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Colloids and Surfaces B: Biointerfaces 160 (2017) 1–10

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

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rotocols

orous NH2-MIL-125 as an efficient nano-platform for drug delivery,maging, and ROS therapy utilized Low-Intensity Visible lightxposure system

runkumar Rengaraj a,1, Pillaiyar Puthiaraj b,1, Nam-Su Heo a, Hoomin Lee a,eung Kyu Hwang a, Soonjo Kwon c, Wha-Seung Ahn b,∗, Yun-Suk Huh a,∗

Department of Biological Engineering, Biohybrid Systems Research Center (BSRC), Inha University, Incheon, 22212, Republic of KoreaDepartment of Chemistry and Chemical Engineering, Inha University, Incheon, 22212, Republic of KoreaDepartment of Biological Engineering, Integrated Tissue Engineering, Inha University, Incheon, 22212, Republic of Korea

r t i c l e i n f o

rticle history:eceived 24 May 2017eceived in revised form 3 August 2017ccepted 5 September 2017vailable online 6 September 2017

eywords:etal organic frameworksH2-MIL-125oxorubicinrug delivery

a b s t r a c t

Metal-organic frameworks are a novel class of organic-inorganic hybrid polymer with potential applica-tions in bioimaging, drug delivery, and ROS therapy. NH2-MIL-125, which is a titanium-based metalorganic framework with a large surface area of 1540 m2/g, was synthesized using a hydrothermalmethod. The material was characterized by powder X-ray diffreaction (PXRD), thermogravimetric analy-sis (TGA), and transmission electron microscopy (TEM), and N2 isotherm analyses. The size of the polymerwas reduced to the nanoscale using a high-frequency sonication process. PEGylation was carried outto improve the stability and bioavailability of the NMOF. The as-synthesized nano-NH2-MIL-125/PEG(NMOF/PEG) exhibited good biocompatibility over the (Cancer) MCF-7 and (Normal) COS-7 cell line. Theinteraction of NMOF/PEG with the breast cancer cell line (MCF-7) was examined by BIO-TEM analysis andlaser confocal imaging. 2′,7′–dichlorofluorescin diacetate (DCFDA) analysis confirmed that NMOF/PEG

OS therapy produced free radicals inside the cancer cell line (MCF-7) upon visible light irradiation. NMOF/PEGabsorbed a large amount of DOX (20 wt.% of DOX) and showed pH, and photosensitive release. Thiscontrolled drug delivery was attributed to the presence of NH2, Ti group in MOF and a hydroxyl group inPEG. This combination of chemo- and ROS-therapy showed excellent efficiency in killing cancer MCF-7cells.

© 2017 Published by Elsevier B.V.

. Introduction

Cancer is a chronic disease that involves the abnormal evolu-ion of cells with the potential to invade other organs and causespproximately 6 million deaths annually [1]. A report issued byhe International Agency for Research on Cancer (IARC) revealed4.1 million cancer patients around the world in 2012. Of these, 7.4illion and 6.7 million cases were men and women, respectively.

n women, breast cancer is the most common cancer with almost.7 million new patients diagnosed in 2012 [2]. Most breast can-

ers originate in the lobules and ducts that connect the lobules tohe nipple [3]. Over the last few years, there has been a significantevelopment in breast cancer treatment but many cases are diag-

∗ Corresponding authors.E-mail addresses: whasahn@inha.ac.kr (W.-S. Ahn), yunsuk.huh@inha.ac.kr

Y.-S. Huh).1 These authors contributed equally to this work.

ttp://dx.doi.org/10.1016/j.colsurfb.2017.09.011927-7765/© 2017 Published by Elsevier B.V.

nosed in the late stage, and multidrug resistance has increased [4].To solve these problems, a platform that can be used for diagnosis,as well as combined therapy on cancer cells, is needed. Recently,nanotechnology has been used in cancer treatments for more effi-cient early diagnoses, imaging, and targeted therapies [5].

Nanocarriers are important drug-delivery systems that can beused for diagnosis and provide sustainable drug release at the tar-geted sites. Along with drug delivery, these nanomaterials can alsobe used in combination therapies, such as gene therapy, theranos-tic, and ROS therapy [6–8]. Over the last decade, different kindsof nanocarriers have been demonstrated in drug delivery systems,such as dendrimers, lipids, carbohydrate polymers, polymer-coatedmetal nanoparticles, activated carbon, zeolites, amine-modifiedporous silica, metal-organic frameworks (MOFs), and porous cova-lent triazine polymers (CTPs) [9–16]. Among the above carriers,

porous MOF is a new class of material that shows excellent capa-bility as a drug carrier material because of their tunable poresize, high surface area, and well-ordered arrangement of metal

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nd organic linkers. In addition, MOFs are constructed from metalon/metal clusters and bi-/multi-dentate organic linkers via a coor-ination bond [17,18]. This highlights the potential use of thisaterial in other applications such as photoluminescence, MRI

maging, and phototherapy [19–22]. Benefiting from the large rangef optional chemical compositions, introducing the non-toxic ther-peutic luminescent molecules directly as organic spacers in theormation of MOF is feasible.

Owing to the above mentioned properties, researchers haveeported MOF as a nanocarrier for drug delivery applications.or example, Ferey et al. first examined MIL-based MOF for drugelivery applications (MIL-100(Cr)) [23]. Horcajada et al., reported

ron-based MOFs (MIL-100(Fe)) using a range of nontoxic organicinkers and used them as a carrier to deliver different types ofrugs (doxorubicin, ibuprofen, caffeine, urea, and benzophenone).hey achieved a more than 20% loading for the above drugs andsed them for MRI imaging [21]. Scaling down the size of theOFs to the nanoscale regime is a crucial step to deliver drugs

nto the cells and obtain stable and reproducible formulations.he nanoscale MOFs (NMOF) can be synthesized by a range ofechniques, such as solvothermal method, reverse microemulsion,ltrasonic synthesis, and microwave irradiation [24–26]. Ultra-onic synthesis was found to be a high yielding, low cost, andnvironmentally friendly method for the synthesis of NMOF [27].ost MOFs are hydrothermally unstable. To solve this problem,

esearchers have used various non-toxic surfactants to improvehe stability of MOF. Lin et al. prepared silica-coated Mn-NMOFnd demonstrated sustained drug release [28]. Agostoni et al.repared PEG-coated iron-NMOF to increase the stability and pre-ent the agglomeration of nanoparticles by increasing the surfaceharge. This PEGylated MOF showed an increased circulation timeue to the lower immunogenicity of the nanoparticle [29]. This

ong-circulating nanoparticle enters the hyperpermeable angio-enic tumor through preferential extravasation without the use ofargeting molecules [30,31].

Doxorubicin (DOX) is used widely in cancer chemotherapyecause it can induce apoptosis by intercalating within DNA [32].he application of DOX in cancer senescence and p53-therapy haseen discussed. This type of drug can reverse the cancer cells with-ut harming the organs [33]. Other than drugs, reactive oxygenpecies (ROS) are important signaling molecules that play crucialoles in cancer, heart disease, and neuroscience [34–36]. ROS areeactive chemical species containing oxygen, such as peroxides,uperoxide, hydroxyl radicals, and singlet oxygen [37]. ROS can beroduced from coherent and non-coherent light sources consist-

ng of filtered lamps/LED. Subsequently, the increased ROS activatelectron transport, blood flow, adenosine triphosphate nitric oxideelease, and diverse signaling pathways [38]. These signals haveeen used to treat cancer and other diseases. Accordingly, there is

ncreasing interest in rapid and efficient drug delivery as well as inOS induction for combinations of therapies in cancer treatment.

Therefore, a titanium-based metal-organic framework (MIL-25) was prepared for drug delivery and ROS therapy viaoordination bonding. The nanoscale MOF (NMOF) was preparedsing an ultrasonication process and encapsulated with PEG-400o increase the stability and bioavailability. The biocompatibilityf the NMOF/PEG was evaluated using a 3-(4,5-dmethylthiazol--yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cellularptake of NMOF/PEG in breast cancer cells (MCF-7) was evidencedy Bio-TEM imaging. The cancer drug DOX was then introducedo the NMOF/PEG using a physisorption method. The ability ofOS induction by NMOF/PEG was examined using a DPPH assay.

his combined efficiency of drug release and ROS induction byMOF/PEG was studied over MCF-7 by confocal imaging usingCFDA and MTT assay.

B: Biointerfaces 160 (2017) 1–10

2. Results and discussion

NH2-MIL-125 (MOF) was prepared via a coordination chem-istry using 2-aminoterephthalic acid and titanium isopropoxideas the benchmark (see scheme. S1) and characterized by PXRD,BET (N2 isotherm analyses), TGA, field emission scanning electronmicroscopy (FESEM), TEM, and UV-vis spectroscopy (Figs. 1 and 2and Fig. S1). Scheme 1 presents a schematic representation of thesynthesis of NMOF/PEG-DOX along with the results. The MOF solu-tion was irradiated directly for 15 min using an ultrasonic generatorequipped with a titanium horn transducer (S-450A, Branson, USA).After 15 min, the solution was passed through a 0.2 �M polyvinyli-dene fluoride filter to collect the NMOF. The NMOF was added withPEG-400 and sonicated for 20 min. The sonication process is usedwidely for encapsulation, and it helps achieve the desired nanopar-ticle size [39]. Fig. S1 presents the various stages of NMOF/PEGbased on the time (5, 10, 15 and 20 min) under constant sonica-tion. The TEM image showed that fragmentation began at 5 min.The fragmented NMOF combined after 5 min due to the energyobtained during sonication and the hydrophobic nature of the PEG.After 10 min, NMOF was mixed with the PEG and after 20 min NMOFcoated the PEG to achieve a particle size of approximately 200 nm.This solution was centrifuged at 10000 rpm for 15 min to removethe excess PEG and washed twice with PBS. The synthesized NMOFand NMOF/PEG were confirmed by FT-IR, XRD, TGA, FE-SEM, andTEM (Fig. 1 and 2 and Fig. S2).

Fig. 1a shows the sharp XRD peaks for MOF, indicating that thematerial has good crystallinity. Powder XRD revealed no changesin crystallinity and peak intensity for NMOF, suggesting that theintegrity of the framework had been preserved and was highly sta-ble after the preparation of NMOF. After the encapsulation of PEGover NMOF, the crystallinity was decreased slightly. The BET sur-face area and pore size of the MOF, NMOF, and NMOF/PEG wereevidenced from the N2 sorption isotherm at 77 K (Fig. 1b). Beforeisotherm analysis, the sample was pretreated for 12 h at 150 ◦Cunder high vacuum conditions. The NH2-MIL-125 isotherm dis-played strong N2 gas uptake in the low-pressure region with a typeI isotherm, suggesting the permanent microporosity of the mate-rial. The specific BET surface area of NH2-MIL-125 was 1540 m2 g−1

with a pore size of 0.63 nm. After sonication, the surface area ofNMOF was increased to 1720 m2 g−1. After the PEGylation process,a considerable decrease in surface area to 155 m2 g −1 was observed,indicating that the PEG chains strongly covered the surface of NMOFand impregnated the pores. Fig. S2 presents the FT-IR spectra ofNMOF and NMOF/PEG. The peaks at 3326 and 3440 cm−1 wereassigned to the symmetric and asymmetric stretching bands of theH N H bonds in NMOF, respectively. In addition, the characteristicsymmetric and asymmetric stretching vibrations of the −O C-O- bonds were observed at 1350 and 1550 cm−1, respectively,indicating the presence of deprotonated carboxylate ions and theformation of NMOF. After encapsulation with PEG, the H N Hbands appeared broadly at 3350 and 3460 cm−1, due to the pres-ence of hydroxyl group in the PEG. The characteristic CH2- bandfrom PEG was observed strongly at 2850 cm−1, indicating that PEGwas encapsulated on the surface of the NMOF. The amount of car-bon, hydrogen, and nitrogen was analyzed by EA, as listed in TableS1. After encapsulation with PEG, the amount of carbon increasedslightly with a concomitant decrease in the amount of nitrogen.This EA result also proved that PEG was strongly encapsulated intothe NMOF. TGA of MOF and NMOF (Fig. 1c) showed that the frame-work was stable up to 300 ◦C and reached approximately 25 wt%residual mass at 1000 ◦C, whereas NMOF/PEG degradation started

at 325 ◦C and reaches around 32 wt% residual mass at 1000 ◦C due tothe incorporation of PEG onto NMOF. These results clearly showedthat the approximately 7 wt% of PEG was loaded onto NMOF. TheUV absorption spectrum (Fig. 1d) of MOF revealed an absorbance

A. Rengaraj et al. / Colloids and Surfaces B: Biointerfaces 160 (2017) 1–10 3

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ig. 1. Characterization and properties of MOF, NMOF, NMOF/PEG and NMOF/PEG-nalysis, and (d) UV–Vis absorbance and photoluminescence spectra (insert) of NM

t approximately 340 nm, which was attributed to the n–�* tran-ition of the nitrogen lone pair electron in 2-aminoterephthaliccid [40]. The part of the transition electrons produce emissiont 450 nm (1ig. 1 d (insert)), and the remaining excited electronsere produced ROS in the environment. Garcia et al. proposed that

hese ROS species can be produced by i) charge separation withhe development of positive holes on the organic ligands contain-ng carboxylate/amino groups and ii) a change in the electronictate of Ti from 4+ to 3+ due to the trapping of electrons duringhotolysis [41]. After sonication, the absorption of NMOF was red-hifted slightly to 350 nm, due to the presence of particles withifferent sizes, which can absorb high-energy light. The emissiont 450 nm and the other remaining excited electrons produced ROSn the NMOF, which may be more beneficial to cancer theraphy.

DLS revealed the presence of polydisperse NMOF with a meanarticle size of 299 nm (Fig. S3b). When the NMOF/PEG was excitedt 350 nm, strong emission was observed at approximately 450 nmFig. 1d), When the wavelength was increased continuously, thereas a small right shift, and the PL intensity decreased. This was

ttributed to the monodispersed form of NMOF/PEG, which has 350 nm excitation wavelength and produces 450 nm photolu-inescence along with ROS species. DLS analysis of NMOF/PEG

evealed a bimodal distribution, which further confirmed theonodispersed form of NMOF/PEG (Fig. S3c).

Fig. 2 presents SEM and TEM images of the MOF (Fig. 2a, b),MOF (Fig. 2c, d), and PEGylation (Fig. 2e, f). SEM and TEM showed

hat the MOF had a cubic shape with a size of 600 nm. DLS showedhat the mean particle size of NMOF was 299 nm, after sonicationFig. S3b). EDX element mapping TEM was performed to map the, N, and Ti distribution in the NMOF (Fig. S4). After PEGylation, the

) XRD patterns, b) N2 isotherm analysis of the surface area of the materials, c) TGAG under different excitation.

particle became uniform in size, as confirmed by UV spectroscopy,and DLS revealed a mean size of 208 nm with a spherical shape(Fig. 2e, f, and Fig. S3). The poor colloidal stability of the uncoatedNMOFs in aqueous media is the main drawback for their use in drugdelivery systems. The stability of MOF, NMOF and NMOF/PEG in thecolloidal system was examined by zeta potential, as listed in TableS2. The zeta potential of MOF was 3.78, which means that it can-not disperse in water. In contrast, the zeta potential of NMOF was−10.59, which was increased to −27.98 after the PEGylation pro-cess. This shows that PEG was strongly encapsulated into the MOFdue to the (i) hydrophobic interaction of PEG with the organic link-ers in the MOF and (ii) the ionic interactions between the hydroxylgroup of the PEG and Ti metal ions in the MOF. The effects of pH onthe stability of NMOF/PEG were examined by zeta potential anal-ysis. Fig. S5 shows that the stability of NMOF/PEG was increasedwith increasing pH.

NMOF/PEG was used for anti-cancer drug delivery and ROStherapy because of its high colloidal stability, surface area, pho-toluminescence properties and ROS induction upon visible lightirradiation. Before the drug loading studies, the biocompatibilityof the porous materials was analyzed. To estimate the cytotox-icity of MOF, NMOF, and NMOF/PEG an MTT cell viability assaywas carried out using COS-7 and MCF-7 cells with different con-centrations of materials for 24 h, as shown in Fig. 3a and b. Cellswithout the addition of MOF, NMOF, and NMOF/PEG were used asthe control. As shown in Fig. 3a and b, NMOF/PEG exhibited less

toxicity at four concentrations in the range of 10–200 �g/ml com-pared to MOF and NMOF. When the concentration of NMOF/PEGwas increased to 200 �g/ml, the cytotoxicity in COS-7 and MCF-7was less than 20%. NMOF/PEG was less cytotoxic to both cells even

4 A. Rengaraj et al. / Colloids and Surfaces B: Biointerfaces 160 (2017) 1–10

Fig. 2. Morphology of MOF, NMOF and NMOF/PEG. SEM analysis of a) MOF, b) NMOF, and c) NMOF/PEG. TEM analysis of d) MOF, e) NMOF, and f) NMOF/PEG.

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t higher concentrations than MOF and NMOF. This was attributedo the larger size of MOF and more ROS present in the NMOF (seeater in ROS studies) than that reported elsewhere [42]. In addition,he distribution of NMOF/PEG in the MCF-7 cell was analyzed byio-TEM images (Fig. 3c, d). In the case of the control (Fig. 3c; with-

ut NMOF/PEG) treatment, the condensed form of the nucleus and aery small number of peroxisomes were observed in the cytoplasmith a well-ordered cell structure. In contrast, after incubating the

OF/PEG, and NMOF/PEG-DOX.

cells with NMOF/PEG for 6 h, a few particles were attached to thecell membrane, and more particles were dispersed throughout thecytoplasm via endosome formation.

The porous and biocompatible nature of NMOF/PEG was usedfurther to load DOX molecules. The drug loading was carried out

simply by stirring in an aqueous solution, and the loading efficiencywas measured by UV–vis spectroscopy. Drug molecules providedwith a suitable size can be absorbed by the porous core interacting

A. Rengaraj et al. / Colloids and Surfaces B: Biointerfaces 160 (2017) 1–10 5

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ig. 3. Cytotoxicity analysis of MOF, NMOF and NMOF/PEG in a) COS-7 b) MCF-7. Eaith NMOF/PEG.

ith the nanoparticle matrix through hydrophobic and H-bondingnteractions between the DOX and organic linker of the nanoparti-les. The observed DOX loading efficiency was approximately 55%ith a saturated loading of 200 �g/mg on NMOF/PEG. The absorp-

ion of drug molecules into the NMOF/PEG was characterized byT-IR, BET surface area measurements, and DLS. The IR spectrum ofOX-loaded NMOF/PEG (Fig. S3) revealed, the characteristic C Oond stretching band for at 1650 cm−1 and the −OH stretchingibration for DOX at around 3400 cm−1, which indicate the suc-essful loading of DOX onto the NMOF/PEG. After DOX absorption,he surface area of the NMOF/PEG was reduced to 14.4 m2g−1 from55 m2g−1 (Fig. 1b), which also confirmed the incorporation ofhe guest molecule (DOX). After drug absorption, the particles sizencreased to 244 nm as shown by DLS (Fig. S3d). The percentageOX release was calculated using both the excitation and emission

pectra of DOX (Fig. 4b and Fig. S6). DOX was released steadily upo 55% at pH 7.4 within 24 h, whereas 65% of DOX was releasednder irradiation conditions (Fig. 4b). This was attributed to thebsorption of energy by the organic linkers in the MOF under irra-iation, so that the interaction between the organic linkers andOX was slightly weaker than under the normal condition. Pre-ious reports showed that tumor tissue (pH 5.5 − 6.0) are morecidic than normal tissues (pH 7.4) and blood. Therefore, the ratef drug release was observed under pH 5.2/normal and irradiationonditions. When the pH was changed to 5.2, the percentage drugelease also increased under both normal and irradiation condi-

ions. These results show that the specific interactions betweenhe drug molecules and the skeletons of the MOFs could not onlynhance the loading capacity but also prolong the release process.

ue represents the mean ± SD (n = 3). Bio-TEM images of MCF-7 c) control d) Treated

This stepwise release is due to the pore sizes of the porous solids andthe interactions from hydrogen bonds and �-� stacking betweenDOX and the organic part of the skeleton. Therefore, pH sensitiveand photosensitive porous MOF materials can be valuable candi-dates for the delivery of anticancer agents and enhancing the cancertreatment efficiency.

To evaluate the ROS present in the MOF, NMOF, and NMOF/PEG,they were analyzed using a DPPH assay with/without the irra-diation of visible light (350–700 region filter) (Fig. S7). Fig. S7ashows that the NMOF possessed a larger amount of ROS than MOFand NMOF/PEG, due to the smaller particle size and larger sur-face area. In addition, ROS production increased dramatically whenNMOF/PEG was irradiated with a visible light source (Fig. S7b).When the distance and exposure time were increased, the amountof ROS produced also increased. The amount of ROS produced wasoptimized by varying the distance between the cell culture and lightsource, and the exposure time (Fig. S7c and Fig. 4c). The optimalconditions were 10 min and 15 cm, and the NMOF/PEG concen-tration was varied (10 �g/ml- 50 �g/ml). As shown in Fig. 4c, theamount of ROS increased with increasing NMOF/PEG concentrationunder constant irradiation with a 15 cm distance for 10 min. Fig. 4dpresents the overall mechanism of NMOF/PEG as a drug deliverycarrier and ROS therapy. NMOF/PEG reduced the burst release ofthe drug (DOX) significantly and exhibited pH sensitive and photo-sensitive drug release. The larger amount of drug released into thecancer cells was attributed to the acidic nature of the cancer cells

compared to the normal cell line. The drug release can also be con-trolled using visible-light exposure system. This exposure system

6 A. Rengaraj et al. / Colloids and Surfaces B: Biointerfaces 160 (2017) 1–10

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ig. 4. NMOF/PEG a) Drug (DOX) absorption, b) pH sensitive & photosensitive drugight irradiation system and d) Mechanism of NMOF/PEG-DOX for drug delivery and

an also be used to produce ROS species to signal the cancer cellso undergo apoptosis.

To evaluate the feasibility of NMOF/PEG for combination ther-py (DOX release and ROS), the cytotoxicity of NMOF/PEG-DOXo MCF-7 was analyzed in the presence or absence of visi-le light irradiation. Statistical analysis (t- test) was used toxamine the significances of cancer cytotoxicity over different con-entrations of DOX, NMOF/PEG-DOX, and NMOF/PEG-DOX ∼ ROSFig. 5). The analysis showed that 2 �g of DOX killed approxi-

ately 8% of the cancer cells whereas NMOF/PEG-DOX (10 �g/mlf NMOF/PEG loaded with 2 �g of DOX) and NMOF/PEG-DOX ∼ ROSilled approximately 14 and 36% respectively, which indicate thathe NMOF/PEG-DOX ∼ ROS is more effective (***p < 0.001) at lowerOX concentration. This was attributed to the higher levels ofOS production, which interacts with the tumor suppressor pro-ein (p53) and regulates the apoptotic process [43]. On the otherand, 10 �g/ml of DOX alone killed 47% of the cancer cells whereasMOF/PEG-DOX (50 �g/ml NMOF/PEG/DOX loaded with 10 �g ofOX) and NMOF/PEG-DOX ∼ ROS killed more than 84 and 90%f cancer cells, respectively. The effective cytotoxicity of bothMOF/PEG-DOX and NMOF/PEG-DOX ∼ ROS was attributed to theffective delivery of larger amount of DOX into the cancer cells. Thiselivery approach and ROS induction can be used to induce cancerell death and prolong the senescence activity.

To examine the cellular colocalization of NMOF/PEG nanopar-

icles on human breast cancer MCF-7 cells, the NMOF/PEG,MOF/PEG-DOX and NMOF/PEG-DOX-ROS were incubated withCF-7 (treated with DCFDA), as shown in Fig. 6. Two types of

lters (400–500, 600–700 nm) were used in the fluorescence imag-

e, c) ROS level at different concentrations of NMOF/PEG upon with, without visibletherapy.

ing microscope. As a control, the morphology of the confluentcell monolayer was normal. After incubation with NMOF/PEG, thecells retained their confluence, and punctate fluorescence wasobserved in the region of 400–500 nm, suggesting NMOF/PEGentrapment within intracellular vesicles; this was validated byBio-TEM. After incubating the cells with NMOF/PEG-DOX, the cellconfluence decreased, and the fluorescence signal from NMOF/PEG(400–500 nm) and DOX (600–700 nm) was observed. Under irradi-ation of NCTP/PEG-DOX with the addition of soluble-DCFDA, ROSwere produced and the soluble form of DCFDA was reduced toinsoluble-DCFDA, which was observed as a yellow color calorimet-rically (Fig. S9).

3. Conclusion

NMOF/PEG was synthesized and applied as both a potentialphotosensitizer and pH-responsive nanocarrier for cancer ther-apy and imaging. The NMOF/PEG was synthesized using a fast and“green” procedure. The PEG coating stabilized the nanostructurein the body fluids, and could be functionalized further with thetargeting ligands. This nanocarrier exhibited excellent biocompat-ibility over both cancers and noncancer cell line. Of significance,the NMOF/PEG–DOX complex features a DOX-loading capacity of200 �g/mg, due to the high specific surface area of MOF, hydropho-bic interaction and �-� stacking of NMOF/PEG and DOX. The

photoluminescence, pH-dependent drug release properties of thiscarrier were found to be suitable for bioimaging and drug deliv-ery over cancer cells. The NMOF/PEG-DOX with ROS inductionhad a significantly higher cytotoxic effect on the cancer cells than

A. Rengaraj et al. / Colloids and Surfaces B: Biointerfaces 160 (2017) 1–10 7

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MOF/PEG-DOX and DOX alone. The results highlight the potentialse of NMOF/PEG as a low-toxic and biocompatible 2D nanomate-ial for cancer therapy and bioimaging.

. Materials and method

.1. Materials

2-Aminoterephthalic acid, biphenyl, N,N-dimethylformamideDMF), methanol, titanium (IV) isopropoxide, DOX, DCF-DA, DPPH,hosphate buffered saline (PBS), fetal bovine serum (FBS), MTTssay, Roswell Park Memorial Institute medium (RPMI), andll other chemicals used in this study were obtained fromigma–Aldrich.

.2. Synthesis of NH2-MIL-125

According to the literature, NH2-MIL-125 was prepared using-aminoterephthalic acid and titanium (IV) isopropoxide [44]. In

typical preparation, 20 mmol of 2-aminoterephthalic acid wasissolved in a DMF: methanol mixture (v/v: 40/20) at room temper-ture with constant stirring. After complete dissolution, 10 mmolf titanium (IV) isopropoxide was added to the reaction mixtureith stirring. The resulting mixture was then poured into a 100 mL

eflon-lined autoclave and placed in a convection oven at 150 ◦C for6 h. The final yellow color solid mixture was cooled slowly to roomemperature and filtered. The resulting solid was washed with DMFnd methanol to remove the unreacted molecules, and then driedt 100 ◦C under vacuum to eliminate the solvents from the pores.

.3. Synthesis of NMOF/PEG400

To prepare nano-size NH2-MIL-125 (NMOF), 500 mg of the as-ynthesized MOF was sonicated using rod-type titanium horn with

60% duty cycle at 60 kHz in 25 mL of PBS at room temperature forpproximately 15 min. The sonicated sample was filtered through

0.2 �m PVDF filter. Finally, 20 mL of the filtrate containing NMOFas mixed with three mL of PEG400 and sonicated at room tem-

erature for 20 min. PEG-400 was selected because of its loweroxicity and widespread use in the pharmaceutical industry [45].fter 20 min of sonication, the uncoated PEG-400 was removed byentrifugation and washed twice with 20 mL of PBS. Finally, the

OS on MCF-7 cell line using t-test. Each value represents the mean ± SD (n = 3).

precipitated NMOF/PEG400 was collected and dried at 100 ◦C for4 h.

4.4. Reactive oxygen species of MOF, NMOF, and NMOF/PEG

A DPPH assay was carried out to quantify the amount of ROS inthe nanocarriers (MOF, NMOF, and NMOF/PEG) [46]. In a 96 wellplate, the nanoparticles (100 �L) incubated with 100 �L of DPPH(10 �M DPPH in methanol). After 30 min, the color changed frompurple to yellow. By following the above procedure, the ROS weremeasured at different concentrations (10–40 �g/ml) of NMOF/PEGwith and without irradiation. The release of ROS was measured byUV–vis (UV-vis) absorbance spectroscopy.

4.5. Cytotoxicity of MOF, NMOF, and NMOF/PEG

The cytotoxicity was determined using an MTT assay on afibroblast (COS-7, ATCC

®CRL-1651TM) and breast cancer cell line

(MCF-7, ATCC®

HTB-22TM). The cells were plated onto 9-well platesat a density of 1 × 104 per well and incubated at 37 ◦C under ahumidified atmosphere containing 5% CO2 for 24 h. The cells werethen washed with pH 7.4 PBS. MOF, NMOF, and NMOF/PEG at vari-ous concentrations (10, 50, 100, and 200 �g) was then added withRPMI to the wells. After 24 h incubation, an MTT assay was con-ducted.

4.6. Bio-TEM analysis

The intracellular distribution of NMOF/PEG was confirmed byBio-TEM image analysis. The following procedure was used to pre-pare the samples for Bio-TEM analysis. The cells were incubatedfor 2 h with or without NMOF/PEG and washed with PBS to removethe excess NMOF/PEG from the medium. In addition, the cells wereimmersed in 0.1 M cacodylate buffer for 1 h, and fixed overnight ina solution containing 2.5% glutaraldehyde. The samples were thenprocessed for 1 h using 1% osmium tetroxide. After dehydrationwith alcohol (70%, 80%, 90% and 100% respectively), the sample wasembedded in propylene oxide. The samples were then embedded

in an epoxy resin and dried for 24 h at 60 ◦C. The embedded cellswere then cut into thin sections using a microtome (Reichert no.318423, Austria) and mounted on copper grids. Finally, the sampleswere stained with 1% lead citrate and 2% uranyl acetate.

8 A. Rengaraj et al. / Colloids and Surfaces B: Biointerfaces 160 (2017) 1–10

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ig. 6. Optical microscopy images of MCF-7 cells under different conditions: (a) uMOF/PEG-DOX after irradiation. The left column shows the bright field images. The

ight column presents overlay images of the bright field and fluorescence images.

ange of 330–380 nm and 450–490 nm for the NMOF/PEG and DOX channels, respe

.7. Drug loading and release behaviors

In a typical procedure, 2 mg of NMOF/PEG was dispersed in 4 mLBS containing DOX at various concentrations (0.2-1.0 mg/ml) atoom temperature for 24 h. The DOX-loaded samples (NMOF/PEG-OX) were then collected by centrifugation, washed three timesith PBS (pH = 7.4) and freeze-dried for 24 h. The procedure for

oading the DOX drug onto NMOF/PEG was repeated 4 times toonfirm the loading. To estimate the DOX loading capacity, thenbound DOX in the filtrate solution (supernatant), was deter-ined by UV-vis-spectroscopy and compared with the calibration

urve of a DOX standard solution.In the DOX release process, two identical NMOF/PEG-DOX sam-

les (1 mg) were immersed in a 10 mL of pH 7.4/5.2 PBS with aellulose membrane (Spectra/por, MWCO12-14KDa) and stirred at7 ◦C. The amount of DOX released was examined at different times.he amount of drug release upon irradiation was also investigated.

t different times, 1 mL of PBS was removed from the NMOF/PEG-OX samples solution (1 mg in 10 mL PBS). The same volume of

resh corresponding PBS buffer was then added. The DOX release

ted, (b) NMOF/PEG (c) DOX-loaded NMOF/PEG, and (d) incubated with DCFDA ofal 2 columns present the fluorescence images of NMOF/PEG and DOX channels. Theorescence images were acquired by fluorescence microscopy under an excitation.

rate was obtained by observing the �max at 480 nm. All measure-ments were carried out in triplicate.

4.8. Estimation of cellular killing ability

The MCF-7 (ATCC HTB-22) cells were cultivated in 9-well platesat a density of 1 × 104 cells per well for 24 h at 37 ◦C. When thecells reached confluence, different concentrations of DOX (0.9, 1.8,2.7, 3.6, and 4.5 �g mL−1) and NMOF/PEG-DOX (10, 20, 30, 40,and 50 �g mL−1) diluted with RPMI medium were added to the 9well plates. The cells were then treated with DOX, NMOF/PEG-DOXeither in the presence or absence of radiation for 15 min, and thecells were then incubated at 37 ◦C for 24 h. Subsequently, an MTTassay was conducted at 480 nm using a microplate reader (Molec-ular Devices, Sunnyvale, CA) and analyzed by computer-assistedanalysis (Softmax Pro; Molecular Devices).

4.9. Confocal fluorescence imaging

The MCF-7 cells were incubated with DOX, NMOF/PEG, andNMOF/PEG-DOX for 24 h at 37 ◦C. The cells were then washed

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hree times with PBS and examined by confocal laser scanningicroscopy (Nikon C2) to capture the fluorescence of NMOF/PEG

nd NMOF/PEG-DOX. The nuclei were not stained with the otheruorescent materials because NMOF/PEG is photoluminescent. Toonfirm the ROS produced by NMOF/PEG, hydrolyzed DCF-DA wasdded to the MCF-7 cell line according to the following procedure. A0 �M sample of DCF-DA was hydrolyzed with 4 mL of 0.01N NaOH.he clear solution was diluted with 20 mL of PBS, and the solutionas then incubated with the MCF-7 cells treated with NMOF/PEG

vernight (24hr or 16hr) at 37 ◦C under an atmosphere containing% CO2. The cells were then observed under a 485 nm light sourceo confirm the ROS activity (590 nm) (Fig. 6).

.10. Characterization

X-ray diffraction (XRD, Rigaku) patterns of MOF, NMOF, andMOF/PEG was performed using Cu K� radiation. The interactionetween the drug and carrier was analyzed by Fourier trans-orm infrared (FTIR) spectroscopy. A Flash-1112- Thermo Scientificlemental analyzer was used to determine the elemental composi-ion of the materials. Field emission scanning electron microscopyFESEM, HITACHI S-4300) and transmission electron microscopyJEOL, JEM-2010F–200 kV) were performed to observe the mor-hology of MOF, NMOF, and NMOF/PEG. The thermal stability andEG loading were analyzed by thermogravimetric analysis (TGA,G 209 F3 tarsus) under an argon atmosphere at a heating ratef 5 ◦C/min. The Brunauer, Emmett, Teller (BET) specific surfacereas of the materials were analyzed by N2 adsorption-desorptionsotherms at 77 K using a BELsorp-Max (BEL, JAPAN) and the poreize was calculated using a nonlocal density functional theory (NL-FT) model. The UV–vis spectra were recorded on a Jasco V-650

pectrophotometer. The photoluminescence (PL) at room temper-ture was recorded on a Jasco FP-6500 spectrofluorometer. Theeta potential was measured using a Malvern Zetasizer Nano-Znstrument with a laser wavelength of 633 nm at 25 ◦C under amoluchowski approximation. A Philips CM-10 electron micro-cope was used for Bio-TEM analysis.

cknowledgments

Wha-Seung Ahn acknowledges gratefully the financial supportrom the National Research Foundation of Korea funded by the

inistry of Education (Grant number: NRF-2015R1A4A1042434)or Basic Science Research Program. This work was also sup-orted by the National Research Foundation of Korea grant fundedy the Ministry of Science, ICT and Future Planning of Korea2014R1A5A1009799).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.colsurfb.2017.09.11.

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