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Carbon Dots Embedded Magnetic Nanoparticles @ Chitosan @Metal Organic Framework as a Nanoprobes for pH Sensitive Targeted Anticancer Drug Delivery Angshuman Ray Chowdhuri, Tanya Singh, Sudip Kumar Ghosh, and Sumanta Kumar Sahu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03988 • Publication Date (Web): 15 Jun 2016 Downloaded from http://pubs.acs.org on June 16, 2016

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ACS Applied Materials & Interfaces

Carbon Dots Embedded Magnetic Nanoparticles @ Chitosan @Metal Organic Framework as a Nanoprobes for pH Sensitive Targeted Anticancer Drug Delivery Angshuman Ray Chowdhuri1, Tanya Singh2, Sudip Kumar Ghosh2, Sumanta Kumar Sahu*1

1 Department of Applied Chemistry, Indian School of Mines, Dhanbad 826 004, Jharkhand, India 2 Department of Biotechnology, Indian Institute of Technology, Kharagpur 721 302, West Bengal, India * Corresponding author. E-mail: [email protected], [email protected]; Fax: +91 326-2307772; Tel: +91 3262235936

Abstract Recently, nanoscale metal organic frameworks (NMOFs) have been demonstrated as a promising carrier for drug delivery, as they possess many advantages like large surface area, high porosity and tunable functionality. However, there are no reports about the functionalization of NMOFs, which combines cancer-targeted drug delivery/imaging, magnetic property, high drug loading content and pH-sensitive drug release into one system. Existing formulations for integrating target molecules into NMOF are based on multi-step synthetic process. However in this study, we report an approach that combines NMOF (IRMOF-3) synthesis and target molecule (Folic acid) encapsulation on the surface of chitosan modified magnetic nanoparticles in a single step. A noticeable feature of chitosan is control and pH responsive drug release for several days. More importantly, doxorubicin (DOX) was incorporated into magnetic NMOF formulation and showed high drug loading (1.63 g DOX g-1 magnetic NMOFs). To demonstrate the optical imaging, carbon dots (CDs) are encapsulated into the synthesized magnetic NMOF, thereby endowing fluorescence features to the nanoparticles. These folate targeted magnetic 1 ACS Paragon Plus Environment


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NMOF possess more specific cellular internalization towards folate-over expressed cancer (HeLa) cells in comparison to normal (L929) cells. Key Words: Carboxymethyl chitosan; Fluorescent carbon dots; Folic acid; Magnetic nanoscale metal organic framework; pH responsive doxorubicin release.

1. Introduction A main challenge for cancer therapy is the engineering of a smart nanocarrier that can competently encapsulate therapeutic molecules at high load, control with pH sensitive release at target sites and behave as MRI contrast agent. Magnetic nanoparticles with porous structure have received tremendous attention with their potential applications in various areas including catalysis, biomedicine, environmental remediation and imaging due to their large surface areas, well-defined pore structures and pore sizes, as well as magnetic separability.1-4 Recently, the magnetic porous nanomaterials have shown tremendous potential in drug delivery systems (DDSs) because of its fascinating properties, such as good biocompatibility, ease of functionalization, high drug loading capacity, and controllable release.5-7 In particular anticancer drug delivery, magnetic nanoparticle possess diverse properties like photothermal agent, MRI contrast agent, multimodal imaging, and real-time monitoring approach to destroy tumor tissue.810

As a new class porous material, nanoscale Metal Organic Frameworks (NMOFs) has generated

marvelous interest owing to their potential application in many areas, especially for bioactive molecule delivery. NMOFs have drawn considerable attention in drug delivery due to their ultrahigh porosity, large surface areas and tunable functionality.11-13 Compared to typical porous materials (e.g.,silica), the synthetic procedure of NMOFs is much simpler and highly efficient. In the past two years, a number of groups have developed NMOFs conjugated magnetic nanoparticle for drug delivery.14-16 However, each of these magnetic NMOF has its own

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limitations. An ideal magnetic NMOF to meet all requirements of drug carriers integrating with structures and surface properties are still needed to be developed. In most cases, researchers have reported the core-shell structured magnetic nanoparticles (MNPs)@NMOF to prepare magnetic NMOFs for drug delivery. In our previous work, we have demonstrated the potential utility of magnetic NMOF in magnetic resonance imaging with targeted drug delivery.17 Recently, many groups have delivered different chemotherapeutics by either direct incorporation into a NMOF or post synthetic covalent attachment to a presynthesized NMOF.18,

19

These chemotherapeutics were encapsulated with modest efficiency.

Therefore, most resulting NMOF based composites exhibit fast drug release, which limits their applicability. For this, some research groups have designed NMOF by modification with different polymers. Ren et al. have synthesized Polyacrylic acid modified zeolitic imidazolate framework-8 for pH responsive drug delivery.20 Recently Bian et al. have reported polyacrylic acid modified magnetic zeolite imidazolate framework for drug delivery.15 Rieter Et al. have decorated nanoscale metal organic framework surface functionalized with variable thickness of silica and polyvinylpyrrolidone for controlled release and sensing.21 All above mentioned magnetic NMOF or only MOF have been surface modified with polymer for drug delivery. However, there are no reports about the functionalization of magnetic NMOFs for targeted imaging or therapy (either in vitro or in vivo), which would have a great significance in drug delivery. The Conjugation of targeting ligand is an important issue for anticancer drug delivery systems. Therefore, it is a challenge to facilely synthesize magnetic NMOF, especially to carry more drugs, target towards the cancer cells with controlled release and attractive superparamagnetic property, which could bring new applications in biomedicine. In this work, we are utilizing a different strategy to deliver chemotherapeutics that is combination of Fe3O4



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nanoparticle@chitosan core into MOF shell to construct a nanoporous material for drug delivery system. Specifically, in this study magnetic NMOF drug carrier has been fabricated by incorporating biodegradable natural polymer chitosan. Chitosan is a biocompatible and biodegradable natural polysaccharide derived from chitin, the second most abundant polysaccharide on earth after cellulose. Chitosan (CS) is increasingly used for various biomedical applications including drug and gene delivery, tissue engineering, wound healing, and antimicrobial property.22-27 We hypothesize the incorporation of chitosan not only enhances the degradability but also improves the drug loading amount, drug release efficiency, and pH sensitivity of the particles. Among the various ligands explored for targeted drug delivery, folic acid (FA) has emerged as an attractive ligand for potential cancer cell specific targeting because folate receptor (FR) is highly expressed in various malignant cancers compared to normal cells.28 The encapsulation of folic acid by drug delivery system is achieved using a lengthy process that includes steps: 1) amine functionalization of the delivery system 2) activation of folic acid and 3) incorporation of folic acid, which is not dissolved in aqueous medium. This process is both costly and time consuming. A strategy to overcome these drawbacks is to combine MOF synthesis and folic acid encapsulation on the surface of magnetic nanoparticle in a one-pot process. In the present study, we have decorated a newly designed magnetic IRMOF-3 nanoparticles loaded with DOX for combined cancer-targeted drug delivery and optical imaging. This platform is composed of three steps, as shown in scheme-1. Firstly, super paramagnetic iron oxide nanoparticles were coated with an O-carboxymethyl chitosan (Fe3O4@OCMC). Here carboxymethyl chitosan is used for pH responsive release of the drug molecules inside the acidic tumor endosomes with initiate apoptosis and the Fe3O4 core can be used as a T2-weighted


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magnetic resonance imaging (MRI) contrast agent as well as magnetically guided drug delivery. Secondly, IRMOF-3 was developed with encapsulation of folic acid in one-pot on the surface of Fe3O4@OCMC nanoparticle (Fe3O4@OCMC@IRMOF-3/FA). Finally doxorubicin was conjugated into the magnetic NMOFs by a physical encapsulation. For drug delivery applications, fluorescence probe has been conjugated to carrier for tracking the biological process.8,

17, 29, 30

Among numerous fluorophores, quantum dots were frequently used for cell

imaging due to their photostability and sharper spectra.31, 32 Recently, carbon dots, a new kind of fluorescent imaging agent, have been used in drug delivery vehicle due to their promising properties, including high water solubility, easy preparation, excellent biocompatibility and low toxicity.33,

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Taking advantage of these exceptional properties, highly fluorescent CDs were

conjugated on the surface of Fe3O4@OCMC@IRMOF-3/FA nanoparticles for optical imaging. For the first time, high fluorescent biocompatible carbon dots are conjugated on the surface of nanoscale magnetic metal organic framework for cancer cell imaging. The potential utility of such a nano delivery system in optical imaging and anticancer therapy was also demonstrated in vitro.



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2. Results and Discussion

Scheme-1. Schematic presentation of the synthetic procedure for the folic acid encapsulated magnetic NMOFs as a targeted DOX carrier. 2.1. Surface morphology, EDAX and elemental mapping analysis Actual shape and morphology of the OCMC coated magnetic nanoscale metal organic frameworks were determined by the FESEM study, as presented in the figure 1. The FESEM image of bare Fe3O4 nanoparticles shows a spherical shape with a high agglomeration due to high magnetization [Figure 1 (a)]. After the OCMC modification upon the Fe3O4 particles seemed to have a spherical smooth surface with a quite higher particle size [Figure 1 (b)]. FESEM image presents well dispersed, spherical particles when IRMOF-3 is conjugated on the surface of Fe3O4@OCMC nanoparticle as shown in figure 1 (c). A unique flower like morphology with a particle size 200±10 nm is obtained when single step in situ folic acid targeted magnetic NMOFs is formulated [Figure 1 (d)]. The FESEM image of synthesized high fluorescent carbon dots having a particle size less than 10 nm is shown in supporting information. 6 ACS Paragon Plus Environment

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The elemental mapping and EDAX study confirmed corresponding multicomponent nanoparticles with the presence of expected elements is shown in the supporting information [figure S3 and figure S4]. Only iron (Fe) and oxygen (O) were found in the Fe3O4 nanoparticles; iron (Fe), oxygen (O), nitrogen (N) and carbon (C) were present in Fe3O4@OCMC nanoparticles and iron (Fe), oxygen (O), zinc (Zn), nitrogen (N) and carbon (C) were exist in folic acid encapsulated magnetic Fe3O4@IRMOF-3. In case of EDAX, all over the scanning region of binding energies, no obvious peak for another element was obtained. This outcome indicated the synthesized products to be of high purity.

Figure 1. FESEM image of (a) Fe3O4 MNPs (b) Fe3O4@OCMC (c) Fe3O4@OCMC@IRMOF-3 and (d) magnetic nanoscale Fe3O4@OCMC@IRMOF-3/FA.



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2.2. TEM and DLS study The genuine microstructure of the Fe3O4, Fe3O4@OCMC and Fe3O4@OCMC@IRMOF3/FA magnetic nanoparticles were investigated by HRTEM, as shown in figure 2. Figure 2 (a) displays the TEM micrograph of bare magnetic Fe3O4 nanoparticles. Results depicts agglomerated magnetic nanoparticles having pre modification particle size of 15±5 nm. After modification with OCMC on Fe3O4 nanoparticles, the particle size is enhanced as well as a thin coverage of polymeric layer is observed. The less agglomeration indicated successful wrapping of OCMC upon super paramagnetic Fe3O4 nanoparticles. The coating with OCMC prevents the agglomeration and can be helps to grow the metal organic framework shell on the magnetic nanoparticles uniformly. On the other hand, one-pot folic acid threated magnetic metal organic framework nanoparticles are showing a unique morphology with size less than 250 nm having micro porous structure, as shown in figure 2 (c). The successful formation of MOF on the surface of Fe3O4@OCMC is confirmed by TEM image and conjugation of folic acid is confirmed by FTIR and UV-Vis study. Figure 2 (d, e, f) displays the corresponding selected area electron diffraction (SAED) patterns of the uncoated superparamagnetic Fe3O4 nanoparticles, OCMC coated Fe3O4 nanoparticles and Fe3O4@OCMC@IRMOF-3/FA. SAED pattern demonstrates well-defined characteristics polycrystalline planes of Fe3O4 nanoparticles is shown in figure 2 (d), which are comparable with the XRD result. In case of Fe3O4@OCMC and Fe3O4@OCMC@IRMOF-3/FA both crystalline and amorphous phase was observed due to the formation of OCMC and MOF layer on the surface of Fe3O4 as shown in figure 2 (e and f).


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As a supplementary of the TEM results to investigate the average hydrodynamic diameter of the synthesized nanoparticles, DLS analysis is further carried out in water medium, as shown in figure S10 (SI).

Figure 2. TEM images and corresponding SAED patterns of (a and d) Fe3O4 MNPs, (b and e) Fe3O4@OCMC and (c and f) Fe3O4@OCMC@IRMOF-3/FA, respectively. 9 ACS Paragon Plus Environment


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2.3. FTIR study The presence of surface functional groups of magnetic nanoparticles, metal organic framework modification, conjugations of the targeting ligands and carbon dots were confirmed by FTIR spectroscopy. The FTIR spectra of as-synthesized Fe3O4 nanoparticles and OCMC is shown in supporting information (SI), as well as Fe3O4@OCMC, Fe3O4@OCMC@IRMOF-3, Fe3O4@OCMC@IRMOF-3/FA and pure folic acid were illustrated in figure 3. The peak at 585 cm-1 engendered by a Fe-O stretching mode inside the Fe3O4 nanoparticles as shown in figure S1 (SI), while peaks at 3450 cm-1 responses for O-H band.35 FTIR spectrum of OCMC (Figure S1 (SI)) displayed a broad band at around 3450 cm-1 (hydroxyl/ amine groups), 1610 cm-1 (amine /carbonyl group’s stretching vibrations), 1315 cm-1 (C–O stretching) and 1073 cm-1 (C-O-C stretching vibrations) which are compared with the previously reported literatures.36,37 The FTIR spectrum of Fe3O4@OCMC having both the characteristic peaks of bare Fe3O4 and OCMC, confirms the successful coating of OCMC on the Fe3O4 nanoparticles, as shown in figure 1 (a). Furthermore appearance of bands at 3350 cm-1 and 3450 cm-1 (stretching vibration of amino group), 900 cm−1 and 1250 cm−1 (C-H stretching vibrations), 1550 cm−1 and 1650 cm-1 (deformation of benzene ring) confirms the formation of IRMOF-3.17,

38,

39

The

Fe3O4@OCMC@IRMOF-3 shows not only the characteristic peaks of Fe3O4@OCMC but also the IRMOF-3 peaks, confirms the successful coverage of Fe3O4@OCMC by IRMOF-3, as shown in figure. In the case of one-pot synthesized Fe3O4@OCMC@IRMOF-3/FA, most of the characteristic peaks of folic acid at 3546, 3400, 3310, 3116, 1690, 1600, 1480, 1410, 1330, 1185, 1108, 834 cm-1 confirms the efficacious attachment of folic acid into the chitosan modified magnetic metal organic frameworks. Attachment of highly fluorescent carbon dots towards the


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Fe3O4@OCMC@IRMOF-3/FA are also confirmed by FTIR spectroscopy, shown in figure S2 (SI).

Figure 3. FTIR spectra of (a) Fe3O4@OCMC, Fe3O4@OCMC@IRMOF-3 and IRMOF-3, as well as of (b) Fe3O4@OCMC@IRMOF-3/FA and free folic acid. 11 ACS Paragon Plus Environment


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2.4. UV- Vis study Besides FTIR analysis, the formation of folic acid conjugated NMOFs on magnetic nanoparticles were again characterized by UV-Vis spectroscopy is shown in figure 4(a). The assynthesized Fe3O4 and Fe3O4@OCMC nanoparticles could not show any characteristics UV-Vis absorbance. Nanoscale IRMOF-3 shows a characteristics UV absorbance at 330 nm. This predominant band is also present in the Fe3O4@OCMC@IRMOF-3, which confirms the successful formation of magnetic NMOFs. Pure folic acid shows two UV-Vis absorbance band one at 280 nm due to n−π* transitions and another at 360 nm for π−π* transitions.40 This perfect existence of these two bands in synthesized Fe3O4@OCMC@IRMOF-3/FA confirms the successful attachment of folic acid into the magnetic NMOF system. 2.5. Thermogravimetric analysis The formation of OCMC wrapping on Fe3O4 magnetic nanoparticles and construction of metal organic framework shell with targeting ligand is further determined by TGA analysis. The TGA thermograms of synthesized uncoated Fe3O4 nanoparticles, Fe3O4@OCMC and Fe3O4@OCMC@IRMOF-3/FA are presented in figure 4 (b). The comparative weight loss at different temperature could be explained on the basis of decomposition of the various surface functionalities from the synthesized nanoparticles. In case of bare Fe3O4 nanoparticles, there is no obvious weight loss is observed. The characteristics TGA thermogram of Fe3O4@OCMC shows weight loss after 250 °C due to the disappearance of OCMC part from the Fe3O4 nanoparticles.41 When the folic acid encapsulated nanoscale IRMOF-3 is coated on the Fe3O4@OCMC nanoparticles, thermogram shows two stage of weight loss, as shown in figure. The first step weight loss may be due to the folic acid and OCMC and second stage of weight loss is for decomposition of metal organic framework from the magnetic core. The obtained 12 ACS Paragon Plus Environment

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TGA results confirmed the step wise coverage of OCMC and folic acid conjugated NMOFs on the Fe3O4 nanoparticles.

Figure

4.

(a)

UV-Vis

spectra

of

Fe3O4

MNPs,

Fe3O4@OCMC,

IRMOF-3,

Fe3O4@OCMC@IRMOF-3, Fe3O4@OCMC@IRMOF-3/FA and free folic acid (b) TGA thermograms

of

Fe3O4

nanoparticles,

Fe3O4@OCMC

Fe3O4@OCMC@IRMOF-3/FA. 13 ACS Paragon Plus Environment

and

of

magnetic

nanoscale


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2.6. XRD Analysis The crystal structures and the phase purity of the magnetic nanoparticles and carboxymethyl chitosan modified magnetic MOFs were determined by powder XRD, as shown in figure 5 (a). XRD spectrum depicts six characteristics diffraction peaks at 2θ = 30.01°, 35.40°, 43.19°, 53.30°, 57.09°, 62.82° and 74.36° were allocated to magnetic Fe3O4 nanoparticles prepared by coprecipitation method. The 2θ values are corresponding to the (220), (311), (400), (422), (511), (440) and (622) reflection plane indices respectively.17,

35

Entirely the reflection

peaks observed in the magnetic products can be easily indexed to a crystalline cubic spinel structure of Fe3O4 with a lattice constant a = 8.410 A° and a space group Fd3m (227) (JCPDS No. 85-1436). In the case of Fe3O4@OCMC a slight broad band at around 2θ = 22°-27° can be assigned due to the amorphous OCMC wrapping upon the Fe3O4 nanoparticles, as shown in figure. Other peaks are remain same, confirms OCMC coating does not change any crystalline phase of the magnetic nanoparticles. A prominent broad XRD diffraction pattern near about 2θ ∼22° depicts the formation of amorphous nanoscale IRMOF-3 in a mixed solvent solvothermal method.38 Besides the corresponding characteristics peaks of Fe3O4@OCMC, the diffraction peaks of amorphous IRMOF-3 can be detected for the Fe3O4@OCMC@IRMOF-3. Folic acid attached Fe3O4@OCMC@IRMOF-3 shows exactly similar X-Ray Diffraction peaks as above [figure 5 (a)]. This result demonstrates that the crystalline phases of magnetic NMOFs are also remain intact when targeting molecule folic acid is directly attached by a single step. The weak reflection XRD peak intensity of Fe3O4 in the hybrid system was probably due to a low content of magnetic materials as well as coating of polymer and NMOFs. The comparison of powder XRD pattern indicates that the successful deposition of carboxymethyl chitosan and IRMOF-3 upon the magnetic nanoparticles.


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2.7. Magnetization study of the nanoprobes The evaluation of magnetic response of synthesized magnetic hybrid nanosystems were studied by SQUID. Variations of magnetization with respect to the change of applied magnetic field for synthesized nanoparticles are shown in figure 5 (b). Magnetization vs magnetic field plot of the nanoparticles shows zero remnant magnetization and no coercivity can be proof the superparamagnetic behavior of all synthesized nanoparticles at 25 °C. The normalized saturation magnetization values derived from corresponding hysteresis loop of the synthesized bare Fe3O4 and Fe3O4@OCMC and Fe3O4@OCMC@IRMOF-3/FA are found to be 78 emu/g, 66.5 emu/g and 51 emu/g respectively. The reduction of magnetization value is due to the presence of coating agent like non-magnetic OCMC and metal organic framework on the surface of Fe3O4 nanoparticles. The decrease in magnetization is depends upon the reduction of magnetic dipolar interactions. Nevertheless, the synthesized hybrid nanoprobe exhibits expected magnetic response can say suitability of our multifunctional nanoprobe as a promising MRI contrast agent and magnetic guided drug delivery.



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Figure 5. (a) Powder X-ray diffraction patterns of Fe3O4, Fe3O4@OCMC, IRMOF-3, Fe3O4@OCMC@IRMOF-3 and Fe3O4@OCMC@IRMOF-3/FA (b) Magnetization versus magnetic field plot at 300 K for Fe3O4, Fe3O4@OCMC and Fe3O4@OCMC@IRMOF-3/FA.


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2.8. PL study Now a day, carbon dot possesses great advantages over conventional dyes in cellular imaging because of easy synthesis, low cost, biocompatible and stability in different media. So, tagging of carbon dots towards NMOFs can be a new horizon in optical imaging. Here CDs were synthesized by our previously reported method and immobilized on the surface of magnetic NMOF for cell imaging. To understand the fluorescence behavior of CDs and CDs tagged Fe3O4@OCMC@IRMOF-3/FA, PL study was performed with different excitation as shown Figure 6. The results show that the CDs and Fe3O4@OCMC@IRMOF-3/FA-CDs exhibit excitation dependent sharp emission behavior. From the photoluminescence spectra of only CDs, it was observed that it possess sharp emission peak centered at 450 nm when it was exited at 300 nm. The PL intensity varied with the variation of excitation wavelength from 300 nm to 410 nm and the maximum emission spectrum was obtained at the excitation wavelength of 380 nm. Bright blue luminescence was observed by naked eye under UV (365 nm) light in aqueous solution [Figure not shown]. When the CDs were tagged with the Fe3O4@OCMC@IRMOF-3 fluorescent intensity is quite decreases but having a potentiality for cellular imaging. The excitation wavelength-dependent PL properties of the Fe3O4@OCMC@IRMOF-3/FA@CDs are probably due to different emissive trap and different sizes of the generated carbon dots on the surface of the composite.42



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Figure 6. Photoluminescence spectra of the (a) synthesized carbon dots and (b) carbon dots conjugated Fe3O4@OCMC@IRMOF-3/FA at different excitations.


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2.9. Cytotoxicity study by MTT assay In vitro cytotoxicity of the synthesized Fe3O4@OCMC@IRMOF-3/FA and DOX encapsulated Fe3O4@OCMC@IRMOF-3/FA nanoparticles were evaluated for determination of the targeted cancer therapy by checking biocompatibility through standard MTT assay on both cancer cell (HeLa) and normal cell (L929). Figure 7 (a) and (B) demonstrate the cytotoxicity profile of HeLa cells and L929 cell lines treated with five different concentrations of nanoparticles. The synthesized Fe3O4@OCMC@IRMOF-3/FA nanoparticles without DOX shows nontoxic toward both of the cell having greater than 80% cell viability up to 100 µg/mL, as shown in figure. So, the magnetic NMOFs may be applicable in biological applications. On the other hand DOX encapsulated Fe3O4@OCMC@IRMOF-3/FA nanoparticles caused toxicity toward both normal and cancer cells. But the toxic effect in the cancer cells were more prominent than the normal cells. 25 µg/mL of DOX loaded NMOFs caused 15 % death of the L929 cells, however the equal quantity of NMOFs caused greater than 50 % inhibition of the HeLa cells. After increment of drug dose, as folate receptor present in cancer cells the death of HeLa cell is more in comparison to L929. Half maximum inhibition concentrations (IC50) of DOX-loaded NMOFs for HeLa and L929 cells were found to be 10 µg/mL and greater than 100 µg/mL respectively. The selective inhibition of cancer cells (HeLa) compared to normal cells (L929) may be due to the folate receptor mediated endocytosis of drug loaded NMOFs caused by folic acid. Free DOX having almost equal toxicity to both normal and cancer cell (figure not shown) but DOX loaded folic acid modified nanosystem decreases the healthy normal cell death. So, our newly synthesized targeted magnetic NMOFs having potential applicability for anticancer

drug

delivery.

At

the

same

time

MTT

assay

of

the

DOX

loaded

Fe3O4@OCMC@IRMOF-3 lacking the FA was studied on the both normal and cancer cell lines showing a weak toxicity as shown in figure 7 (c) and 7 (d). 19 ACS Paragon Plus Environment


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Figure 7. Cell viability of folic acid conjugated nanoprobes in (a) L929 and (b) HeLa cell lines and without folic acid conjugated nanoprobes in (c) L929 and (d) HeLa cell lines.

2.10.

Intercellular uptake study The intracellular uptake efficiency and the folic acid targeting ability are investigated by

confocal imaging of the HeLa cells at different time interval, as shown in figure 8. A high fluorescent biocompatible carbon dots were attached to the magnetic NMOFs as a fluorescent tag and the nucleus of the individual cells were stained by PI to identify the cells. The HeLa cells were treated with 5 µg/mL of carbon dots tagged magnetic NMOFs for 15 min, 30 min, 45 min and 60 min for the determination of time dependent uptake of the carbon dots conjugated 20 ACS Paragon Plus Environment

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Fe3O4@OCMC@IRMOF-3/FA. The carbon dots labelled folic acid encapsulated NMOFs were easily internalized into the cancer cells through receptor-mediated endocytosis and produced bright blue signal inside the cells. As the time increases the fluorescent intensity increases, confirms the more nanoparticles were internalized. The quantitative cellular uptake of the Fe3O4@OCMC@IRMOF-3/FA is shown in supporting information [figure S17].



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Figure 8. CSLM image of Hela Cells incubated with 5 µg/mL Fe3O4@OCMC@IRMOF-3/FACDs for 15 min, 30 min, 45 min and 60 min at 37 °C with corresponding bright field and merge field [Scale bar = 20 µm], Nuclei were stained with PI.

2.11.

Cellular morphology study and apoptosis To observe the inhibition of HeLa cell proliferation and change of cell morphology, we

have visualized the DAPI stained nuclear morphology through the incubation with DOX loaded NMOFs. Results revealed that the nucleus of the cells was condensed and fragmented in a dose dependent manner, as shown in figure 9. This result suggested that DOX released from NMOFs inside the cell and start to proper work which inhibited the cell growth and encouraged apoptosis. The quantitative data of apoptosis study is shown in supporting information [figure S18].


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Figure 9. Apoptosis study of HeLa cells treated with DOX loaded Fe3O4@OCMC@IRMOF3/FA in bright field and corresponding DAPI stained image [(a-b) control, (c-d) 5 µg/mL, (e-f) 10 µg/mL, (g-h) 15 µg/mL]. Arrows in the figures indicate apoptotic nuclei.



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2.12. In vitro drug release study Carboxymethyl chitosan was conjugated towards the magnetic NMOFs to increase the drug loading efficacy and also for the improvement of pH responsive drug release performance. Doxorubicin has been selected as model anticancer drug to examine the loading and release profile. Here, about 163 mg of DOX were encapsulated into 100 mg of NMOFs and encapsulation efficiency into nanoscale Fe3O4@OCMC@IRMOF-3/FA were found to be 96 %. In vitro cumulative doxorubicin release experiments were carried out in a pseudo physiological pH aqueous media prepared by PBS at pH 7.4 and intercellular cancer cell pH 5.5 at 37 °C, as shown in figure S16. For both pH the cumulative release was monitored up to 8 days. The 22.5 % and 26.72 % of DOX was released after 12 h and 24 h respectively at pH 7.4, while 47.92 % and 55.1% of drug released after 12 h and 24 h at pH 5.5. This result reveals that a pH dependent doxorubicin release from the chitosan modified magnetic metal organic framework. At pH 5.5 an immediate release of 29.15 % drug was observed at first 3 h. The higher amount of drug release and burst release rate of doxorubicin may be due to the two reason, one is the slight breakage of the vehicles and other is higher solubility of DOX at lower pH. Interstitial fluid in tumor environment is mildly acidic than normal tissue. So we can say that drug release can be quicker into the slightly acidic tumor cells than in any normal cells or in blood media. It is presumed that the pH responsive drug delivery may be very useful to eliminate the side effects in human body. The NMOFs are also very stable in physiological pH media (PBS, pH 7.4) for about one month, indicating very useful smart materials for in vivo drug delivery.

3. Conclusion In summary, we have successfully designed a new class of hybrid magnetic NMOF which exhibited high stability, safety and biocompatibility in vitro. It shows enhanced doxorubicin 24 ACS Paragon Plus Environment

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loading efficiency, cancer cell targeting and pH responsive drug release. One key finding is that, the nanoscale IRMOF-3 formation and tumor-targeting folic acid encapsulation on the surface of Fe3O4 nanoparticle in a single step. More importantly, for the first time, we have demonstrated carbon dots encapsulated magnetic NMOFs for the optical bio-imaging. We believe that this magnetic NMOF formulation strategy is general and can be useful to deliver many other anticancer drugs. 4. Experimental Section: 4.1. Materials and Chemicals used Chitosan polymer (CS) (Mw = 100,000-300,000, degree of deacetylation ≥ 90% was determined by free amine groups) and anticancer drug doxorubicin hydrochloride were purchased from Sigma Aldrich. Ferric chloride (FeCl3), ferrous sulphate (FeSO4), and N, Ndimethyl formamide (DMF), NH4OH, Zinc nitrate dihydrate (Zn(NO3)2·6H2O, 99%), monochloroacetic acid, isopropanol were acquired from Merck India. 2-amino terepthalic acid (NH2-H2BDC, 99%), folic acid (FA), polyvinyl pyrrolidone (PVP) was obtained from TCI fine chemicals. Pure ethanol was acquired from Merck Germany. Sodium hydroxide (NaOH) was purchased from Merck India. N, N- dimethyl formamide (DMF) was purified by vacuum distillation. N-hydroxysuccinimide (NHS), dialysis membrane (MWCO 14 KD), trinitrobenzene sulfonic acid (TNBS), 1-[3 (dimethylamino) propyl] - 3-ethylcarbodiimide hydrochloride (EDC), propidium iodide (PI), 4/-6- diamidino-2-phenylindole (DAPI), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and RNase were procured from Sigma Aldrich Chemicals, USA. Fetal bovine serum (FBS) and Minimum essential medium (MEM) were purchased from Himedia, India, and Hyclone, USA respectively. Millipore water was used in the entire experiment.



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4.2. Synthetic procedure of O-carboxymethyl chitosan (OCMC) OCMC were synthesized by our earlier described protocol with a little modification.43 In Brief, the chitosan (1.0 g) was swelled in 30 mL of 40% sodium hydroxide solution at 0 °C for 24 h. After that the chitosan was washed with isopropanol by three times and dissolved in 30 mL of isopropanol. 3.0 g of monochloroacetic acid was dissolved in 5 mL of isopropanol and added into the chitosan solution drop wise for 30 min and the reaction was continued for 12 h at 35 °C. After that, the reaction was stopped by adding 25 mL 70% ethyl alcohol. The obtained sodium salt of O-carboxymethyl chitosan product was separated followed by washing with dry ethanol and vacuum dried at 40 °C.

4.3. Synthesis of magnetic nanoparticles Superparamagnetic Fe3O4 nanoparticles were synthesized according to earlier described protocol with a slight alteration.17 In a brief, anhydrous FeCl3 (0.324 g) and FeSO4·7H2O (0.274 g) were dissolved in 40 mL Millipore water at 25 °C under argon atmosphere. The mixture solution was gently stirred and the reaction temperature was enhanced to 75 - 80 °C under same inert condition. Consequently, ammonia solution (5 mL, 25%) was added very slowly into the flask and stirred for another 60 min to allow the perfect spherical growth of the magnetic nanoparticles. The synthesized nanoparticle solution was then cooled down to room temperature and the resulting particles were separated from solution by a magnetic concentrator followed by repeated washing with 50:50 water-ethanol mixture (v/v) followed by drying at 60 °C in a vacuum oven for 6 h.


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4.4. Preparation

of

O-carboxymethyl

chitosan

coated

magnetic

nanoparticles

(

Fe3O4@OCMC) For physical immobilization of OCMC on the surface of magnetic nanoparticles, 100 mg of OCMC was dissolve in 90 mL Millipore water. After that 50 mg of Fe3O4 was dispersed in 10 mL water and added to the OCMC solution. The reaction was continued for 12 h at 40 °C. The product was separated by magnetic decantation and washed several times with water to remove the unbound OCMC. The obtained product was dried at 60 °C for 12 h in a vacuum oven.

4.5. One-pot synthesis of folic acid encapsulated metal organic framework on Fe3O4@OCMC (Fe3O4@OCMC@IRMOF-3/FA) To synthesize core-shell folic acid impregnated Fe3O4@OCMC@IRMOF-3 in a one-pot, firstly 0.25 g PVP was solubilize in a mixture of solvent including pure ethanol (6 mL) and pure DMF (4 mL). Then, 20 mg of as-synthesized Fe3O4 nanoparticles were well-dispersed in the above solution. After that, 112 mg of Zn (NO3)2 and 28 mg of NH2-H2BDC were dissolved in DMF (4 mL) were added drop wise into the Fe3O4@OCMC solution. Then, the total solution was sonicated for 20 min. Consequently, the solution was placed into a Teflon-lined stainless steel autoclave and heated at 100 °C for 6 h (heating rate 1.5 °C/min). Finally, the acquired deep reddish brown colored product was collected by magnetic separation. The product was re dispersed in 25 mL pure DMF and heated at 100 °C and dialyzed against distilled water (MWCO 14 k Da) for 12 h to remove the unbound organic ligands, folic acid and PVP. Finally, the product was washed with ethanol-water mixture and dried in vacuum at 60 °C for 12 h.



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4.6. Synthesis of carbon dots conjugated Fe3O4@IRMOF-3@OCMC/FA nanoparticles (Fe3O4@ OCMC@ IRMOF-3/ FA-CDs) Firstly, highly fluorescent acid group functionalized carbon dots was synthesized in a single step hydrothermal method by our previously reported procedure.44 In brief, 0.3 g of citric acid and 0.754 g of (NH4)2HPO4 (1:4 molar ratio) were taken in 20 mL water. Acquired mixture was transferred into a Teflon-lined stainless steel autoclave and heated in an oven at 180 °C (heating rate 1 °C/min). After completion of 4 h reaction, autoclave was cool down and dark brown colored solution of CDs was dialyzed with a dialysis tube (MWCO 2 k Da). High fluorescent carbon dots embedded folic acid targeted nanoscale metal organic framework

(Fe3O4@OCMC@IRMOF-3/FA)

was

synthesized

by

activation

of

acid

functionalized carbon dots and followed by addition of Fe3O4@OCMC@IRMOF-3/FA nanoparticles through EDC/NHS chemistry. EDC (30 mg) and NHS (30 mg) was added to the previously prepared carbon dot solutions and kept the solution for 3 h in dark condition to activate the acid groups. After that, 15 mg of Fe3O4@OCMC@IRMOF-3/FA nanoparticles were dispersed to it and continue the reaction for 12 h in dark. Then the prepared Fe3O4@OCMC@IRMOF-3/FA-CDs nanoparticles were magnetically separated and dialyzed (MWCO 2 K Da) against Milli- Q water to remove the unbound carbon dots.

4.7. Doxorubicin loading In the current drug delivery system, doxorubicin was taken as a model anticancer drug for the estimation of loading efficiency and release patterns. Firstly, Fe3O4@OCMC@IRMOF-3/FA nanoparticles were washed with phosphate-buffered saline (pH 7.4) and Milli-Q water. 5 mg Fe3O4@OCMC@ IRMOF-3/FA nanoparticles were introduced into 5 mL of 1.7 mg/mL DOX solution and retained in an orbital shaker for 12 h in dark. The drug loaded Fe3O4@OCMC@ 28 ACS Paragon Plus Environment

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IRMOF-3/FA nanoparticles were collected by magnetic decantation. The supernatant solution was collected to determine the drug loading content (LC) and drug encapsulation efficiency (EE) of DDS from UV-Vis absorbance at 481 nm. A calibration curve was plotted by taking a different concentration of DOX solution to determine the amount of DOX encapsulated into the nanoparticles. The photographic picture of free DOX and after DOX loading in the magnetic NMOFs is shown in electronic supplementary Information (Figure S6). The drug LC and EE were determined by the following equations.

Weight of drug in nanoparticles

×

Drug loading contents (%) =

100

Weight of nanoparticles taken

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

×

100

Weight of total drug injected

4.8. In vitro doxorubicin release To determine the impregnated drug discharge from the core of the polymer modified nanoscale magnetic metal organic framework, the in vitro cumulative DOX release study were carried out. The release was checked in two different pH to demonstrate the pH responsive performance of the nanoparticles at 37 °C. First 2.5 mg of DOX loaded nanoparticle were taken in physiological pH condition (pH ∼7.4) and incubated at 37 °C then similar experiment were performed in lysosomal pH condition (pH ∼5.5). Cumulative percentage of DOX release was evaluated spectrophotometrically (at 481 nm) from standard curve of free DOX at regular time interval.



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4.9. Cell Lines The cells cultivated for in vitro experiments L929 (murine fibroblast) and human cervix adenocarcinoma (HeLa) cell lines were obtained from the National Centre for Cell Sciences (NCCS), Pune, India and grown in MEM and DMEM (Dulbecco's modified Eagle medium) medium, respectively, with 10% fetal calf serum, penicillin (100 U.mL-1), streptomycin (100 mg/mL), 4×10-3 M L-glutamine at 37 °C in a 5% CO2 and 95% air humidified atmosphere.

4.10.

Cytotoxicity study by MTT Assay

HeLa cells were trypsinized and counted using hemocytometerand then seeded in 96-well micro plate at 5×10 4 cells/mL in DMEM complete medium. The cells were incubated at 37 ºC in 5% CO2 for 24 h to allow adherence. The medium was replaced with fresh DMEM incomplete medium containing various concentration of free nanoparticles and doxorubicin loaded nanoparticles viz. 5, 10, 25, 50 and 100 µg/mL for 24 h. After incubation, the cells were washed with 1X PBS and 100 µL of MTT solution (1 mg/mL) was added to each well. After 3 h of incubation, MTT solution was removed and replaced with 200 µL of DMSO. Absorbance was read at 570 nM using microplate reader (Thermo Scientific Multiskan Spectrum, USA). Each sample was assayed in triplicate, and control samples include cells with DMSO. 45, 46, 47 4.11. To

Intracellular uptake study visualize

the

intercellular

uptake

efficiency

of

carbon

dots

tagged

Fe3O4@OCMC@IRMOF-3/FA by cancerous HeLa cell line, 5x104 cells / well were seeded in 24 well plate and was allowed to adhere for 6-8 h at 37 ºC and 5% CO2. The cells were incubated with 5 µg/mL of Fe3O4@OCMC@IRMOF-3/FA-CDs for different time interval. Thereafter, the cells were fixed with 70 % alcohol for 2 h at 4 ºC followed by RNase A treatment in 2X SSC buffer at 37 ºC for 30 min. Finally, nucleus was stained with propidium iodide (2µg/mL). To 30 ACS Paragon Plus Environment

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study the cancer cell specificity of the drug, the entire experiment was performed on a noncancerous cell line, L929 as a control.47, 48 4.12.

Apoptosis and DAPI staining for nuclear morphology study

To understand the effect of DOX loaded nanoparticle carrier on doxorubicin-induced apoptosis morphologically in HeLa cells, we performed the DAPI staining.49 HeLa cells were treated with different concentrations of DOX loaded nanoparticles for 24 h. After treatment, the cells were washed with PBS buffer and fixed with 4% paraformaldehyde for 10 min at 37 °C and permeabilized with 0.1% Triton X-100. The fixed cells were then washed with PBS and stained with DAPI (300 nM) for 10 min at 37 °C. The cells were again washed thrice with PBS and investigated under a confocal microscope.

4.13.

Characterization

All surface functional groups within nanoparticles system were examined by using Fourier transform infrared spectroscopy (FTIR) (Agilent Carry 660 instrument) at ambient temperature. The all samples were scanned with ATR method between 400 cm-1 to 4000 cm-1. To examine the surface morphology of the synthesized nanoparticles, field emission scanning electron microscopy (FESEM) analysis was performed by using a Supra 55 through an air lock chamber with energy dispersive spectroscopy (EDAX) and elemental mapping. High resolution transmission electron microscopy (HTEM, JEOL 3010, Japan operated at 300 keV) was used to determine the particle size and microstructure of the synthesized hybrid nanosystems. The samples were well dispersed in absolute ethanol by ultra-sonication and drop casted on a carbon coated copper grid. The magnetic properties of Fe3O4, nanoscale Fe3O4@OCMC@IRMOF-3 and Fe3O4@OCMC@IRMOF-3/FA were measured by using SQUID instrument (Evercool SQUID



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Magnetometer) at 25.0 ± 0.5 °C. Crystallographic state and phase of the nanoparticles were determined by powder X-ray diffraction (PXRD) with an Expert Pro (Phillips) X-ray diffractometer using Cu Kα. Thermo gravimetric analysis (TGA) was carried out by TGA, 2850 thermogravimetric analyzer (TA instruments) at N2 atmosphere. Measurements of NMOFs hydrodynamic diameter was performed on freshly prepared samples using a Zetasizer 3000 HS (Malvern Instruments). The fluorescent intensity of the carbon dots conjugated magnetic NMOFs was measured by fluorescence spectrometer (Perkin Elmer LS 55). UV-Vis absorption measurements were performed on a Shimadzu 220V (E) UV-Vis spectrophotometer for confirming the attachment of FA with the magnetic NMOFs and again to determine the DOX concentration in the NMOFs. Cellular uptake and apoptosis study were carried out under Olympus FV-1000 confocal microscope with excitation at 360 nm. 4.14.

Statistical analysis

Entire statistical analysis was performed using a statistical pack, OriginPro8, Northampton, MA 01060, USA) with student’s t tests, p < 0.05 as a limit of significance. The analysis of variance was accomplished by a one-way ANOVA using a Graph Pad Prism 6 software programme package.

Supporting Information (SI) Synthetic procedures of IRMOF-3 nanoparticles and Fe3O4@OCMC@IRMOF-3; Quantification of amine number on the surface of the magnetic NMOFs; FTIR spectra of OCMC, Fe3O4, Fe3O4@OCMC@IRMOF-3-CDs, Fe3O4@OCMC@IRMOF-3/FA-CDs and CDs; Elemental mapping and EDAX spectra of Fe3O4 MNPs, Fe3O4@OCMC@IRMOF-3/FA and Fe3O4@OCMC; TEM, SEM and SAED pattern of synthesized CDs; Camera photo and UV-Vis


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spectra of free DOX and after DOX loading; Stability of all synthesized nanoparticles; DLS spectra of nano magnetic MOFs; Quantitative data of cellular uptake and apoptosis.

Acknowledgements This work was financially supported by the DST, Government of India (SB/FT/CS068/2013) and Indian School of Mines, Dhanbad.



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