Synthesis of multifunctional upconversion NMOFs for targeted antitumor drug delivery and imaging in triple negative breast cancer cells

Accepted Manuscript Synthesis of multifunctional upconversion NMOFs for targeted antitumor drug delivery and imaging in triple negative breast cancer cells Angshuman Ray Chowdhuri, Dipranjan Laha, Soumen Chandra, Parimal Karmakar, Sumanta Kumar Sahu PII: DOI: Reference:

S1385-8947(17)30345-5 http://dx.doi.org/10.1016/j.cej.2017.03.008 CEJ 16604

To appear in:

Chemical Engineering Journal

Received Date: Revised Date: Accepted Date:

9 January 2017 2 March 2017 3 March 2017

Please cite this article as: A.R. Chowdhuri, D. Laha, S. Chandra, P. Karmakar, S.K. Sahu, Synthesis of multifunctional upconversion NMOFs for targeted antitumor drug delivery and imaging in triple negative breast cancer cells, Chemical Engineering Journal (2017), doi: http://dx.doi.org/10.1016/j.cej.2017.03.008

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Synthesis of multifunctional upconversion NMOFs for targeted antitumor drug delivery and imaging in triple negative breast cancer cells Angshuman Ray Chowdhuri1, Dipranjan Laha2, 3, Soumen Chandra1, Parimal Karmakar2, Sumanta Kumar Sahu*1 1 Department of Applied Chemistry, Indian institute of Technology (ISM), Dhanbad 826004, Jharkhand, India 2 Department of Life Science and Biotechnology, Jadavpur University, 188, Raja S C Mallick Road, Kolkata 700032, India 3 Division of Molecular Medicine, Bose Institute, Kolkata, 700054, India. * Corresponding author. E-mail: [email protected], [email protected]; Fax: +91 326-2307772; Tel: +91 3262235936

ABSTRACT Estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2

lacking triple negative breast cancers (TNBC) are the leading cause of death. The successful transport of chemotherapeutics in TNBC with receptor mediated targeting and image guided treatment is a serious challenge for cancer therapy. In this work, folic acid encapsulated nanoscale metal organic framework (NMOFs) is developed on the surface of upconversion nanoparticles (UCNPs) as a targeted and pH responsive anticancer drug carrier. To construct the assembled core-shell drug delivery system (DDS), NaYF4: Yb 3 +, Er 3+ is taken as UCNPs for its outstanding luminescence properties, then a folic acid encapsulated NMOFs based on tetravalent metal Zr (IV) is directly developed on UCNPs [labeled as UCNP@UIO-66(NH2)/FA]. Excitingly, UCNP@UIO-66(NH2)/FA are nontoxic towards TNBC cells (MDA-MB-468) and normal cell lines (NIH3T3). The anticancer drug doxorubicin (DOX) is encapsulated into UCNP@UIO-66(NH2)/FA with high drug loading efficiency (1.42 g DOX per g NMOFs) and 1

shows pH responsive drug release. The DOX loaded UCNP@UIO-66(NH2)/FA successfully enters in to the MDA-MB-468 cells through a folate receptor mediated endocytosis and exhibits a higher cytotoxicity than normal cells. Flow cytometry and nuclear apoptosis studies suggest the present DDS can be potential applicable in breast cancer therapy to reduce the side effects. KEYWORDS: Nanoscale metal organic frameworks, upconversion, folic acid, triple negative breast cancers, pH responsive release.

1. INTRODUCTION In recent years, researchers have paid increasing attention to develop nanomedicine, which is a platform of choice for various advantageous properties [1]. An extensive range of nanomedicines have been developed for diverse medical applications, especially for anti-cancers because the cancer is still one of major threats to human health. Among the different type of cancers, breast cancer is the most common type of cancer. The molecular markers estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) distinguish breast cancer into several subgroups. Tumors that lack of the above three markers are commonly termed as triple-negative breast cancer (TNBC). Unfortunately, no effective standard therapy and appropriate drugs have been approved for TNBC treatment. Therefore, the development of novel effective therapeutic approaches against TNBC is urgently anticipated. Till now, numerous smart drug delivery systems were developed in different type of cancers including TNBC, which play a significant role for the development of anti-cancer nanomedicines [2-5]. In particular, these smart drug delivery systems possess diverse properties like highly biocompatible, efficient cellular uptake, therapeutic molecule release at targeted site with stimuli responsive behaviors. However, some tedious multi steps were involved to obtain

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the functionalized smart DDS with small amount drug loading content. Therefore, a strategy need to develop the crucial aspects for construction of smart DDS like (i) facile synthesis of biocompatible nanocarrier conjugated with targeting ligand (ii) the nanocarrier that can retain high loading extents of the therapeutic molecules with stimuli responsive release (iii) a tracking process that can allow for real-time imaging over extended periods of time. Here we have addressed these issues by developing a nanocarrier triggered by NMOFs conjugated with upconversion nanoparticle. Up to now, several anticancer nanomedicines based on organic (liposome, polymer micelles, and protein nanoparticles) [6-8] and inorganic (Fe3O4, mesoporous silica, silver, gold) [9-12] nanoscale DDS have been well reported. Among them, some possess more unique advantages due to their special structures, stable chemico-physical properties, and low toxicities. Recently, NMOFs belongs to inorganic-organic hybrid DDS have attracted considerable attention since the first discovery in the late 1990s due to their striking features like large surface areas, ultrahigh porosity, and tunable functionality [13-15]. The formation of NMOFs with wellordered crystalline solids, tunable pore sizes and geometries is attributed to the bridge of metal clusters and organic ligands through either covalent or coordinate bonds. Thereafter, these NMOFs could be functionalized and utilized for diverse bio applications like (i) photodynamic therapy (e.g., DBC-UIO [16], PCN-224 [17], Hf-TCPP [18], and TBC-Hf [19]); (ii) photothermal therapy (e.g., UIO-66@PAN [20], Fe3O4/ZIF-8-Au [21]); (iii) bio-imaging [22] (e.g., Fe3O4/Au/ZIF-8 [23]);

(iv) MRI contrast agent (e.g., Fe3O4@IRMOF-3/FA [24],

Fe3O4@UIO-66 [25], Mn3(BTC)2(H2O)6 [26]); (v) angiogenesis (e.g., Fe-MIL-101 [27]); (vi) antibacterial agents (e.g., [Co4(H2O)2(TDM)(H2O)8] [28], BioMIL-5 [29], ZIF-67 [30]); and (vii) gene delivery (e.g., hexagonal-plate like UIO [31]). Among the NMOFs reported for drug

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delivery applications, UIO-66 is an excellent one because of its high biocompatibility and biosafety. Recently, Zhao et al. have synthesized a core-shell like magnetic UIO-66 (Fe3O4@UIO-66) and successfully demonstrated the compound as a magnetic resonance imaging contrast agent as well as doxorubicin delivery carrier [25]. Tan et al. studied the biocompatibility and drug release from UIO-66(NH2) [32]. Recently, Tavra et al. and Tai et al. reported nanoscale UIO-66(NH2) for the calcein and 5-fluorouracil delivery [33, 34]. However, there are no reports about the functionalization of UIO-66 for targeted drug delivery, which would have a great significance in cancer therapy. In this work, UIO-66(NH2) is functionalized with targeting ligand in a single step, obtaining a nanoscale platform useful in imaging, targeted drug delivery, as well as pH responsive drug release. In recent years, integration of inorganic nanoparticles with NMOFs to form composite has emerged as one of the significant research areas, which will further enhance the utility of the NMOFs based materials. Numerous inorganic nanoparticles have been effectively incorporated within NMOFs matrix, such as upconversion nanoparticles, magnetic nanoparticles, palladium nanoparticles, silver nanoparticles, zinc oxide nanoparticles and gold nanoparticles for diverse applications [24, 35-40]. Among them, core-shell type composite materials of upconversion NMOFs have drawn considerable attention due to their excellent properties. Under near infrared (NIR) excitation, lanthanide doped UCNPs emit NIR to visible light with narrow emission peak possess great potential in labelling biological entities such as high photostability, very low autofluorescence background, resistance to photobleaching, as well as deep light penetration depth in biological tissues, which are mainly suitable for biomedical imaging [41, 42]. A number of excellent articles have reported the progress of UCNPs based nanomedicines [43]. Surface modification on UCNPs would play a vital role in their cellular uptake through the plasma

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membrane. Consequently, the investigations on the effects of surface modifications are essential not only for controlled drug delivery with high therapeutic capacity but also for the engineering of UCNPs for targeted cell labelling. However surface modification on the UCNPs may be damage the florescence property. Therefore, the strategy of coating NMOFs shells on UCNPs cores is able to combines the merits of both components for the applications in biomedicine. In this work, we have developed a facile and efficient approach to synthesize a porous NIR to visible type upconversion nanocomposite, composed of a NaYF4: Yb+3, Er+3 core and a microporous UIO-66(NH2) shell, as a multifunctional smart drug delivery system as shown in scheme 1. This microporous shells could not only retain the intensity of upconversion luminescence but also enable these nanocomposites as drug delivery carrier, due to its high surface area and accessible micropores for huge amount of drug loading. To realize targeted drug delivery, we have conjugated UIO-66(NH2) with folic acid on the surface of UCNPs in a single step, which enabled the target drug carrier to cancer cells. Simultaneously, the nanocomposites show the pH responsive drug release. The synthesis process, structure, and morphology of the porous upconversion nanocomposites were investigated in details. Therefore, the as-developed upconversion NMOFs offer a new possibility in exploring as not only for targeted alternative drug delivery vehicles but also simultaneously multimodal imaging.

2. MATERIALS AND METHODS

2.1. Chemicals and media used The rare earth chlorides (99.9%, trace metals basis) [e.g., yttrium chloride (YCl3), ytterbium chloride (YbCl3), and erbium (III) chloride (ErCl3)], zirconium (IV) chloride, and N, N- dimethylformamide (DMF) were obtained from Sigma Aldrich. 1-octadecene (> 99%), 5

ammonium fluoride (NH4F, > 99.9%), oleic acid (> 99%), and 2-aminoterephthalic acid (NH2BDC, > 90 %) were purchased from TCI fine chemicals. Zinc nitrate hexahydrate (Zn (NO3)2·6H2O, 99%) and sodium hydroxide (NaOH) were purchased from Merck India. Pure methanol and ethanol were acquired from Merck Germany. DMF, ethanol, and methanol were dried by using reported procedures. Dialysis membrane having MWCO 12 K Da, 4/-6diamidino-2-phenylindole

(DAPI),

RNase,

3-(4,

5-dimethylthiazol-2-yl)-2,

5-

diphenyltetrazolium bromide (MTT), anticancer drug doxorubicin (DOX), and folic acid (FA) were acquired from Sigma Aldrich, USA. Minimum essential medium (MEM) were brought from Himedia, India and Hyclone, USA respectively. Millipore water was used in the entire experiment. Essentially, analytical or above grade chemicals and reagents were used in this study. DMEM, fetal bovine serum (FBS), and penicillin/streptomycin (100 units.mL-1) were bought from Sigma Aldrich (USA).

2.2. Synthesis of NaYF4:Yb3+, Er3+ upconversion nanoparticles (UCNPs) The NaYF4: Yb3+, Er3+ upconversion nanoparticles (UCNPs) were prepared by coprecipitation method following the previous protocols with slight modification [44, 45]. Briefly, 0.3 g of YCl3, 0.1 g of YbCl3, and 0.01 g of ErCl3 were added into mixture of 1-octadecene (15 mL) and oleic acid (20 mL) under stirring condition followed by heating at 120 ºC to completely solubilize the rare earth precursors. After that, 0.2 g of NaOH and 0.296 g of NH4F were dissolved in 20 mL of pure methanol and added into the above solution. The mixture solution was vigorously stirred for 1 h at 60 ºC. Then, the temperature was enhanced to 100 ºC to remove methanol. Finally the mixture solution was heated to 320 ºC for 1 h under argon atmosphere for the complete development of upconversion nanoparticles. When the final product was obtained,

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the mixture was cooled naturally and the product was separated by centrifugation. The obtained products were washed in ethanol and dried in a vacuum oven at 60 ºC for 12 h.

2.3. Synthesis of folic acid conjugated upconversion nanoscale metal organic frameworks [UCNP@ UIO-66(NH2)/FA] Folic acid conjugated upconversion nanoscale metal organic framework was developed in a single step by the following procedure. Briefly, 50 mg of previously synthesized NaYF4:Yb3+, Er3+ nanoparticles were dissolved in 20 mL of dry DMF and then vigorously sonicated for 30 min by a probe type ultrasonicator. Then the metal organic framework precursors were added. 50 mg of ZrCl4 and 38.75 mg of NH2-BDC were dissolved in 10 mL of dry DMF and mixed with the UCNPs solution. After that 15 mg of folic acid was dissolved in 5 mL of dry DMF separately and added to the previous solution. Then, the mixture solution was transferred to a Teflon lined stainless steel autoclave and placed in a hot air oven at 100 ºC for 12 h. After complete formation of UCNP@UIO-66(NH2)/FA, the autoclave was naturally cooled down to room temperature and products were separated by centrifugation followed by dialysis as well as washing with DMF and ethanol-water mixture then dried in vacuum oven at 50 ºC for 12 h. Finally the pure product was stored at cooled place (4 ºC) for further use.

2.4. Synthesis of UCNP@UIO-66(NH2) and UIO-66(NH2) UCNP@UIO-66(NH2) and only UIO-66(NH2) were synthesized for comparison study. For synthesis of UCNP@UIO-66(NH2), previous protocol was followed as UCNP@UIO66(NH2)/FA without addition of folic acid molecules. For synthesis of only UIO-66(NH2), reported procedure was followed with a slight modification [25]. Briefly, 75 mg of ZrCl4 and 58 mg of NH2-BDC was dissolved in a 20 mL dry DMF. After proper mixing, the mixture was transferred in a Teflon lined stainless steel autoclave and placed in a hot air oven at 100 ºC and 7

the reaction was continued for 12 h. After the complete formation of product, autoclave was cooled naturally and separated by centrifugation, washed several times by first DMF then ethanol-water mixture. Finally the products was dried at 50 ºC in a vacuum oven for 12 h.

2.5. Drug loading into UCNP@UIO-66(NH2) /FA An anticancer drug doxorubicin was loaded into the folic acid conjugated upconversion metal organic frameworks [UCNP@UIO-66(NH2) /FA] to determine the drug loading amount and anticancer efficacy by the following procedures [46, 47]. Initially, a DOX solutions was prepared by adding different concentration of DOX in 5 mL of water. Then 5 mg of UCNP@UIO-66(NH2)/FA were added to the previously prepared DOX solution. After that, the solution was placed on an orbital shaker for 12 h in a dark condition. The DOX loaded UCNP@ UIO-66(NH2)/FA was separated and unbound DOX was removed by washing three times with water. After separation of DOX loaded UCNP@UIO-66(NH2)/FA, the supernatant solution was taken for the determination of drug encapsulation efficiency (DEE) and drug loading content (DLC) by using UV-Vis spectroscopy at 481 nm. For the determination of the DOX encapsulation efficiency in to the present drug delivery system, a calibration plot was used by taking a different concentration of free DOX. Finally, the DOX loaded UCNP@UIO66(NH2)/FA was kept in dark condition at 4 ºC for further experiments. The DLC and DEE was calculated by the following two equations.

Weight of drug in nanoparticles

×

Drug loading contents (%) =

100

Weight of nanoparticles taken

Weight of drug in nanoparticles Drug entrapment efficiency (%) = Weight of total drug injected 8

×

100

2.6. In vitro drug release In vitro cumulative drug release profile was checked to conform the amount of impregnated DOX discharge with respect to predetermined time interval from the core of the upconversion metal organic frameworks [46, 47]. Actually, the drug release study was carried out in pH ∼7.4 and pH ∼5.5 at 37 °C to establish the pH responsive behavior of present drug delivery system. Firstly, 3 mg of DOX loaded UCNP@UIO-66(NH2)/FA was dispersed in pH ∼7.4 (physiological pH condition) followed by incubation in dark condition. After regular time interval, the 3.0 mL of supernatant was taken out and same volume of fresh PBS (pH ∼7.4) was added to maintain the equal volume. The exactly same trials were carried out in pH ∼5.5 (lysosomal pH condition). The amount of DOX release at regular time interval was quantified by checking the absorbance at 481 nm from standard curve of free DOX.

2.7. Cell lines & culture Human triple negative breast cancer cells (MDA-MB-468) and murine fibroblast (NIH3T3) cells were cultured in DMEM media supplemented with 10 % FBS and 100 U.mL− 1 penicillin-streptomycin in 5 % CO2 at 37 °C. At 85 % confluence, cells were harvested and subcultured according to experimental requirements. Cells were seeded for 24 h prior to the treatment with UCNP@UIO-66(NH2), UCNP@UIO-66(NH2)/FA, DOX loaded UCNP@UIO66(NH2), and DOX loaded UCNP@UIO-66(NH2)/FA. All the treatments were performed at 37 °C at a density allowing exponential cell growth.

2.8. Cell viability evaluation in normal cells and breast cancer cells The viability of NMOFs treated cells ware measured by MTT assay as described previously [46-48]. In brief, cells were seeded in 24 well plates at 1 × 10 4 for 24 h. Then, we compared the cytotoxicity of UCNP@UIO-66(NH2), UCNP@UIO-66(NH2)/FA, DOX loaded 9

UCNP@UIO-66(NH2), and DOX loaded UCNP@UIO-66(NH2)/FA against MDA-MB-468 and NIH3T3 cells under equivalent concentrations of DOX for 24 h. After incubation, cells were washed twice with PBS and incubated with MTT solution (450 μg.mL-1) for 3-4 h at 37 °C. The resulting formazan crystals were dissolved in a MTT solubilization buffer and the absorbance were measured at 570 nm by using a microplate reader (Biotek, USA). Each point was assessed in triplicate.

2.9.

Cellular morphology study

Human triple negative breast cancer cells (MDA-MB-468) and murine fibroblast (NIH3T3) cells were grown on a 35 mm plate for 24 h. After that, cells were treated with 5 μg.mL-1 of UCNP@UIO-66(NH2)/FA up to five days at 37 °C. Cells were washed with 1× PBS solution for three times. The morphology of these cells was observed after each one days under microscope (Leica, Wetzlar, Germany).

2.10. Intracellular uptake study MDA-MB-468 and NIH3T3 cells were grown on cover slip at 37 ºC for 24 h. After that, cells were treated with UCNP@UIO-66(NH2), UCNP@UIO-66(NH2)/FA at 5 μg.mL-1 for 1 h, 2 h, 4 h, and 6 h. Then, the cells were washed with 1× PBS and mount the coverslip. Finally, the fluorescent image was captured by fluorescence microscope (Leica).

2.11. DAPI staining for nuclear morphology study We further moved to verify apoptosis of DOX loaded UCNP@UIO-66(NH2)/FA treated MDA-MB-468 cells. After exposure of different concentration of DOX loaded UCNP@UIO66(NH2)/FA, cells were washed three times with 1 × PBS and stained with 4′, 6-diamidino-2phenylindole (DAPI) in Vectashield (0.2 g.mL−1, Vector Laboratories Inc.). The percentage of 10

cells with rupture and decondensed nuclei were counted under a fluorescence microscope (Leica) after 24 h.

2.12. Cell cycle analysis For cell cycle analysis, 1 × 106 cells/mL were treated with DOX loaded UCNP@UIO66(NH2) and DOX loaded UCNP@UIO-66(NH2)/FA at LD50 dose. After 24 h incubation, the cells were washed with 1 × PBS and fixed with chilled 70% ethanol and kept for 2 h at 4 °C. Prior to stain with 50 μg.mL-1 propidium iodide (PI, Sigma), the cells were incubated for 1 h with 100 μg.mL-1 of DNAse free RNAse A (SRL, India) at 37 °C [49]. The cell cycle was analyzed with a Becton Dickinson (FACS Calibur) flow cytometer, equipped with an air-cooled 20 mW argon laser. 25,000 events were counted at each data point.

2.13. Characterization Fourier transform infrared spectroscopy (FTIR) was performed on Agilent Carry 660 instrument at 25 °C for the determination of surface functional groups and the appropriate conjugations of the NMOFs through ATR method between 400 cm-1 to 4000 cm-1. Field emission scanning electron microscopy (FESEM), elemental mapping, and EDAX analysis were carried out on Supra 55 (Zeiss) instruments. The exact particle size and the core-shell type microstructures of the synthesized NMOFs were analyzed by High resolution transmission electron microscopy (HRTEM) on JEOL 3010, Japan at 300 keV. UV-Vis spectra were recorded to confirm the folic acid attachment and to determine the amount of drug loading inside the NMOFs as well as the drug release pattern on Shimadzu 1800. Hydrodynamic diameters and zeta potentials of the NMOFs were measured through dynamic light scattering (DLS) techniques on nanoseries ZS, Malvern Zetasizer instruments. The surface area and pore size distribution of the synthesized NMOFs and drug loaded NMOFs were determined by microporous BET surface 11

area analyzer (Quantachrome Instruments). Expert Pro (Phillips) X-ray diffractometer using Cu Kα was used to determine the crystallographic state and phase of the NMOFs. Perkin Elmer LS 55 fluorescence spectrometer associated with 980 nm external laser was used to determine the upconversion photoluminescence intensity of the UCNP@UIO-66(NH2)/FA. Folic acid encapsulation as well as DOX loading into the UCNP@UIO-66(NH2)/FA were further confirmed

on

TGA,

2850

thermogravimetric

analyzer

(TA

instruments)

through

thermogravimetric analysis (TGA) under N2 atmosphere.

2.14. Statistical analysis Statistical investigations were carried out on Graphpad Prism 6 and OriginPro8 in addition with student’s t tests (limit of significance, p < 0.05). The variance analysis was performed using one-way ANOVA by Graphpad Prism 6 software.

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Scheme 1. Representation of the synthetic procedure for the folic acid encapsulated upconversion NMOFs [UCNP@UIO-66(NH2)/FA] as a targeted anticancer drug carrier and pH responsive drug delivery in human triple negative breast cancer cells (MDA-MB-468).

3. RESULTS AND DISCUSSION

3.1. FESEM, EDAX, and elemental mapping analysis Surface morphology and the perfect shape of the UCNPs, UCNP@UIO-66(NH2) and the folic acid conjugated UCNP@UIO-66(NH2) were examined by FESEM analysis, as shown in figure 1. Only bare as synthesized UCNPs are uniform in shape, monodispersed, and particle size of 40±5 nm, as displayed in figure 1 (a) and (b). Figure 1 (c) reveals that after treatment with the NMOFs precursors at 100 °C, a UIO-66(NH2) layer was formed upon the UCNPs, results increase in particle size of 170±10 nm with a uniform spherical shape. A corresponding high resolution picture is shown in figure 1 (d). Here, we had tried to developed a targeted drug delivery system by conjugating folic acid in to the UCNP@UIO-66(NH2) in a single step during the growth of UIO-66(NH2) upon the UCNPs. When folic acid encapsulated UIO-66(NH2) is conjugated on the surface of UCNPs in a single step, there is no change in morphology and size was observed. FESEM image of doxorubicin loaded UCNP@UIO-66(NH2)/FA is shown in figure S7 (a) [supporting information (SI)]. The surface morphology of UCNP@UIO66(NH2)/FA in different pH medium after 24 h stirring at 37 °C is shown in figure S12 (SI). FESEM images of DOX loaded UCNP@UIO-66(NH2)/FA after drug release in pH 7.4 and pH 5.5 at 37 °C up to 12 h and 24 h are exposed in figure S15 & S16 respectively. After surface morphology study, the EDAX and elemental mapping analysis were carried out to confirm the presence of exact elements in the corresponding synthesized NMOFs as presented in figure S1 & S2 respectively (SI). It is observed that sodium (Na), yttrium (Y), 13

fluorine (F), ytterbium (Yb), and erbium (Er) are found in the UCNPs. On the other hand in UIO66(NH2), carbon (C), nitrogen (N), and Zirconium (Zr) are present. In case of UCNP@UIO66(NH2)/FA, Na, Y, F, Yb, and Er are found for the UCNPs part as well as C, N, O, Zr are due to the presence of UIO-66(NH2) and folic acid. Throughout the scanning range of binding energies, no extra elements belonging to impurities are identified in EDAX spectrum.

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Figure 1. FESEM image of (a) UCNPs, (c) UCNP@UIO-66(NH2), and (e) UCNP@UIO66(NH2)/FA, as well as (b, d, and f) represents the corresponding high resolution pictures of a, c, and e respectively.

3.2. TEM and DLS study For investigation of complete microstructures, hydrodynamic particle size and the change of particle size at different stage of NMOFs, HRTEM and DLS study were carried out, as shown in figure 2. From HRTEM micrograph of bare UCNPs [figure 2 (a)], it is confirms that the particles are well dispersed with uniform in size of about 40±5 nm. When folic acid conjugated UIO-66(NH2) [UIO-66(NH2)/FA] is developed upon the UCNPs by a hydrothermal method, the particle size is improved about 180±20 nm, as shown in figure 2 (b). From HRTEM image, it confirms that the thickness of the NMOFs shell is greater than 50 nm. This huge assembly of NMOFs shell upon the UCNPs would enable to enhance the drug loading amount as well as regulate the drug release pattern [25, 34, 50]. In this case, the possible mechanism of formation of core-shell type nanostructure is initially Zr4+ can adsorb on the surface of UCNPs. Then the desired amount of NH2-BDC is interact with the adsorbed Zr4+ on UCNPs and influence the growth of UIO-66(NH2), results nanoscale upconversion core-shell metal organic frameworks. After complete formation of UCNP@UIO-66(NH2)/FA, an anticancer drug DOX was loaded into the UCNP@UIO-66(NH2)/FA. The HRTEM image of DOX loaded UCNP@UIO66(NH2)/FA is shown in figure S7 (b) [SI]. Figure 2 (c) & (d) represent the corresponding selected area electron detraction (SAED) patterns of UCNPs and UCNP@ UIO-66(NH2)/FA respectively. SAED pattern of UCNPs shows a characteristics crystalline planes but in case of UCNP@ UIO-66(NH2)/FA, some amorphous nature with crystallinity is observed due to the formation of UIO-66(NH2)/FA shell upon the UCNPs.

15

DLS experiments were carried out in water medium (pH-7.0) to determine the hydrodynamic particle size of the UCNPs and UCNP@ UIO-66(NH2)/FA as a supplementary of HRTEM results. The average hydrodynamic diameter of UCNPs and UCNP@UIO-66(NH2)/FA are found to be 105±5 nm and 250±10 nm, as shown in figure 2 (e) & (f) respectively. When DOX is loaded inside the UCNP@UIO-66(NH2)/FA, the particle size is slightly increases, as shown in figure S7 (c) [SI]. On the other hand, DLS spectra of DOX loaded UCNP@UIO66(NH2)/FA in different pH medium and the stability study after several weak (change in particle size) are illustrated in figure S13 & S14 separately [SI]. Corresponding higher particle size is observed in DLS than HRTEM because HRTEM study was carried out in dry state while DLS analysis performed in water medium (pH-7.0) [37].

Figure 2. HRTEM image of (a) UCNPs, [inset: high resolution image] and (b) UCNP@UIO66(NH2)/FA, as well as corresponding SAED patterns and DLS spectra of (c and e) UCNPs and (d and f) UCNP@UIO-66(NH2)/FA respectively. 16

3.3. FTIR analysis The characteristics functional groups on the surface of synthesized UCNPs, UIO66(NH2), UCNP@UIO-66(NH2), and their conjugations with the folic acid were identified by FTIR study, as shown in figure 3. Here, NaYF4: Yb3+, Er3+ nanoparticles were synthesized in oleic acid medium, so few oleic acid may be capped upon the UCNPs which is confirmed by the FTIR peak positions. From figure 3 (a) it is clearly observed that, a characteristic broad peak at around 3420 cm-1 can be assigned for the existence of stretching vibrations of –OH groups on the surface of the UCNPs [51]. Peaks at around 1650 cm-1 and 1430 cm-1 were appeared for the presence of asymmetric and symmetric type stretching vibrations of the –COOH group respectively [51, 52]. The symmetric and asymmetric stretching vibrations of methylene group of the long alkyl chain presence in the oleic acid generates two additional peaks at 2930 cm-1 and 2860 cm-1. In case of UCNP@UIO-66(NH2), peaks at 1570 cm-1, 1430 cm-1, 1385 cm-1 are comes from the UIO-66(NH2) part, which is exactly same position as it is in UIO-66(NH2), as shown in figure 3 (a) [25]. Again the peaks at 3440 cm-1 and 3320 cm-1 can be assigned for the asymmetric and symmetric stretching absorptions of the primary amine groups present in the NH2-H2BDC ligands in the UIO-66(NH2). This result confirms the successful conjugation of the UIO-66(NH2) with the UCNPs. After successful synthesis of UCNP@UIO-66(NH2), we had tried to develop a folic acid conjugated UCNP@UIO-66(NH2) in a single step. The characteristic FTIR spectrum of free folic acid is exposed in figure 3 (b). In case of UCNP@UIO-66(NH2)/FA, the maximum prominent peaks of folic acid (3520, 3300, 3090, 2929, 1690, 1465, and 1230 cm1

) are present, as shown in figure 3 (b). Most interesting thing is that after conjugation of folic

acid in a single step, no characteristic peak position of UIO-66(NH2) and FA is shifted, confirms that the complete electrostatic interaction (physical) of folic acid and the UCNP@UIO-66(NH2). There is no possibility of any covalent bonding or coordination of folic acid with Zr4+. 17

Figure 3. FTIR spectra of (a) UCNPs, UCNP@UIO-66(NH2), and UIO-66(NH2), as well as (b) folic acid (FA) and UCNP@UIO-66(NH2)/FA.

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3.4. Upconversion photoluminescence study Upconversion photoluminescence spectra of UCNPs (NaYF4:Yb3+, Er3+) and UCNP@ UIO-66(NH2)/FA were recorded on 980 nm NIR laser excitation for evaluation of NIR to Vis upconversion emission behavior of synthesized NMOFs, as shown in figure 4. Green emission band at around 520-550 nm corresponding to 2H11/2 → 4I15/2 and 4S3/2 → 4I15/2 energy transitions as well as red emission band at about 650-660 nm is observed due to 4F9/2 → 4I15/2 energy transition of Er3+ ions of NaYF4:Yb3+, Er3+ (UCNPs) [53]. In this case, initially NIR light (980 nm) is absorbed by Yb3+ ions then the energy is transferred to the nearby Er3+ ion. After getting the energy, Er3+ ions are excited to the 4I11/2 level. Again second 980 nm photon by the excited Yb3+ can populate the 4F7/2 level of Er3+, afterward Er3+ will relax nonradiatively to the 2H11/2 and 4

S3/2 levels. Finally resulting in the green (520 nm, 2H11/2 → 4I15/2; 540 nm, 4S3/2 → 4I15/2) and red

(654 nm, 4F9/2 → 4I15/2) emission, respectively [54]. The upconversion emission intensity of UCNP@UIO-66(NH2)/FA is little bit decreases due to the thick shell of UIO-66(NH2)/FA upon UCNPs. The digital camera photo images of UCNP@UIO-66(NH2)/FA in presence of 980 nm laser are shown in figure S6 [SI].

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Figure 4. Emission spectra of UCNPs and UCNP@UIO-66(NH2)/FA upon 980 nm laser excitation.

3.5. Surface area measurements Microporous Brunauer-Emmett-Teller (BET) analysis was performed to determine the surface area of synthesized upconversion nanoscale metal organic frameworks, as shown in figure 5. The surface area of UCNP@UIO-66(NH2)/FA is found to be 932 m2.g-1 with 1.10 nm pore width. This high surface area can be responsible for the high amount of drug loading [25, 47]. After DOX loading, we have again carried out the surface area measurement to prove the drug loading inside the micropores of UCNP@UIO-66(NH2)/FA. Surface area of DOX loaded UCNP@UIO-66(NH2)/FA is decreases to 186 m2.g-1. The pores of UCNP@UIO-66(NH2)/FA 20

are also blocked by the DOX molecules as expected and average pore size is reduces to about 0.95 nm. Decrement of surface area and porosity reveals huge amount of DOX encapsulation inside the UCNP@UIO-66(NH2)/FA.

Figure 5. (a) Nitrogen adsorption-desorption isotherms as well as (b) corresponding pore size distributions of UCNP@UIO-66(NH2)/FA and DOX loaded UCNP@UIO-66(NH2)/FA.

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3.6. XRD study The purity of phase as well as crystalline structures of synthesized UCNP@UIO66(NH2)/FA were confirmed and compared with UCNPs and UIO-66(NH2) by powder XRD (PXRD) analysis, as shown in figure S3 [SI]. The characteristics PXRD patterns of synthesized UCNPs (NaYF4: Yb3+, Er3+) exhibits good crystallinity. The PXRD of synthesized UCNPs were well agreed with the calculated values of standard hexagonal β-NaYF4 phase (JCPDS 16-0334), indicates the synthesized UCNPs having crystalline hexagonal phase [55]. On the other hand, PXRD patterns of UIO-66(NH2), shows all characteristic peaks (mainly 2θ = 7.5° & 8.5º), which are well matched with the previous reports [25, 34, 56]. It is interesting to note that, the characteristic peaks of UCNPs in addition with the typical diffraction peaks of UIO-66(NH2) can be identified in case of UCNP@ UIO-66(NH2)/FA. Again, after one step fabrication of folic acid conjugated UIO-66(NH2) upon UCNPs, no evident change of crystalline phase is observed, confirms a physical interaction between folic acid and UIO-66(NH2). PXRD patterns examination of synthesized NMOFs designates a fruitful development of upconversion metal organic frameworks.

3.7. Thermogravimetric analysis Thremogravimetric analysis (TGA) was performed to confirm the attachment of folic acid with in the upconversion NMOFs as well as encapsulation of DOX inside the UCNP@UIO66(NH2)/FA. The TGA thermograms of UCNP@UIO-66(NH2), UCNP@UIO-66(NH2)/FA, and DOX loaded UCNP@UIO-66(NH2)/FA are shown in figure S4 [SI]. The construction of NMOFs upon UCNPs, folic acid attachments, and the DOX encapsulation into the UCNP@UIO66(NH2)/FA could be explained on the basis of step wise relative weight loss due to the 22

elimination of different functionalities with respect to temperatures. In all cases, the weight loss up to 100 ºC can be due to the loss of the adsorbed moistures from the pores of NMOFs. The metal organic frameworks and the organic molecules folic acid absolutely decomposes from the UCNPs till temperature 600 ºC. After 700 °C, the percentage of weight loss of UCNP@UIO66(NH2) is 63.5 % and 57.5 % in case of UCNP@UIO-66(NH2)/FA, confirms more than 3 % folic acid content in UCNP@UIO-66(NH2)/FA. This folic acid amount in UCNP@UIO66(NH2)/FA has the vital influence on the performance of targeting to cancer cells. Again for DOX loaded UCNP@UIO-66(NH2)/FA, 56 % weight loss at same temperature region approves the successful encapsulation of DOX molecules with in the UCNP@ UIO-66(NH2)/FA.

3.8.

Cytotoxicity study by MTT assay The cytotoxicity of UCNP@UIO-66(NH2), UCNP@UIO-66(NH2)/FA, DOX loaded

UCNP@UIO-66(NH2), and DOX loaded UCNP@UIO-66(NH2)/FA were tasted in vitro through MTT assay, as shown in figure 6. The MDA-MB-468 and NIH3T3 cells were treated with different concentrations (0-100 µg.mL-1) of NMOFs. Firstly, up to 100 µg.mL-1 of UCNP@UIO66(NH2) and UCNP@UIO-66(NH2)/FA exhibit greater than 80 % cell viability towards both normal and cancer cells. Results suggest that the synthesized NMOFs can be appropriate as drug delivery vehicles. But, DOX loaded UCNP@UIO-66(NH2)/FA shows more toxicity in case of MDA-MB-468 cells than NIH3T3 cells while approximately similar toxicity observed both in case of DOX loaded UCNP@UIO-66(NH2) treated MDA-MB-468 cells and NIH3T3 cells. This observation clearly demonstrates that the cancer cell targeting of UCNP@UIO-66(NH2)/FA is more effective towards human TNBC. Approximately 30 % NIH3T3 cell death caused by 25 µg.mL-1 of DOX loaded UCNP@UIO-66(NH2)/FA. On the other hand, the same amount of DOX loaded UCNP@UIO-66(NH2)/FA caused greater than 60 % inhibition of the MDA-MB-

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468 cells. With increase in dose of DOX loaded UCNP@UIO-66(NH2)/FA, the percentage of cancer cells death increases than normal cells due to the presence of folate receptors in TNBC. Half maximum inhibition concentration (IC50) of DOX-loaded UCNP@UIO-66(NH2)/FA in case of MDA-MB-468 is found to be 8 μg.mL-1. Only DOX shows nearly similar type of toxicity in MDA-MB-468 and NIH3T3 cells (figure not shown). So, DOX loaded UCNP@UIO66(NH2)/FA may be used as a candidate towards targeted cancer therapy in TNBC.

Figure 6. Cell viability of folic acid conjugated NMOFs (UCNP@UIO-66(NH2)/FA and DOX loaded UCNP@UIO-66(NH2)/FA) in (a) NIH3T3 and (b) MDA-MB-468 cells and without folic acid conjugated NMOFs [UCNP@UIO-66(NH2) and DOX loaded UCNP@UIO-66(NH2)] in (c) NIH3T3 and (d) MDA-MB-468 cells. 24

3.9.

Cellular morphology study MDA-MB-468 cells were treated with UCNP@UIO-66(NH2)/FA for 5 days to visualized

any change of cellular morphology by using fluorescence microscope, as shown in figure 7. Simultaneously, UCNP@UIO-66(NH2)/FA treated NIH3T3 cells are shown in S17. Evidently no change in morphology is observed, suggests nontoxic behaviour of synthesized upconversion NMOFs towards both normal and cancer cells.

Figure 7. Cellular morphology study of MDA-MB-468 cells incubated with 5 μg.mL-1 of UCNP@UIO-66(NH2)/FA up to five days at 37 °C [Each scale bar represents 5 µm].

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3.10. Cellular uptake study Time dependent intracellular uptake of synthesized NMOFs in folate receptor positive human triple negative breast cancer cells (MDA-MB-468) were studied to demonstrate the targeting ability of UCNP@UIO-66(NH2)/FA, as shown in figure 8. From the microscopic image, it was observed that with increase the time, folic acid modified UCNP@UIO-66(NH2) remarkable increase the fluorescence intensity inside the MDA-MB-468 cells in comparison to the UCNP@UIO-66(NH2). Result suggests the greater internalization of UCNP@UIO66(NH2)/FA through a endocytosis into folate receptor overexposed TNBC cells. Intracellular uptake of UCNP@UIO-66(NH2) and UCNP@UIO-66(NH2)/FA in NIH3T3 cells is shown in figure S18 [SI]. It can say that UCNP@UIO-66(NH2)/FA having a potential to transport doxorubicin selectively in folate receptor positive breast cancer cells.

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Figure 8. Fluorescence microscopic image of MDA-MB-468 cells incubated with UCNP@UIO66(NH2)/FA after 1 h, 2 h, 4 h, and 6 h at 37 °C [each scale bar represents 10 µm].

27

3.11. Analysis of nuclear morphology by DAPI staining One step further, we have stained MDA-MB-468 cells by DAPI to visualize the apoptotic morphology after treatment with different concentrations (0-15 µg.mL-1) of DOX loaded UCNP@UIO-66(NH2)/FA by florescence microscope, as shown in figure 9. Here, undamaged morphology is observed in case of control MDA-MB-468 cells but apoptotic morphology with broken nuclei are noticed in treated cells, confirms cell expiry. The percentage of disjoined cells was gradually increased with increase the concentration of drug loaded NMOFs. So, DOX loaded UCNP@UIO-66(NH2)/FA capably deliver DOX molecules inside the MDA-MB-468 cells, inhibits the cancer cell growth and accelerated apoptosis.

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Figure 9. Apoptosis study of MDA-MB-468 cells treated with DOX loaded UCNP@UIO66(NH2)/FA in bright field and corresponding DAPI stained image [(a-b) control, (c-d) 5 μg.mL1

, (e-f) 10 μg.mL-1, (g-h) 15 μg.mL-1]. The apoptotic nuclei are shown by pink colored arrows. 29

3.12. Cell cycle analysis Finally, the apoptosis was further confirmed by FACS analysis, as shown in figure 10. From the FACS study, it was observed that the Sub G0 population was higher in DOX loaded UCNP@UIO-66(NH2)/FA treated cells in compare to control, UCNP@UIO-66(NH2)/FA, and DOX loaded UCNP@UIO-66(NH2) treated cells. The percentage of sub G0 population of each NMOFs treated cells are shown in S19 [SI]. This increment of Sub G0 population clearly indicates that DOX loaded UCNP@UIO-66(NH2)/FA possessed more toxicity associated with apoptotic cell death [46, 57].

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Figure 10. Flow-cytometry analysis of cell cycle phase distribution. Distribution of MDA-MB468 cells treated with (a) Control, (b) UCNP@UIO-66(NH2)/FA, (c) DOX loaded UCNP@UIO66(NH2),and (D) DOX loaded UCNP@UIO-66(NH2)/FA for 24 h.

3.13. In vitro DOX release study A nanoscale metal organic framework, UIO-66(NH2) was fabricated on the surface of UCNPs to achieve huge amount of DOX loading due to high surface. In this case, 142 mg DOX molecules are encapsulated inside the 100 mg of UCNP@UIO-66(NH2)/FA and drug encapsulation efficiency of UCNP@UIO-66(NH2)/FA is found to be 96.82 %. Cumulative release of DOX molecules from UCNP@UIO-66(NH2)/FA was checked in pseudo physiological pH medium (pH 7.4) and intercellular cancer cell medium (pH 5.5) up to one week in vitro at 37 °C, as shown in figure S20 [SI]. About 30 % & 40 % DOX are discharged from UCNP@UIO66(NH2)/FA after 12 h & 24 h respectively in pseudo physiological pH medium at pH 7.4. At the same time interval, approximately 65 % & 72 % DOX release are observed in cancer cell medium at pH 5.5. So, synthesized NMOFs would quickly deliver DOX molecules into cancer cells than normal cells, as the cancer cell environments are slightly acidic. This pH dependent release performance is very useful to reduce normal cell death in cancer therapy. The stability of DOX loaded UCNP@UIO-66(NH2)/FA in physiological pH media (PBS, pH 7.4) up to three weeks are shown in figure S14 [SI], designating usefulness of present DDS. The change of surface morphology after DOX release at 12 h and 24 h in both pH medium are shown in figure S15 & S16 [SI].

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4. CONCLUSION In conclusion, we have successfully designed and fabricated a core-shell upconversion nanoscale metal organic frameworks, which accumulation of targeting to TNBC, high drug loading capacity, and pH responsive drug release for breast cancer therapy. More importantly, there is no any obvious change in fluorescence intensity after surface modification UCNPs with NMOFs which convert near infrared to visible upconversion luminescence. The folic acid encapsulation would enhance the intercellular uptake of NMOFs inside MDA-MB-468 cells by the receptor mediated endocytosis. The anticancer drug doxorubicin has been successfully encapsulated in the upconversion NMOFs with high loadings (1.42 g DOX g-1 NMOFs) and realized controlled release inside TNBC regions with reduction of the normal cell death. Further work is currently on-going to use the upconversion NMOFs for in vivo studies.

Supporting Information (SI) EDAX spectrum and elemental mapping of UCNPs, UCNP@UIO-66(NH2)/FA, and UIO-66(NH2); PXRD patterns of UCNP@UIO-66(NH2)/FA, UCNPs, and UIO-66(NH2); Thermogravimetric analysis (TGA) of UCNP@UIO-66(NH2), UCNP@ UIO-66(NH2)/FA, and DOX loaded UCNP@UIO-66(NH2)/FA; Zeta potential of UCNPs, UCNP@UIO-66(NH2), and UCNP@UIO-66(NH2)/FA at pH 5, pH 7, and pH 9; Digital camera photo image of UCNP@UIO-66(NH2)/FA in presence of 980 nm laser; FESEM, HRTEM, and DLS of DOX loaded UCNP@UIO-66(NH2)/FA; UV-Vis spectroscopy of free DOX and DOX after loading; Camera photo image of solid state UCNP@UIO-66(NH2), UCNP@UIO-66(NH2)/FA, DOX loaded UCNP@UIO-66(NH2) and DOX loaded UCNP@UIO-66(NH2)/FA, free DOX and DOX after loading into UCNP@UIO-66(NH2)/FA; Stability study of UCNPs, UCNP@UIO-66(NH2), 32

and UCNP@UIO-66(NH2)/FA; FESEM image (change of morphology)

of synthesized

UCNP@UIO-66(NH2)/FA in different pH after 24 h stirring at 37 °C; DLS spectra of DOX loaded UCNP@UIO-66(NH2)/FA in different pH medium; Stability of DOX loaded UCNP@UIO-66(NH2)/FA after several weeks in PBS at pH 7.4; FESEM image of synthesized UCNP@UIO-66(NH2)/FA after DOX release up to 12 h and 24 h in pH 7.4 and pH 5.5 at 37 °C; Cellular morphology study of NIH3T3 cells incubated with UCNP@UIO-66(NH2)/FA up to five days at 37 °C; Intracellular uptake of UCNP@UIO-66(NH2) and UCNP@UIO-66(NH2)/FA by NIH3T3 cells for 2 h and 6 h at 37 °C; In vitro cumulative DOX release profiles for DOX loaded UCNP@UIO-66(NH2)/FA measured at pH 5.5 and 7.4 at 37 ºC at different time interval; Graphical representation of quantitative apoptosis by DOX loaded UCNP@UIO-66(NH2)/FA.

Acknowledgements This work was financially supported by the DST, Government of India (SB/FT/CS068/2013) and Indian School of Mines, Dhanbad. We are thankful to Dr. Kaushal Kumar, Department of applied physics, IIT (ISM) Dhanbad, India and Prof. Sudip Kumar Ghosh, IIT Kharagpur, India for florescence and photoluminescence studies.

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Graphical Abstract

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Highlights

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Upconversion NMOFs are developed for targeted drug delivery.

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The synthesized NMOFs possess high surface area and less cytotoxicity.

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This core shell nanoparticles reduce auto-fluorescence and enhance photo stability.

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Upconversion NMOFs enhanced doxorubicin loading and exhibits pH responsive release.

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