Rose Bengal attached and dextran coated gadolinium oxide nanoparticles for potential diagnostic imaging applications

Accepted Manuscript Rose Bengal attached and dextran coated gadolinium oxide nanoparticles for potential diagnostic imaging applications

Shailja Kumar, Virendra Kumar Meena, Puja Panwar Hazari, Surinder Kumar Sharma, Rakesh Kumar Sharma PII: DOI: Reference:

S0928-0987(18)30123-4 doi:10.1016/j.ejps.2018.03.008 PHASCI 4437

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

3 August 2017 5 March 2018 6 March 2018

Please cite this article as: Shailja Kumar, Virendra Kumar Meena, Puja Panwar Hazari, Surinder Kumar Sharma, Rakesh Kumar Sharma , Rose Bengal attached and dextran coated gadolinium oxide nanoparticles for potential diagnostic imaging applications. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017), doi:10.1016/j.ejps.2018.03.008

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ACCEPTED MANUSCRIPT Rose Bengal attached and dextran coated gadolinium oxide nanoparticles for potential diagnostic imaging applications Shailja Kumara, Virendra Kumar Meenaa,b, Puja Panwar Hazarib, Surinder Kumar Sharmac Rakesh Kumar Sharmaa* a

Department of Pharmaceutical Sciences, Lovely professional University, Jalandhar Punjab India

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Institute of Nuclear Medicine and Allied Sciences, DRDO, Ministry of Defense, Delhi, India

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Nanotechnology and Drug Delivery Research Lab, Department of Chemistry, University of Delhi, Delhi, India.

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Corresponding author (*): Dr. Rakesh Kumar Sharma ([email protected]) Ph 91 9310050453

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Abstract

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We report here, reverse micelle mediated synthesis of multifunctional dextran (dex) coated Gd2O3 nanoparticles (NPs) carrying rose bengal (RB) dye for magnetic resonance and optical imaging. The diameter of these RB attached dex coated Gd2O3 NPs (Gd-dex-RB NPs) was found to be ~17nm as measured by TEM. NMR line broadening effect on the surrounding water protons affirmed the paramagnetic nature of these NPs. Optical properties of Gd-dex-RB NPs were validated by UV-Vis and fluorescence spectroscopy. Time dependent release profile of RB from NPs at two different pH of 7.4 and 5.0 revealed that these NPs behave as slow releasing system. In-vitro study revealed that NPs are efficiently taken up by cells and show optical activity in cellular environment. In vitro cell viability (SRB) assay was performed on cancerous (A-549, U87) and normal (HEK-293) cell lines, showed the absence of cytotoxic effect of Gd-dex-RB NPs. Therefore, such multifunctional NPs can be efficiently used for bio-imaging and optical tracking.

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1 Introduction

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Key Words- Rose Bengal, Reverse Micelle, Gadolinium Oxide Nanoparticles, Contrast Agent, Optical Imaging.

Possession of numerous unique properties at nano-scale enables nanomaterials to exhibit novel theranostic (therapeutic & diagnostic) applications. Additionally, NPs work as an excellent nanocarriers for various drug molecules or therapeutic agents which can either be incorporated within the particles or attached to its surface [1][2]. A lot of research is advancing to develop multimodal NPs in ‘advance’ medicine field which could target more than one issue in tandem. Nanoscale magnetic material, such as iron oxide and gadolinium oxide, has been actively researched for their novel applications in bio-medical field. These magnetic NPs can be conjugated with other functional molecules, such as fluorophore, biomolecules or drugs, for imaging, diagnosis and therapy [3]. A combination of magnetic resonance imaging (MRI) and optical imaging in a single entity would render simultaneous multiple diagnoses in biological specimen.

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Both MRI and optical imaging techniques are non-invasive. Such imaging modalities are complementary rather than competitive in nature. Multifunctional nanomaterial incorporating both paramagnetic ions (responsible for MRI signal) and a fluorophore can be synchronously detected by fluorescence microscopy [4]. Such nanomaterial offers the advantages of both the techniques such as high sensitivity from fluorescence microscopy with profound 3-dimensional images of biological system through MRI analysis. Also the limitations of both the imaging modalities are compensated by each other like minute information of anatomical background obtained by fluorescence microscopy and low sensitivity of MRI [5][6].

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MRI is a crucial tool in clinical diagnosis. In order to enhance tissue contrast in MRI, contrast agent (CA) is added. Gadolinium oxide based NPs work as T1 CA which produce bright contrast in MR images by shortening the longitudinal relaxation time [7]. Seven unpaired electrons present in 4f orbital of Gd(III) ion produce strong magnetic moment. Due to small size and large surface to volume ratio, large number of Gd(III) ions present on the surface of Gd2O3 NPs produce large longitudinal relaxation of water protons present in its vicinity [8]. Park et al. [9] synthesized Gd2O3 NPs of ~1nm size by utilizing three differentGd(III) ion precursors namely gadolinium chloride hydrate, gadolinium acetate hydrate and gadolinium acetylacetonate hydrate. They showed that the particles efficientlyproduced high in vivo contrast in T1 MR images of rat’s brain tumor. Large r1is attributed to huge surface to volume ratio of NPs along with cooperative inducing surface of gadolinium ions for relaxation of water protons.Faucher et al. [10] developed polyethylene glycol coated Gd2O3 NPs of mean diameter 1.3nm via a fast and efficient method for molecular and cellular MR imaging. Their study demonstrated that particles provide strong contrast enhancement in T1-weighted MR imaging and visualization, of labelled cells which are implanted in vivo, can be done.

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Dual imaging agents are critical for diagnosis and treatment in near future as they can provide complementary information for diagnosing diseases with improved certainty. For instance, collaboration of MRI with fluorescence imaging is crucial because of the high sensitivity and good spatial resolution provided by MRI and strong fluorescence intensity provided by fluorescence imaging [11]. Numerous dyes have been either attached or entrapped with NP based CA for this purpose. Xu et al. [11] reported synthesis of fluorescein polyethyleneiminecoated Gd2O3NPs for MRI and cell labeling. Use of these dye loaded NPs as CA resulted in a strong contrast enhancement in the MR images of a rat having liver tumor. Also treatment of DU145 cells with NPs resulted in strong fluorescence. Bridot et al. [12] prepared core shell paramagnetic Gd2O3 NPs covered with polysiloxane shell carrying a fluorophore and carboxylated PEG attached to its surface. These NPs were utilized for in vivo fluorescence and MR imaging. The NPs exhibited higher proton relaxivities as compared to commonly used CAs and could be followed by fluorescence imaging. PDT is widely identified as an effective approach for bacterial photoinactivation and cancer treatment. PDT is based on chemicals (known as photosensitizer, PS) which can absorb and transfer energy to other chemical compounds which in turn generate highly reactive metastable

ACCEPTED MANUSCRIPT species. PS in excited state can interact with O2 in ground state forming reactive oxygen species. These reactive oxygen species (especially singlet oxygen) cause cell death by damaging DNA and plasma membrane [13].Rose Bengal is a xanthene dye having intense absorption in visible region. Rose Bengal is a type-II photosensitizer with high singlet oxygen yield making it apt for PDT. In spite of being an effective PS, RB suffers from low intracellular uptake. This issue has been, lately, addressed by using NPs as vehicles to conjugate PS for enhanced cellular uptake without hampering the photodynamic properties of PS [14].

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Herein, we report the preparation of dual modality dex coated Gd2O3NPs carrying RB dye (Gddex-RB) for the simultaneous purpose of MR and optical imaging. Nano size Gd2O3 particles work as CA in MRI and presence of RB dye ensures their application for optical tracking in biological system. In order to impart biocompatibility and non-toxicity to Gd2O3NPs, they are coated with dex [15][16]. Dex also acts as a conjugator for attaching RB to the particle surface. We adopted water-in-oil microemulsion mediated synthesis of the NPs where the aqueous core of the reverse micelle was used as ‘nano’ reactor within which reaction occurs. We anticipate that such multifunctional NPs can further be modified and optimized for photodynamic therapy due to the presence of RB which is an anionic photosensitizer with good singlet oxygen yield [17][18]. 2 Experimental Section

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2.1 Materials

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Sodium bis-(2-ethylhexyl)sulfosuccinate (AOT,96%), Gadolinium nitrate pentahydrate, Ammonia and Ethanol were procured from Acrōs Organics (New Jersey, USA), Central Drug House(Mumbai, India), Rankem (Delhi, India) and Merck (Darmstadt, Germany) respectively. Rose Bengal, Deuterium oxide (D2O) and dextran (75KD) were obtained from Alfa Aesar (Heysham, England and Ward Hill, Canada).Hexane and citric acid were purchased from spectrochempvt. Ltd. (India). Sodium phosphate dibasic anhydrous (Na2HPO4) was purchased from SRL India Ltd. Phosphate buffer saline (PBS), Sulforhodamine B (SRB) and Hoechst Stain were incurred from Sigma Aldrich (St Louis, MO). Tris base and paraformaldehyde were purchased from Merck. Human embryonic kidney cells lines (HEK-293) and lung carcinoma cells lines (A-549) were procured from National Centre for Cell Sciences, Pune India. All chemicals were used as such without any further purification. 2.2 Synthesis of Gd-dex-RB NPs Dye loaded NPs were synthesized in the aqueous core of the reverse micelle of hexane/AOT/water system. Two different microemulsions, A & B, were prepared. Microemulsion A was prepared by adding 100µL of double distilled water (DDW) and 250µL of 0.1M Gd(NO3)3 aq. solution to 15 mL of 0.1M AOT in hexane. In the similar manner, microemulsion ‘B’ was prepared by adding 100µL of DDW and 250µL of 1M NH3 to 15 mL of 0.1M AOT in hexane. In both the microemulsions, molar ratio of water to surfactant (Wo) [19][20] was kept at 13. The microemulsions were magnetically stirred till they became optically clear after which ‘B’ was

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added dropwise to ‘A’ with continuous stirring. After complete addition, resultant microemulsion was stirred for 20 hours which led to the formation of Gd2O3 NPs. Next, 100µL of 1% dex solution was added to microemulsion to coat the surface of the formed particleswith dex. The solution was stirred for another 24 hours at room temperature. Next, dex coated Gd2O3 NPs (Gd-dex NPs) were separated by centrifugation at 8000 rpm and washed several times with hexane and alcohol and were dispersed in 5mL DDW. In order to attach RB onto the surface of Gd-dex NPs, 20µL of 0.05% RB aqueous solution was added to 5mL of NP dispersion and stirred for 24 hours. The RB coated NPs (Gd-dex-RB) were separated by centrifugation at 8000 rpm and finally dispersed in 1mL DDW for further use.

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2.3 Characterizations 2.3.1 Transmission electron microscopy (TEM)

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TEM images of the Gd-dex-RB NPs were taken onTECNAI G2-30 U TWIN instrument (FEI, Eindhoven, Netherlands) which operates at an acceleration voltage of 300 KV. Dilute aqueous dispersion of NPs was prepared and a drop of this solution was deposited on the copper grid. The copper grid was completely dried at room temperature and observed under microscope. 2.3.2 Ultraviolet-visible (UV-Vis) spectroscopy

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All UV-Vis spectrums were recorded, at room temperature, on Shimadzu-1601 UV-vis spectrophotometer operated in the range of 190 nm -1100 nm. 2.3.3 Fluorescence Spectroscopy

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The fluorescence emission spectrums of various samples were recorded, at room temperature, on Cary Eclipse fluorescence spectrophotometer (Varian, Palo Alto, CA). The samples were scanned from 550 nm to 800 nm. 2.3.4 X-ray diffraction (XRD)

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XRD analysis was performed on Rigakuminiflex desktop XRD instrument at room temperature. Scanning of the dry NP sample was done in the 2θ range of 15–700. 2.3.5 Fourier Transform Infrared (FT-IR) Spectroscopy FT-IR spectrums of various samples were taken on RXIFT IR (Perkin Elmer) instrument which has alloy coated nichrome wire as source. Dry samples were dispersed into KBr powder and pressed into a pellet. The KBrpellets were scanned in the range of 400 cm-1 to 4000 cm−1 at room temperature. 2.3.6 Thermo gravimetric analysis (TGA).

ACCEPTED MANUSCRIPT TGA of powered samples were performed on Perkin Elmer DTA/TGA/DSC instrument in nitrogen atmosphere. The samples were scanned in the temperature range of 320C to 10180C. Change in the weight % of the samples with temperature was studied. 2.3.71H-Nuclear magnetic resonance (1H-NMR). 1

H-NMR spectrum of NPs in D2O (solvent) was recorded using 400 MHz spectrometer (JNMECX-400P, JEOL, Tokyo, Japan).

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4 Attachment efficiency of Gd-dex-RB NPs.

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2.5 Release kinetics of RB dye at pH 5.0 and 7.4.

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An indirect approach was adopted to estimate the attachment of RB to the NPs surface using UVvisible spectroscopy. After attaching RB to NPs, the solution was centrifuged to separate the unattached dye from NPs. Amount of dye present in supernatant was estimated by measuring its absorbance at 549 nm (characteristic peak of RB dye). Attachment efficiency was determined by deducting the amount of RB dye present in supernatant from the amount of total dye initially added during synthesis.

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Release of the RB dye from Gd-dex-RB NPs was probed in buffers of pH 5.0 and 7.4. In a glass vial, 5.6 mg of Gd-dex-RB NPs were dispersed in 10mL of phosphate buffer of desired pH and kept on docile stirring at room temperature. After regular pre-determined time intervals, 3mL of dispersion was withdrawn and centrifuged to separate the dye released from the particles. Amount of dye released from the NPs was calculated by measuring its absorbance at 549nm in supernatant. The solution was transferred back to the glass vial after noting the absorbance.

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2.6 In-vitro cytotoxicity study through SRB assay

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SRB assay was performed to estimate the cytotoxicity of NPs under study. Cells were seeded at a density of 5000 cells per well in 96 well microtiter plate. Cells were treated with varying concentration of RB, Gd-dex NPs and Gd-dex-RB NPs from 0.55mg/mL – 55 ng/mL for 24 hrs. At time point, 100μL of ice cold 10% (wt/vol) trichloroacetic acid (TCA) was added to each well and were incubated for 1 hr at 4°C followed by washing the plate four times under running tap water. Plates were then air dried and 100μL of 0.057% SRB solution was added in each well for 30 min. Plates were quickly rinsed four times with 1% (v/v) acetic acid and air dried. To dissolve the dye, 200μL of 10mM Tris base solution (pH 10.5) was added to each well and kept on gyratory shaker for 5 min. The color intensity was measured fluorometrically with excitation at 488 nm and emission at 585 nm. Surviving fraction was calculated and plotted for the concentration range 0.55mg/mL – 55ng/mL as a function of time. 2.7 In-vitro cellular uptake

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Firstly, A-549 lung carcinoma cells were seeded onto coverslips in a 6-well plate at a density of 10,000 cells/well. Following it after 24 hrs, the cells were incubated with the RB, Gd-dex NPs and Gd-dex-RB NPs at the concentration of 55ng/mL in culture media for 4 hrs. After the incubation, media was carefully removed and cells were washed with PBS twice, and further fixed with 4% formaldehyde solution (pH-7.0) for 5 mins. After that cells were again washed with PBS and incubated with DAPI solution (20µg/mL) for 2 minutes. The cells were washed twice with PBS gently to remove excess of DAPI stain. Then coverslip containing the cells were picked and placed on glass slide with DPX mounting media and images were taken on inverted fluorescence microscope (UV filter for DAPI, and Red filter for Gd-dex-RB NPs images respectively).

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Further quantitation of the NPs uptake was done by flow cytometery analysis of the Gd-Dex-RB NPs treated A-549 cells. The cells were grown to confluency, and single cell suspension was made and seeded into a 6-well plate with each well having 5x106 cells. The cells were allowed to adhere overnight, once adhered these cells were subjected to different concentration of Gd-Dex-RB NPs (55ng/mL- 55µg/mL) with incubation period of 4 h. After incubation, the cells were scraped and converted to single cell suspension, washed twice with PBS and re-suspended in cold methanol for 5 min. Later, cells were pelleted down and re-suspended in 500µL ofPBS for acquisition. 3 Result and discussion

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Merger of MR and optical imaging in a single entity with enhanced detection of molecular events is the purpose of this reported research work. We adopted microemulsion mediated preparation of dex coated Gd2O3 NPs which are subsequently attached with RB. The particles were found to be of spherical morphology with average diameter of about 17nm as measured using TEM (fig.1). The particles are highly monodispersed in shape and size. The UV-Vis spectrums of RB, Gd-dexRB NPs and Gd-dex NPs are exhibited in fig.2(a). The absorption spectrum of RB solution shows an anticipated characteristic peak at 549nm. This characteristic peak is also observed in the absorption spectrum of Gd-dex-RB NPs thereby confirming the presence of dye on the particles. The UV-Vis spectrum of Gd-DexNPs without RB did not show any peak corresponding to RB. Therefore, this suggests that the peak at 549 nm in particles is entirely due to RB and not arising because of Gd, dex or any other impurity. The optical behavior of RB attached to NPs was further investigated by taking the fluorescence spectrums (fig.2(b)) of the three systems viz RB aqueous solution, Gd-dex-RB NPs and Gd-dex NPs dispersions in water. As can be seen in fig.2(b), RB loaded NPs show appreciable fluorescence emission which highlights their ability to be used in optical imaging whereas Gd-dex NPs do not show any such fluorescence peak. The crystallinity and phase of the NPs were identified by carrying out the powder XRD analysis. The XRD pattern of the NPs is given in fig.3 and it shows distinct peaks corresponding to 221, 222, 400 and 134 planes and indicate the cubic structure of the Gd2O3 NPs [21][22]. Next, the FT-IR spectrums of the four systems vizdex, RB, Gd-dex NPs, and Gd-dex-RB NPs were taken and are shown in fig.4. In case of dex and Gd-dex NPs, peak corresponding to O-H stretching vibrations appeared at ~3445cm-1 for both. A sharp peak at 2925cm-1 corresponding to C-H

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stretching vibration in dex is shifted to 2930cm-1in Gd-dex NPs. Some new characteristic absorption peaks appeared in the case of Gd-dex NPs, like peaks at 1040cm-1 and 1158cm-1 corresponding to the stretching vibration of alcoholic hydroxyls (C-OH). Another noticeable peak appeared in the spectrum of Gd-dex NPs at 1400cm-1 which represented O-H bend [23]. A peak at 1160cm-1 is present for both dex and Gd-dex NPs which represents antisymmetric C-O-C stretching vibration [24]. A small but sharp peak present at 1640 cm-1(which is a characteristic peak of C=O stretching vibration) disappeared completely in the case of Gd-dex NPs. This data proves that the dex was successfully coated on the surface of nanosize Gd2O3 core via electrostatic interactions, Vander Waals force and hydrogen bond. Similar results were also reported by Hong et al. [25]. Next, the spectrum of RB was analyzed and it showed a strong characteristic peak at 1614cm-1which corresponds to C=O stretching vibration. The carbonyl stretching frequency was observed to shift to lower frequency value as compared to normal frequency value (1700cm-1) of carbonyl group due to the presence of more electronegative halogen substituents [26]. The other three bands present at 1545cm-1, 1454cm-1 and 1340cm-1 represents C=C double bonds of the aromatic rings [27]. In the case of Gd-dex-RB NPs, peaks corresponding to C=C have shifted to 1520cm-1 and 1401cm-1. The band corresponding to carbonyl stretching frequency has completely disappeared in the spectrum of Gd-dex-RB NPs. From these observations, we can say that RB molecules are attached to dex through carboxylic group via H-bonding and electrostatic interaction [28][25].

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Thermogravimetric analysis of the Gd-dex-RB NPs, dex and RB was probed from 320C to 10160C in N2 atmosphere to study their thermal behavior (fig.5). Two decomposition stages were observed in the thermogram of dex. The first decomposition stage is around 1300C which depicts loss of water. The next decomposition stage occurs around 2500-3500C which reflects degradation of saccharide structure in dex. At 9580C, dex gets degraded completely. This is in accordance with a report described earlier by Tang et al. [29]. In the thermograph of RB, a steep fall in weight percent was observed around 3500C which continues till 5600C at which 62.5% weight loss occurs. After this point, a slow change in weight loss is observed and finally at 10160C, about 90% degradation of RB takes place. In the case of Gd-dex-RB NPs, an overall 57% decrement in weight loss was observed. A gradual decay with temperature was observed over the entire temperature range with no abrupt changes. Overall decrease in the weight of the NPs can be attributed to the presence of dex and RB on the surface. The particles were found to be more thermally stable in comparison to dex and RB. To substantiate the application of these particles as contrast agent in MR imaging, 1 H-NMR spectrum of these particles was taken in D2O as solvent. For reference, 1H-NMR spectrum of D2O shows a sharp peak of water protons (fig.6(a)). However, in the presence of Gddex-RB NPs in its neighborhood, proton peak broadening is observed in the NMR spectrum (fig.6(b)). Gd has high magnetic moment and long electron spin relaxation time (1 × 10−9s to 1 × 10−8 s) which maximizes dipole-dipole interaction between electrons and the protons present in the vicinity of NPs. Such interactions induce faster proton relaxation and enhance signal intensity in MR images [30]. Now a sharp peak in NMR results when the time taken for the relaxation of proton is greater than the time taken for measurement. According to Heisenberg’s uncertainty

ACCEPTED MANUSCRIPT principle with greater uncertainty in time, greater certainty in frequency is observed. It is represented by equation-

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Here, Δt and Δυ represents uncertainty in time and frequency respectively. A very sharp peak will be observed when the uncertainty in time is high. So the 1H-NMR spectrum of D2O follows uncertainty principle. However, when the NMR spectrum of D2O is recorded in the presence of paramagnetic Gd2O3 moiety, peak broadening is observed because when Gd2O3 is present in the vicinity, relaxation time of water protons reduces. Due to which Δt becomes less and consequently Δυ increases which gives a broad peak. Therefore, we can say that the paramagnetic behavior of Gd2O3 affects the relaxation time of neighboring protons and hence these particles can be aptly used as CA in MR imaging.

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Attachment efficiency (A%) of RB to the Gd-dex NPs was established using UV-vis spectroscopy. An indirect approach was adopted by determining the amount of RB added for surface coating of the particles and the amount of dye remaining in the supernatant. The A% was found to be 96% and the equation used for calculating it is given below-

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Next, the release kinetics of the RB dye from Gd-dex-RB particles was probed to see the release pattern of the NPs(fig.7). Release kinetics is a critical parameter for biomedical application of NP [31]. We investigated time-dependent release profile of RB from NPs in two different buffer solutions of pH 7.4 and 5.0. At pH 7.4, release of 14% and 17.6% was noticed after 18hr and 43hr respectively. It reached to ~29% after 165hrs which remained almost same even after 187hrs. In buffer of pH 5.0, release of ~15% and 18% was observed after 23hrs and 46hrs, respectively, which reached to 30% at 167hrs and no further increase, was noticed even after 189hrs. Overall, release profile of the NPs was found to be more or less same at both the pH.Due to the attachment of fluorophore to the surface of the particles (and not entrapped or encapsulated within the particles) and biodegradability of dex, an overall release of ~30% was observed over a period of 8 days, in both the cases. We can conclude that the NPs can be considered as a slow releasing system at both the pH. To substantiate the optical activity of the fluorophore loaded NPs, fluorescence images of Gd-dex NPs, RB solution and Gd-dex-RB NPs are illustrated in fig.8(a, b and c). Gd-dex-RB NPs and RB solution exhibits fluorescence whereas Gd-dex NPs do not, indicating that the fluorescence is due to RB and not due to Gd and dex. We also investigated the in vitro interaction of the NPs with cells. This interaction between NPs and cells has two important aspects, first the cellular uptake of the NPs by the cells and second is the biocompatibility and non-toxicity of the NPs in cellular environment. Therefore, we first probed the cellular uptake of the NPs by treating the A-549 cell lines with a concentration of

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55ng/mL of Gd-dex-RB NPs, Gd-dex NPs and RB for 4 hours. The results are given in fig.9(a). Cellular uptake data of Gd-dex-RB NPs shows optimum uptake in A-549 cells where RB shows fluorescence in near IR region. Cells treated with RB and Gd-dex NPs do not show optical activity. When the cells were treated with Gd-dex-RB NPs (55ng/mL), they shows two types of red fluorescence, one shown by yellow arrows which shows disperse red fluorescence by NPs inside the cells, while the one shown by cyan arrows indicates the false enhanced positive red fluorescence of NPs aggregate. Fluorescence images of the cells treated with Gd-dex-RB NPs show optical activity of particles in and around the cells. The NPs were not conjugated with any kind of specific ligand which would provide targeted property to certain specific kind of receptors. We proposed, a non-specific kind of internalization mechanism for the conjugate, but most appropriate mechanism would be phagocytosis in membrane enclosed vesicles inside the cell. Quantitative studies on cellular uptake were also performed on A-549 cell line. The uptake study of Gd-dex-RB NPs was conducted at different concentrations, which suggested that NPs uptake was increased gradually with increasing concentration of NPs to which the cells were subjected. The cellular uptake of Gd-dex-RB NPs at different concentrations was studied with flow cytometery analysis [Fig. 9(b)]. The highest concentration used for uptake studies by flow cytometery analysis was 55µg/mL, a concentration greater than this NPs start to exhibit cytotoxicity. Therefore, A-549 cells were cultured, and exposed to different concentrations of GdDex-RB NPs. The upper panel [Fig. 9(b)] shows a gradual increase in ∆MFI (mean fluorescence intensity) with increase in concentration of Gd-dex-RB NPs, which suggests the cellular uptake of the NPs inside the cells. Similarly, Sytox Green-A uptake was also increased with increasing concentration in the treatment

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The cytotoxicity of the Gd-dex-RB NPs was probed via SRB assay performed on cancerous cell lines (A-549 and U87 cells) as well as on normal cell line (HEK cells). All the three cell lines were treated with varying concentrations (0.55mg/mL – 55ng/mL) of RB solution, Gd-dex NPs and Gddex-RB NPs for 24 hours. The results of SRB assay are summarized in fig.10. Up to a concentration of 55ng/mL of RB, Gd-dex-RB NPs and Gd-dex NPs without RB, the surviving fraction of all three cell lines was above 90% as depicted by data point 1 in all the three graphs. Upon increasing the concentration of RB solution and NPs dispersions to 5.5µg/mL, ~86% and ~92% cell viability was observed with HEK and U87 cells, respectively, with all three solutions but with A-549 cells ~75%, ~60% and ~67% surviving fraction was observed for RB, Gd-dex NPs and Gd-dex-RB NPs respectively. When cytotoxic curves were analyzed, the Gd-dex-RB NPs, Gd-dex NPs and RB all were found to be non-cytotoxic to the cells at lower concentrations. At the higher concentration, the Gd-dex-RB NPs show reduced toxicity as compared to the Gd-dex NPs and RB, but if we compare Gd-dex NPs and RB they have similar cytotoxic effect on the cells. At concentration higher than 5.5µg/mL, the particles started to show cytotoxicity. Accordingly, the cellular uptake studies were also done in the non-toxic range of the NPs. At higher concentration of 0.55 mg/mL, RB solution and NPs dispersions were found to be cytotoxic for all three cell lines. Therefore, according to experimental data it is observed that Gd-dex-RB NPs exert negligible toxicity in cellular environment in the concentration range of desired application. Hence these

ACCEPTED MANUSCRIPT particles can be aptly used for bio-imaging. Thus, it is proposed that further modification of the NPs can be done for photodynamic therapy and anti-cancer activity. Such systems will expand the horizon of biomedical diagnosis and treatment in near future. 4 Conclusion

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In this report, we have demonstrated the microemulsion mediated synthesis of multifunctional Gddex-RB NPs of size ~17nm for optical and MR imaging applications. The 1H-NMR line broadening effect of Gd-dex-RB NPs on the surrounding water protons substantiate the paramagnetic nature of these NPs and therefore indicate that these particles can be efficiently used as contrast agent in MRI. Fluorescence spectrum, fluorescence microscopy images and UV-visible spectrum of the particles revealed the efficient optical activity of the particles. Release profile of the NPs in pH 5.0 and 7.2 showed that the particles behave as slow releasing system. These NPs show in vitro cellular uptake and optical activity which substantiates their utility in bioimaging and in vitro optical tracking. In vitro SRB assay performed on cancerous as well as normal cell lines affirmed that the Gd-dex-RB NPs are not cytotoxic. Thus, these results collectively demonstrate the efficiency of these multifunctional Gd-dex-RB NPs to be used for safe bioimaging and tracking purpose.

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Acknowledgment

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The authors are highly thankful to University Science Instrumentation Centre (USIC), University of Delhi and INMAS, DRDO Delhi for allowing us to use the necessary instrumentation facilities.

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Funding: This work was supported by Council of scientific and industrial research (CSIR) [Scheme No.: 02(0153)/13/EMR-II] New Delhi, India.

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Figure 1. TEM image revealed that the size of the NPs is ~17nm. Figure 2. (a) UV-visible and (b) fluorescence spectrums, of RB, Gd-dex-RB NPs and Gd-dex NPs. Figure 3. XRD pattern of the synthesized Gd-dex-RB NPs.

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Figure 4. FT-IR spectra of Gd-dex-RB NPs, Gd-dex NPs, dex and RB. Figure 5. Thermogravimetry analysis of Gd-dex-RB, RB and dex performed in temperature range of 320C to 10160C in N2 atmosphere. Figure 6. NMR spectrum of (a) D2O and (b) Gd-dex-RB NPs dispersed in D2O. Figure 7. Release kinetics of RB from Gd-dex-RB NPs in neutral (7.4) and acidic (5.0) pH.

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Figure 8. Fluorescence images of (a) Gd-dex NPs (b) RB solution and (c) Gd-dex-RB NPs. Figure 9. (a) Cellular uptake study on A549 cell after treating with 55ng/mL of Gd-dex-RB NPs, Gd-dex NPs and RB for 4 hours.

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(b) Flow Cytometery analysis of NPs uptake: (i) First panel shows the quantitative uptake of GdDex-RB NPs in terms of ∆MFI which represents gradual uptake of NPs with increasing concentrations of Gd-Dex-RB NPs. The overlapping curves shows the fluorescence intensity counts at different concentration of NPs with respect to auto control. ∆ MFI values were also plotted against different concentration of Gd-Dex-RB NPs. (ii) Second panel shows the corresponding uptake of Sytox Green-A dye at different concentrations. Figure 10. Cytotoxicity (SRB) assay performed on A549, U87 and HEK-293 cell lines.

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