, 19, 853–859.

5 M. He, P. Huang, C. Zhang, H. Hu, C. Bao, G. Gao, R. He andD. Cui, Adv. Funct. Mater., 2011, 21, 4470–4477.

6 A. Noculak, A. Podhorodecki, G. Pawlik, M. Banski andJ. Misiewicz, Nanoscale, 2015, 7, 13784–13792.

7 J. Zhou, Y. Sun, X. Du, L. Xiong, H. Hu and F. Li, Biomaterials,2010, 31, 3287–3295.

8 Y. Hou, R. Qiao, F. Fang, X. Wang, C. Dong, K. Liu, C. Liu,Z. Liu, H. Lei, F. Wang and M. Gao, ACS Nano, 2013, 7,330–338.

9 L. Ch. Liu, Z. Gao, J. Zeng, Y. Hou, F. Fang, Y. Li, R. Qiao,L. Shen, H. Lei, W. Yang and M. Gao, ACS Nano, 2013, 7,7227–7240.

10 N. J. J. Johnson, W. Oakden, G. J. Stanisz, R. S. Prosser andF. C. J. M. van Veggel, Chem. Mater., 2011, 23, 3714–3722.

11 I. Miladi, G. Le Duc, D. Kryza, A. Berniard, P. Mowat, S. Roux,J. Taleb, P. Bonazza, P. Perriat, F. Lux, O. Tillement, C. Billoteyand M. Janier, J. Biomater. Appl., 2013, 28, 385–394.

12 D. Kryza, J. Taleb, M. Janier, L. Marmuse, I. Miladi,P. Bonazza, C. Louis, P. Perriat, S. Roux, O. Tillement andC. Billotey, Bioconjugate Chem., 2011, 22, 1145–1152.

13 C. Huang, Y. Zhang, H. Yuan, H. Gao and S. Zhang, NanoLett., 2013, 13, 4546–4550.

14 M. Nyk, R. Kumar, T. Y. Ohulchanskyy, E. J. Bergey andP. N. Prasad, Nano Lett., 2008, 8, 3834–3838.

15 R. A. Jalil and Y. Zhang, Biomaterials, 2008, 29, 4122–4128.16 S. A. Hilderbrand, F. W. Shao, C. Salthouse, U. Mahmood

and R. Weissleder, Chem. Commun., 2009, 4188–4190.17 S. Jiang, Y. Zhang, K. M. Lim, E. K. W. Sim and L. Ye,

Nanotechnology, 2009, 20, 155101.18 L. Q. Xiong, T. S. Yang, Y. Yang, C. J. Xu and F. Y. Li,

Biomaterials, 2010, 31, 7078–7085.19 F. Wang, R. Deng, J. Wang, Q. Wang, Y. Han, H. Zhu, X. Chen

and X. Liu, Nat. Mater., 2011, 10, 968–973.20 J. C. Zhou, Z. L. Yang, W. Dong, R. J. Tang, L. D. Sun and

C. H. Yan, Biomaterials, 2011, 32, 9059–9067.21 J. Jin, Y.-J. Gu, C. Wing-Yin Man, J. Cheng, Z. Xu, Y. Zhang,

H. Wang, V. H.-Y. Lee, S. H. Cheng and W.-T. Wong, ACSNano, 2011, 5, 7838–7847.

22 H. Xing, X. Zheng, Q. Ren, W. Bu, W. Ge, Q. Xiao, S. Zhang,C. Wei, H. Qu, Z. Wang, Y. Hua, L. Zhou, W. Peng, K. Zhaoand J. Shi, Sci. Rep., 2013, 3, 1751.

23 L. Pan, M. He, J. Ma, W. Tang, G. Gao, R. He, H. Su andD. Cui, Theranostics, 2013, 3, 210–222.

24 G. K. Das, D. T. Stark and I. M. Kennedy, Langmuir, 2014, 30,8167–8176.

25 E. Wysokinska, J. Cichos, E. Zioło, A. Bednarkiewicz,L. Strzadała, M. Karbowiak, D. Hreniak and W. Kałas,Toxicol. In Vitro, 2016, 32, 16–25.

26 F. Auzel, Chem. Rev., 2004, 104, 139–174.27 A. Prakash, H. Zhu, C. J. Jones, D. N. Benoit, A. Z. Ellsworth,

E. L. Bryant and V. L. Colvin, ACS Nano, 2009, 3, 2139–2146.28 J. Pichaandi, J.-C. Boyer, K. R. Delaney and F. C. J. M. van

Veggel, J. Phys. Chem. C, 2011, 115, 19054–19064.

This journal is © The Royal Society of Chemistry 2016

Paper RSC Advances

Publ

ishe

d on

06

Oct

ober

201

6. D

ownl

oade

d by

Cen

trum

Wie

dzy

i Inf

orm

acji

Nau

k-T

ech

on 1

0/10

/201

6 13

:32:

59.

View Article Online

29 Y. Xuefeng, L. Min, X. Mengyin, C. Liangdong, L. Yan andW. Ququan, Nano Res., 2010, 3, 51–60.

30 O. S. Woleis, Chem. Soc. Rev., 2015, 44, 4743–4768.31 S. Mondini, C. Drago, A. M. Ferretti, A. Puglisi and A. Ponti,

Nanotechnology, 2013, 24, 105702.32 E. Amstad, M. Textor and E. Reimhult, Nanoscale, 2011, 3,

2819–2843.33 L. Zhang, F. X. Gu, J. M. Chan, A. Z. Wang, R. S. Langer and

O. C. Farokhzad, Clin. Pharmacol. Ther., 2008, 83, 761–769.34 Y. Zhang, D. Xu, W. Li, J. Yu and Y. Chen, J. Nanomater.,

2012, 12, 7.35 J. Han, J. Li, W. Jia, L. Yao, X. Li, L. Jiang and Y. Tian, Int. J.

Nanomed., 2014, 9, 517–526.36 H. Chen, X. Zhang, S. Dai, Y. Ma, S. Cui, S. Achilefu and

Y. Gu, Theranostics, 2013, 3, 633–649.

This journal is © The Royal Society of Chemistry 2016

37 A. Ambrosone, P. del Pino, V. Marchesano, W. J. Parak,J. M. de la Fuente and C. Tortiglione, Nanomedicine, 2014,9, 1913–1919.

38 A. Gnach, T. Lipinski, A. Bednarkiewicz, J. Rybka andJ. A. Capobianco, Chem. Soc. Rev., 2015, 44, 1561–1584.

39 L. Q. Xiong, Z. G. Chen, M. X. Yu, F. Y. Li, C. Liu andC. H. Huang, Biomaterials, 2009, 30, 5592e600.

40 M. E. Davis, Nat. Rev. Drug Discovery, 2008, 7, 771–782.41 J. F. Hainfeld, L. Lin, D. N. Slatkin, F. A. Dilmanian,

T. M. Vadas and H. M. Smilowitz, Nanomedicine, 2014, 10,1609–1617.

42 H. Hu, M. X. Yu, F. Y. Li, Z. G. Chen, X. Gao, L. Q. Xiong,et al., Chem. Mater., 2008, 20, 7003e9.

43 H. Hu, L. Q. Xiong, J. Zhou, F. Y. Li, T. Y. Cao andC. H. Huang, Chem.–Eur. J., 2009, 15, 3577e84.

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