Surface Functionalized Multifunctional Gd2O3-Fluorescein Composite

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Surface Functionalized Multifunctional Gd2O3-Fluorescein Composite Nanorods for Redox Responsive Drug Delivery and Imaging Applications Arindam Saha, and Parukuttyamma Sujatha Devi ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00535 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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ACS Applied Nano Materials

Surface Functionalized Multifunctional Gd2O3-Fluorescein Composite Nanorods for Redox Responsive Drug Delivery and Imaging Applications Arindam Saha,* Parukuttyamma Sujatha Devi* Sensor and Actuator Division, CSIR-Central Glass and Ceramic Research Institute, Kolkata700032 (India) *Address correspondence to [email protected], [email protected]

Abstract: We have developed multifunctional Gd2O3-fluorescein based magnetic-fluorescent composite nanorods with small, uniform shape and size distribution and excellent colloidal stability for multimodal imaging and redox responsive drug delivery applications. Gd2O3 nanorods have been synthesized via high temperature colloidal route and composite development has been achieved via one-step polyacrylate coating. This unique coating approach provides controlled surface functional groups along with near zwitterionic surface charge. For redox-responsive drug loading, hydrophilic drug daunorubicin has been modified with disulfide linkage and loaded with the composite nanorods via covalent linkage. High drug loading (~82 %) has been observed with excellent redox responsive release behaviour. These composite nanorods have been used as multimodal imaging probe, fluorescence imaging as well as magnetic resonance imaging (MRI). Fluorescence imaging of the composite nanorods inside cancer-positive KB cells exhibit efficient cellular internalization and drug delivery with high cytotoxic effect. These surface functionalized composite nanorods having small size, excellent colloidal stability, high drug loading capability, redox responsive release behaviour along with multimodal imaging potential can serve as prospective nano-bio material in drug delivery and imaging purposes. Keywords: Composite, Functionalized, redox responsive, imaging, drug delivery, cytotoxicity

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Introduction: Surface engineered multifunctional nanomaterials have attended immense importance in the field of bioengineering and theranostics especially in the field of drug-delivery. Various

nanomaterials

including

silica

nanoparticles,1,2

dendrimers,3,4

solid

lipid

nanoparticles,5,6 micelles,7,8 liposomes9,10 and polymer nanoparticles11,12 have been extensively studied for drug delivery applications since decades. But very few of them have been translated into practical application due to their large size, uncontrolled surface functional groups, poor colloidal stability and lack of specific targeting capability. Targeted and responsive drug delivery is the key focus in recent development of drug delivery vehicles to nullify toxic side effects, to reduce the dose of the administered drugs and to minimize the toxic effects on normal cells.13-17 For specific targeting to disease cells/tissues, conjugation of particular targeting moieties have been explored (e.g. folic acid and RGD peptide for tumor cell targeting, triphenylphosphene (TPP) for mitochondria targeting etc.).18 This bioconjugation, either covalent or via electrostatic assembly, requires extreme control over surface functional groups. Previous studies have demonstrated that the surface density of targeting ligands control the cellular endocytosis pathway as well as dictates the intracellular localization of the nanoparticles.19-23 Thus controlling the number (or density) of surface functional groups play crucial role in cellular targeting and intracellular localization of drug delivery vehicles to maximize its effectiveness. Responsive release of drug molecules from the drug delivery cargo is an equally important factor to control the release kinetics and to avoid the toxic effects on normal cells. Various responsive drug delivery vehicles like pH responsive,7,14,24-26 temperature responsive,24,27-29 redox responsive,15,16,30-33 photo responsive,32,34-35 enzyme responsive

36-38

have been extensively studied with various nanomaterials and with variety of drug molecules, both hydrophobic and hydrophilic. Although pH and temperature responsive drug release is well studied and widely used due to low pH and high temperature (~40oC) in the tumor microenvironment, they are not highly specific and requires proper targeting approach to the desired site. To overcome the specificity issue, currently scientists are more focussed on redox or enzyme responsive drug delivery systems. However, in both the cases, surface engineering of nanomaterials play pivotal role to control this responsive characteristics. Another important aspect for drug delivery vehicle is integrated imaging modality which can be used to track the drug molecules in real time inside cellular compartments. This 2 ACS Paragon Plus Environment

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imaging modality can be fluorescence based, based,

8,26,39-41,43

26,39-42

magnetic resonance imaging (MRI)

positron emission tomography (PET) based

44,45

or X-ray computed

tomography (CT) based.46,47 Currently researchers are exploring multimodal imaging modality since a single imaging modality is not sufficient to provide all the detail information of the cellular microenvironment.39,42,44,48,49 Among these imaging modalities fluorescence imaging is most versatile and widely used among the scientists. Various fluorescence dye molecules and nanoparticles have been explored for bio-imaging studies over the years, including organic dye molecules,50-52 semiconductor quantum dots (CdSe/ZnS, CdS, InP etc.),53-55 doped nanoparticles,26,56,57 carbon nanoparticles,58,59 silicon nanoparticles,60 graphene quantum dots etc.61,62 Among them organic dye molecules are most common and widely used due to its strong emission quantum yield, small Stokes' shift, low toxicity and sharp emission maxima. Although organic dye molecules suffer from photobleaching phenomenon, many ongoing research is involved into improving its photo-stability.50,63-66 Multifunctional composite nanomaterials are currently under focus for drug delivery vehicles. Various multifunctional composite nanomaterials have been investigated in the last two decades which includes magnetic-fluorescent composites,26,39,67-69 plasmonic-fluorescent composites, 50,70,71 magnetic-plasmonic composites 69,71-73 and graphene based composites74,75 for wide range of biomedical applications like multimodal imaging, drug delivery, gene delivery, cell separation, protein enrichment, biosensing etc. The major advantage of these nanocomposites is multiple functionalities in a single entity without compromising its size or colloidal stability. Magnetic-fluorescent composite nanoparticles are most widely studied among these nanocomposites. In this composites iron-oxide nanoparticles are generally used as magnetic component, whereas the fluorescent counterpart varies between organic fluorescent molecules (dyes) and fluorescent nanoparticles. Dye molecules are usually conjugated on the surface of magnetic nanoparticles or incorporated inside the porous polymeric shell on the magnetic nanoparticles.67,68 On the other hand fluorescent nanoparticles are either form heterodimer with the magnetic nanoparticles or they are simultaneously incorporated inside a polymeric/silica shell.67,76-78 There are also a few examples where these two different nanoparticles are linked via covalent linkage either through a spacer molecule or via ligand exchange methods.69 But the major problem of these composite nanomaterials is the low quantum yield due to strong collisional quenching property of iron oxide nanoparticles. Moreover, these nanocomposites have large size, poor colloidal stability and uncontrolled surface functional groups. Another magnetic nanoparticle 3 ACS Paragon Plus Environment


currently in focus is Gd2O3 nanoparticles.

26,79-85

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This is particularly important because of its

ability to serve as T1 contrast agent in magnetic resonance imaging (MRI) due to its seven unpaired electrons (Gd3+). Another important aspect of this nanoparticle is neutron therapy due to its high neutron capture cross-section. But this nanomaterial is less explored in the field of biomedicine due to lack of proper synthetic techniques for precise control of shape and size, lack of composite development methodology, poor colloidal stability and uncontrolled surface chemistry. Currently, various Gd3+ based chelates have been approved and widely used as MRI contrast agents. Eu3+ doped Gd2O3 nanomaterials have been explored recently as magnetic-fluorescent composite for drug delivery and multimodal imaging.26,81-85 Although, proper control over shape and size of the nanoparticle and its surface chemistry is still a major concern. In this work we have synthesized thin and small Gd2O3 nanorods of uniform size distribution via one-pot colloidal synthesis technique at high temperature. These hydrophobic nanorods are then converted to hydrophilic nanorod via one-step polyacrylate coating adapted from our previous work, which provides controlled surface functionality on nanoparticle surface.20,22,26,78 In this polymer coating technique we have introduced fluoresceinmethacrylate monomer to develop magnetic-fluorescent nanocomposite. Fluoresceinmethacrylate monomer was used to impart fluorescence properties to the composite along with other monomers like amine and acid monomers to provide controlled surface functional groups and polyethylene glycol (PEG) monomer to provide improved biocompatibility. The fluorescein molecules are randomly distributed on the polymeric shell at various distances from the core nanoparticle and thus minimizing the fluorescence quenching. Similar type of coating has been previously explored by us to develop plasmonic-fluorescent nanocomposites for multimodal imaging purpose.50 Here we have extensively worked on the controlled surface engineering to provide desired number of surface functional groups on nanoparticle surface. We have attached hydrophilic drug molecule daunorubicin to this nanocomposite via redox responsive disulfide linkage. Drug loading have been found to be ~82 %. Drug release from this nanocomposites have been tested for 48 hours in presence of excess glutathione (GSH) and have been found to be extremely responsive to the redox environment. This is particularly important since all the tumor cells have high concentration of GSH compared to normal cells. Moreover these nanocomposites have been explored for fluorescence imaging on KB cells (subline of HeLa cells). Detailed cellular imaging study and cytotoxicity study substantiate the internalization of the drug molecules and their subsequent responsive 4 ACS Paragon Plus Environment

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delivery. We have also tested its potential as MRI contrast agent by measuring its proton relaxivity. Experimental Section: Materials and methods: Gd (III) nitrate hexahydrate, Oleic acid, 1-Octadecene (ODE) , N(3-aminopropyl)-methacrylamide methacrylate,

Fluorescein

Tetramethylethylenediamine, hydroxysuccinamide

hydrochloride,

methacrylate, Igepal,

(NHS),

Acrylic

acid,

Poly(ethylene

Bis[2-(methacryloyloxy)ethyl]

Ammonium cysteamine

persulfate,

phosphate,

Folic

dihydrochloride,

glycol)

acid,

N-

1-Ethyl-3-(3-

dimethylaminopropyl)carbodiimide (EDC), Fluorescamine, 5,5'-dithiobis(2-nitrobenzoic acid)

(DTNB)

,

Daunorubicin

hydrochloride

(DAUN),

Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium

Epichlorohydrin,

Bromide

(MTT),

3-(4,5cellulose

membrane (MWCO ~12000 Da) and RPMI was obtained from Sigma-Aldrich and used as received. Absolute ethanol, Cyclohexane, Hexane, Chloroform, Dimethyl formamide (DMF) and triethylamine was obtained from Merck Germany and used as received. Dimethyl sulfoxide (DMSO) and Dichloromethane (DCM) were received from Merck and distilled before use. Phosphate buffer saline (PBS) of pH 7.4 was prepared by using buffer capsules (Sigma-Aldrich) at appropriate manner. Preparation of Gd (III) oleate: The oleate complex has been prepared via previously reported method.26 1 mmol Gd(NO3)3 (~ 345 mg) and 4 mmol sodium oleate (~1.2 gram) was mixed with 2 ml water, 13 ml ethanol and 15 ml hexane followed by vigorous stirring at 80oC for 4 hours. After the reaction, the hexane layer was separated in a separatory funnel and washed with water 5-6 times to remove unreacted sodium oleate and salts. Finally the hexane was dried off and the powder of Gd-oleate was collected for further use. Synthesis of Gd2O3 nanorods: The synthesis was carried out in a typical three neck flask system attached with schlenk line.26 In brief, 0.3 mmol Gd-oleate complex was mixed with 0.3 mmol oleic acid (~95 µl) in 1:1 molar ratio and ~10 ml ODE in a three necked flask followed by heating at 300oC and kept there for one hour under inert atmosphere. After cooling,

the

solution

was

washed

with

acetone

and

chloroform/ethanol

by

redispersion/precipitation method twice. The final product was dispersed in cyclohexane for further use. Polymer coating of the nanorods to prepare Gd2O3-Fluorescein (Gd2O3-Fl) composite: Hydrophobic Gd2O3 nanorods were transformed into hydrophilic nanoparticles adopting 5 ACS Paragon Plus Environment


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previously reported polyacrylate coating approach with some modification.26,78 2 ml of hydrophobic nanorod (5 mg/ml) was mixed with 100 µL of an aqueous solution of N-(3aminopropyl)- methacrylamide hydrochloride (~2 mg), 100 µL of an aqueous solution of acrylic acid (~4 µL), 100 µL of an aqueous solution of poly(ethylene glycol) methacrylate (36 µL), 100 µL of an aqueous solution of fluorescein methacrylate (3 mg), 100 µL of an aqueous solution of phosphate cross-linker (bis[2- (methacryloyloxy)ethyl] phosphate) (6 µL), and 100 µL of base (tetramethylethylenediamine) to obtain a 10 ml reverse micelle solution with igepal-cyclohexane. The optically clear solution was purged with nitrogen gas under stirring condition. Polymerization was initiated by adding 100 µL of an aqueous solution of ammonium persulfate (3 mg). The reaction was carried out for 1 hour and then precipitated by adding absolute ethanol. The precipitate obtained was washed with chloroform and ethanol 3-4 times to remove any unreacted monomer or particle. Finally, the water dispersed nanoparticles were carefully dialyzed against fresh water by cellulose membrane (MWCO ~12000 Da). Drug modification with disulfide linkage: This modification has been done by simple conjugation chemistry. Typically ~5 mg daunorubicin hydrochloride was reacted with ~15 µl epichlorohydrin in DCM in presence of ~20 µl triethylamine base. Then it was reacted with ~40 mg cystamine dihydrochloride in DMSO by drop wise addition. The reaction was continued for one hour and finally the product was obtained by vacuum distillation. Conjugation of folate-NHS and drug molecules to the polymer coated nanorods: For folate conjugation with Gd2O3-Fl composite nanorods, we have treated folate-NHS with the surface functionalized nanorods. In brief, solution of folate-NHS with 0.05 M concentration in DMF was reacted with the polymer coated amine functionalized nanorods for overnight in dark. To keep the folate density low on nanoparticle surface, we have adopted the competitive conjugation chemistry with PEG-NHS ligand following our previous work.

20,26

Next the solution was dialysed (cellulose membrane MWCO ~12000 Da) against basic water and fresh water respectively. The dialysed solution was then treated with disulfide modified drug (with surface amine groups) with a concentration of 250 µg/ml molecules with EDC (1mg/ml) and NHS (1 mg/ml) solution. The reaction was carried out for 3 hours and finally the solution was dialysed to remove free drug molecules. Quantum yield measurement: For quantum yield measurement Gd2O3-Fl nanocomposites was excited at 470 nm and compared with fluorescein as standard dye (95% quantum yield). We have measured the absorbance of all samples by keeping the optical density (OD) value 6 ACS Paragon Plus Environment

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between 0.01 to 0.1 and subsequently their emission spectra were recorded. By comparing the OD values and the area of the emission curve between sample and standard we have estimated the quantum yield values. Fluorescamine test: Fluorescamine test was performed to confirm and estimate the primary amine groups on functionalized nanocomposite surface. In brief, 1 ml of polyacrylate coated Gd2O3-Fl nanocomposite was mixed with freshly prepared fluorescamine solution (2mg/ml) in acetone. The emission spectra was then collected by exciting at 400 nm. DTNB test: To confirm the presence of free thiols on nanocomposite surface DTNB test was conducted. DTNB solution was prepared in DMSO (4mg/ml) and then diluted to a final concentration of 0.1 mM by 0.1M Tris-HCl (pH 7.5) buffer. Next 50 µl of sample (Gd2O3-Fl composite, drug loaded Gd2O3-Fl composite and Gd2O3-Fl composite after drug release) was mixed with 950 µl of DTNB solution with appropriate dilution and incubated for 5 minutes. Finally it was observed by UV-visible spectroscopy at 412 nm. Drug release study: For drug release study, small portion of drug loaded samples (typically 200 µl) were taken in a dialysed membrane and dialysed against buffer solutions of pH 7.4 (PBS buffer) containing 1 mM and 5 mM glutathione (GSH) separately, for varying time period, typically from 30 minutes to 72 hours. Drug released in the buffer medium was collected and measured via photoluminescence spectroscopy. Drug release was also monitored by monitoring the free thiols in solution via DTNB test. Control drug release experiment was studied similarly without adding any GSH solution. Cell imaging study: KB cells were cultured in a 24 well plate using a folate free RPMI media for the cell imaging study. After desired confluency was achieved, cells were treated with drug loaded Gd2O3-Fl nanocomposite solutions and incubated for 2 hours at 37oC and 5% CO2 atmosphere. Next the cells were washed with PBS buffer twice followed by addition of fresh RPMI media. The labelled cells were then treated with Hoechst for 10 minutes to stain the nucleus of the cells, and then washed with PBS buffer and finally observed under fluorescence microscope. For responsive drug release study, cells were treated with external GSH along with drug loaded samples before observing under fluorescence microscope. Control experiments were performed using drug loaded samples without folic acid conjugation on KB cells. In another control experiment, folic acid functionalized drug loaded samples were added to KB cells pre-treated with external folic acid. To observe the effect of



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drug loaded sample on normal cells, CHO cells (Chinese Hamster Ovary cells) were treated with folate functionalized drug loaded sample. Cytotoxicity study: Cytotoxicity study was performed via well recognized MTT assay. KB cells were first cultured in a 24 well plate in folate free RPMI media followed by mixing with drug loaded samples and samples without drug of varying concentrations. The samples were incubated for 24 hours at 37oC and 5% CO2 atmosphere. Next the cells were treated with 50 µl MTT solution (~5mg/ml) and further incubated for 3 hours under similar conditions. The formazon crystals thus formed was separated carefully and dissolved in water-DMF (1:1) mixture with SDS. Cytotoxicity was measured from the absorbance of each well of the plate at 570 nm and comparing with the control cells. Cytotoxicity was also performed with free drugs for comparison. Cytotoxity was also studied with and without GSH to compare the effect of responsive drug release. Cytotoxity of CHO cells were also studied as control experiment. Proton relaxivity study: Proton relaxivity study was conducted by dispersing the functionalized, drug loaded nanomaterials in water at different concentrations (0.1mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM) of Gd3+. Instrumentation: XRD analysis was performed on X’Pert Pro MPD X-ray diffractometer (PANalytical) system using CuKα radiation ( λ= 1.5406 Å) at a 2θ degree scan rate of 2o per minute between 10o and 80o. TEM study was performed in a Tecnai G2 30ST (FEI) highresolution transmission electron microscope operating at a voltage of 300 kV. Particle size and zeta potential were measured in a Horiba (SZ-100) analyzer. The photoluminescence properties of the nanoparticles were studied on a steady state spectrofluorometer (QM-40, Photon Technology International, Pvt) using a 150 watt xenon lamp. The UV-Visible absorbance

spectroscopy

were

performed

on

a

UV-Vis-NIR

Spectrophotometer

(SHIMADZU UV-3600). Fourier transform-infrared (FT-IR) spectra were performed between 4000 cm-1 and 400 cm-1 on a NICOLET 380 FTIR spectrometer (Thermo Scientific) using KBr pallet. Magnetic measurement was performed using a Cryogenic Physical Property Measurement System (PPMS). Differential interference contrast microscopy (DIC) images and fluorescence images of cells were performed by Olympus IX81 microscope using imagepro plus version 7.0 software. The MTT assay was carried out in a Synergy Mx monochromator based multimode microplate reader (BioTek). T1 and T2 relaxation times of the sample were measured with Bruker Minispec (mq 60) TD-NMR instrument (60 MHz; 8 ACS Paragon Plus Environment

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1.41 T) at 37°C. The application which determines T1 relaxation times by applying Inversion Recovery pulse sequences, was employed for the T1 measurements. Similarly, the application which determines T2 relaxation times by applying Carr-Purcell-Meiboom-Gill (CPMG) pulse sequences, was employed for the T2 measurements. Relaxivities (r1 and r2) were determined from the slopes of concentration-dependent 1/T1 and 1/T2 changes. Result: Synthesis of Gd2O3 nanorods: Gd2O3 nanorods were synthesized via high temperature colloidal route where the metal precursors are decomposed at 300oC (Scheme 1a).26 Previously prepared Gd-oleate salt and oleic acid were mixed in a 1:1 ratio with ODE, a high boiling non-coordinating solvent. The mixture was then heated at 300oC for one hour under inert atmosphere. The solution colour turns from colourless to dark yellow in this time-period indicating the decomposition of the metal salt precursor. The nanorods thus formed was purified via precipitation-redispersion method. In this method, acetone is used to remove the solvent ODE by precipitating the nanorods from the solvent. The hydrophobic nanorods were then dispersed in minimum amount of chloroform and reprecipitated with large amount of ethanol. This method was repeated twice to make sure minimum free surfactants remain in the solution. To optimize the nanorod synthesis we have varied the reaction time from 30 minutes to 4 hours. Less reaction time forms small spherical particles without any definite shape or size distribution. 2 hours reaction time provide broken rods which are smaller than our optimized reaction conditions. Much higher reaction time offers nanoparticles of different shape and size.26 Characterization of Gd2O3 nanorods: X-ray diffraction (XRD) spectra of the purified sample (Figure 1a) has revealed strong peaks at 2θ 28.56o, 31.71o, 33.03o, 47.47o, 56.5o and 66.19o which corresponds to 222, 321, 400, 440, 622 and 642 crystal planes respectively of cubic Gd2O3 structure (JCPDS card number 12-797) with lattice parameter a=10.8 Ao. Absence of any other peaks in XRD spectra confirms phase pure preparation of cubic Gd2O3 nanorods. From transmission electron microscopy (TEM) analysis (Figure 1b-c) it has been observed that small ultrathin nanorods have formed which are ~25-35 nm in length with ~2 nm width. The nanorods have shown very well shape and size distribution. It has been observed that these nanorods have formed lamellar distribution on the TEM grid with uniform distance from each other. HRTEM image (Figure 1d) exhibits crystal planes with lattice spacing ~3.2 Ao which nearly matches with the 222 crystal plane of XRD. The SAED



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pattern of the nanorods (Figure 1e) exhibits prominent feature of 222 and 440 plane which confirms the findings from XRD spectra. Elemental analysis of the sample (Figure 1f) results in only gadolinium which confirms the absence of any impurity. The ratio of Gd and O atom percent has been found ~1:3 from EDS. Marginally higher oxygen content is due to oleic acid capping ligands. Optical properties of these nanorods have been studied by absorption and emission spectroscopy. In the absorption spectra, there is a strong peak at ~275 nm arises due to f-f electronic transition between 8S7/2 and 6IJ levels of Gd3+ (Figure 2a). No emission was observed in fluorescence spectroscopy except weak blue emission ~350 nm due to oleic acid capped with the oxide nanorod. Magnetic measurement of these nanoparticles have been performed and the M-H curve exhibit paramagnetic behaviour at room temperature. (Figure 2b) This paramagnetism is typical of Gd2O3 host due to its seven unpaired parallel f electrons. The volume magnetic susceptibility (χv) has been found as high as ~3.95 X 10-4. Synthesis of Gd2O3-Fl nanocomposite and their surface functionalization: Surface functionalization of these nanorods were performed via one-step polyacrylate coating which converts the hydrophobic nanorods into hydrophilic one along with desired surface functionality (Scheme 1b).26,78 In this coating approach we have used controlled ratio of amine and acid monomer along with PEG monomer. Amine (0.01 mmol) and acid monomer (0.09 mmol) ratio was kept 1:9 which reduces the toxic surface amine groups but provides sufficient functionality for further conjugation. PEG monomer (0.1 mmol) was kept in sufficient amount to avoid non-specific interactions during cellular internalization and to increase biocompatibility. To impart fluorescence in the composite, we have used small amount of fluorescein methacrylate monomer (0.0075 mmol). All the monomers were separately taken in reverse micelle formed by igepal-cyclohexane mixture and mixed altogether with the hydrophobic nanorod solution to form a clear solution. The polymer coating was performed under inert atmosphere, in presence of organic base. Ammonium persulfate was used as initiator for polymerization. Once it is initiated, polymerization commences from the surface of the nanorods. In this one-pot polymerization approach, monomers are randomly oriented on the nanorod surface with random distribution of fluorescein molecules in the polymer backbone. This also provides controlled number of amine and acid functionality on the nanorod surface. The polymerization was quenched after one hour by adding absolute ethanol, which being polar breaks the reverse micelle. The hydrophilic, surface functionalized nanorods get precipitated. The coated nanorods were


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washed with ethanol and chloroform to remove any hydrophobic impurities. Finally, the nanorods were dialysed against fresh water to remove unreacted monomers. We have modified the surface of this coated nanorods with folic acid which act as specific targeting ligand.42 It is well known that many tumor cells (cancer positive cells) over-express folate receptors. Thus, folic acid can specifically target tumor cells without affecting the normal cells. For this surface conjugation, folic acid is converted to NHS modified folic acid (Folate-NHS) adopting a previously reported technique.42 Amine groups on nanorod surface was confirmed by fluorescamine titration. Folic acid was conjugated via well known amine-NHS coupling between the amine groups on the coated nanorod surface and the -NHS group in modified folic acid. To control the surface folate density on nanoparticle surface we have used competitive conjugation chemistry along with PEG-NHS ligand following our previous report.26 Here we have used 1:9 molar ratio of folate NHS and PEG-NHS to keep the folate density low on nanoparticle surface. The conjugation was continued for overnight in dark and finally dialysed against basic water followed by fresh water for 4-5 times to get rid of unreacted folic acid. Folic acid attachment was confirmed from fluorescence spectroscopy as well as from DLS measurements (SI, Figure S1, S2). Characterization of surface functionalized Gd2O3-Fl nanocomposite: The surface functionalized nanorods have been studied via TEM to observe their shape and size distribution (Figure 3a-c and SI, Figure S3). As evidenced from TEM, the hydrophilic nanorods maintain their shape and size distribution even after polymer coating although their lamellar distribution has been lost. This is due to the polymer coating, which replaces the well distributed long chain fatty acids from the nanorod surface. From the EDS spectra (Figure 3d), the atomic ratio between Gd and O has been found ~1:10. This increased oxygen content is due to the polymer backbone. The hydrodynamic size of the hydrophilic nanorods were measured by DLS, which was found around 35-50 nm (Figure 3e) and does not change much after folic acid functionalization (~40-70 nm) (SI, Figure S2). However, higher amount of folic acid (~25 µM/mg of sample) conjugation leads to agglomeration with large hydrodynamic diameter (~70-100 nm) (SI, Figure S2). Zeta potential measurement reveals the surface charge of the hydrophilic nanorods at ~-17 mV. This surface charge accounts for higher concentration of surface acid groups rather than amine groups. This nearly neutral surface charge play pivotal role in cellular internalization studies as it minimizes the unwanted non-specific interactions with the cellular proteins. We have also studied the X-ray photoelectron spectroscopy (XPS) of the surface functionalized composite (SI, Figure S4). In 11 ACS Paragon Plus Environment


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the deconvoluted spectra of C1s, the binding energy of ~284.4 eV arises due to -C-C- linkage of the polymer backbone, whereas the binding energy at ~285.6 arises due to -C-O-C- type of ether linkage of PEG molecules. In O1s spectra, binding energy at ~530.5 eV arises mostly due to Gd2O3 component and carboxyl groups of the polymer backbone. The binding energy at ~532.2 eV is solely due to ether linkages of PEG molecules. In case of N1s spectra, there is only one peak at binding energy ~399.3 eV which is due to the primary amine (-NH2) groups of the polymer backbone. From XPS, elemental analysis has been carried out by integrating the peak area. It has been found from that analysis that the atom percent ratio between N and O is ~1:12, which supports the fact that the samples are functionalized with more number of surface acid groups compared to amine groups. The surface charge of the functionalized nanorods also changes from -17 mV to ~-25 mV after folic acid conjugation which accounts for the higher surface acid groups after folate conjugation. Optical properties of the hydrophilic nanorods were studied by UV-Visible and fluorescence spectroscopy. In UV-Visible spectral characteristic it has found that the overall spectra is broadened along with a new peak at ~485 nm due to absorbance from fluorescein molecules (Figure 4a). The broadening is due to polymer coating. Fluorescence spectra was measured by exciting the hydrophilic nanorods at 450 nm. A strong green emission centered ~525 nm is observed due to emission from fluorescein molecules (Figure 4b). Although the emission is somewhat decreased after folic acid conjugation, due to quenching ability of folic acid, it is sufficient for cellular labeling applications (SI, Figure S5). Quantum yield of the nanocomposites was measured ~20 % by comparing with the standard fluorescein dye. Lifetime measurement of the nanocomposite was performed and it was observed that the composite exhibited two different average lifetime (two exponential fitting) as compared to simple fluorescein dye which gives single exponential fitting (Figure 4c). This is due to the distribution of the fluorescein molecules on the polymer backbone, maintaining different distances from the core oxide nanoparticles. To study the surface functionality of the nanocomposites FTIR study has been performed (Figure 5). A broad peak at ~3431 cm-1 is observed due to -OH stretching frequency of PEG molecules. Two sharp peaks at ~2926 cm-1 and 2856 cm-1 arises due to C-H asymmetric and symmetric stretching respectively. The sharp peak at ~1719 cm-1 arises due -C=O stretching frequency of the acid functional groups. A small hump at ~1602 cm-1 is observed for -N-H deformation from amine functional groups. Two sharp peaks at ~1570 cm-1 and ~1385 cm-1 are due to asymmetric and symmetric -CO2-1 stretching frequency of acid functional groups respectively. The sharp and strong peak at 12 ACS Paragon Plus Environment

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~1491 cm-1 is mainly because of -CN and amide II stretching of primary amine functional groups. A small peak at ~1454 cm-1 is due to -C-H asymmetric deformation vibration of ethers arises from PEG functional groups. The sharp peak at ~1300 cm-1 is due to -OH deformation of acid functional groups. The peaks at ~1253 cm-1 and ~1190 cm-1 are due to asymmetric and symmetric stretching of C-O-C groups of PEG molecules. The peak at ~946 cm-1 is arises due to C-P rocking vibration and the peak at ~556 cm-1 is due to -P=O stretching arises from the phosphate crosslinkers. Magnetic measurement of the coated particles exhibits paramagnetic behaviour as evidenced from M-H curve with volume magnetic susceptibility (χv) 2.63 X 10-4. The decreased value in magnetic susceptibility is due to the incorporation of organic mass (Figure 2b) from the coated polymer. Colloidal stability study: Colloidal stability of the nanocomposites play crucial role in bioimaging and drug delivery applications. For practical application purpose nanocomposites should not agglomerate during biological study and must retain their colloidal stability for longer period. We have studied the colloidal stability of these nanocomposites in three different pH buffers, pH 5.0 (citrate buffer), 7.4 (phosphate buffer) and 10 (carbonate buffer) (Figure 6 and SI, Figure S6). Nanocomposites show excellent colloidal stability in all the conditions for more than three months with a little increase in their hydrodynamic diameter. Although in acidic pH stability is less, but it remains ~80-100 nm even after three months. Colloidal stability was also studied in cell culture medium (RPMI) which does not show any adverse effect on the stability (SI, Figure S7, S8). This is evidence from this fact that nonspecific interaction with the proteins have been avoided in our case due to nearly zwitterionic surface charge. However, colloidal stability of the nanocomposites decreases with the increase in surface folate concentration due to its hydrophobic nature. We have also studied the zeta potential of the functionalized sample. At pH 7.4 the zeta potential was observed ~17 mV which changes to -38 mV at pH 10.0 and ~10 mV at pH 5.0. This data supports the fact that more number of surface acid groups are exposed on the nanoparticle surface as compared to amine groups. Drug loading and release study: For drug delivery study we have used hydrophilic drug daunorubicin as model drug. We have chosen daunorubicin since it is a well studied drug and its biochemical activity has been tested. For specific drug delivery purposes, responsive delivery is the current state of the art. We have developed a redox responsive drug delivery system. Tumor cells have highly reducing environment due to higher concentration of reduced glutathione. To develop this drug delivery system daunorubicin is first modified with 13 ACS Paragon Plus Environment


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primary amine terminated disulfide bond (Scheme 1b). For this purpose, drug molecules are initially mixed with epichlorohydrin solution in DCM in a 1:1 molar ratio in presence of weak base triethylamine. Next, epichlorohydrin was added drop wise to the drug molecules to ensure 1:1 linkage with the drug molecules. Finally, DCM was evaporated and the modified drug molecules were collected as orange solid. Next, the epoxide modified drug molecules are treated with cysteamine dihydrochloride solutions. Here also we used 1:1 ratio although cysteamine dihydrochloride used in slightly less amount, so that all of the reagent is consumed. Here, cysteamine dihydrochloride was added drop wise to the modified drug molecules in DMSO. The reaction was continued for 6 hours. Finally the product has been extracted from diethyl ether. Drug loading to the nanocomposites was performed via EDC coupling between -COOH groups on nanocomposite surface and free primary amine groups of the drug molecules (Scheme 1). Change of zeta potential from ~-25 mV to ~-5 mV confirms the successful drug attachment on nanoparticle surface. To optimize the drug loading we have used large amount of modified drug molecules and incubated for 24 hours time under dark at ~20oC. After drug loading, the drug loaded samples were dialysed against fresh water to remove any free drug molecules. Drug attachment was confirmed via UVVisible and fluorescence spectroscopy (Figure 7a, b). In UV-Vis spectra (Figure 7a), drug loaded composite show broadened absorbance at ~485 nm and small hump at ~540 nm. This is characteristics of drug molecule DAUN, since pure DAUN has strong absorbance in ~480490 nm and weak absorbance at ~540 nm. In emission spectra (Figure 7b), upon excitation at 450 nm, a new peak at ~580 nm arises along with the fluorescein emission at ~520 nm. This new peak is due to the emission from drug molecules. Moreover, zeta potential measurement show surface charge ~-5 mV which is due to the unavailability of surface acid groups after drug conjugation. From UV-Visible spectroscopy loading efficiency has been calculated ~82.4 % whereas encapsulation efficiency has been found ~30 µg/mg of nanocomposite (SI, Figure S9). Drug release study was performed under reducing environment using GSH. GSH reduces the disulfide bond present between drug molecules and nanocomposites and subsequently drug release have been observed (Figure 7c and SI, Figure S10). All the experiments were performed under physiological pH. Without any added GSH, only ~15% drug release has been observed which was saturated within 24 hours time period. In presence of 1 mM GSH, ~70% drug release has been observed which is increased to ~82% in presence of 5 mM GSH. In both the cases maximum drug release has been observed in first 10 hours 14 ACS Paragon Plus Environment

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and then gradually saturated over 24 hours time period. This drug release was also monitored via DTNB test for free thiol (SI, Figure S11). In presence of glutathione, drug molecules gradually released leaving behind free thiol groups which has been detected via DTNB test for the same time period of drug release. This result also supports the redox responsive drug release profile. Cellular imaging and toxicity study: Gd3+ is a well known and efficient T1 contrast agent for MRI. We have tested our nanocomposite to evaluate its efficiency as T1 contrast agent in MRI via proton relaxation measurement. This measurement had been performed along with our previous samples of Eu doped Gd2O3 nanoparticles.26 Proton relaxation study has confirmed the high relaxivity value (r1) of 26.22 mM-1s-1 which is high enough to be utilized for clinical purposes (Figure S12). Since the composites have strong green emission originates from fluorescein molecules, they can be used as potential fluorescence imaging material. Cellular imaging was performed on KB cells, which is a well studied folate-positive cancer cell line. Folate modified drug loaded nanocomposites were treated to the KB cells in folate free RPMI media. Folate free RPMI media make sure that the folate receptors remain free for targeting by the drug loaded samples. After 2 hour incubation, cells were washed with PBS buffer and stained with Hoechst for nucleus staining. After further washing with a PBS buffer twice, the labelled cells were studied under a fluorescence microscope (Figure 8). Under UV excitation, blue emission from the stained nucleus was observed (Figure 8b). When we excite the same region with blue light, green emission from the nanocomposites were clearly visible (Figure 8c). After careful observation and merging the fluorescence images it is confirmed that the nanocomposites localizes near the perinuclear regions (Figure 8d). This is because of low folate density on nanoparticle surface, which critically modify the cellular internalization pathway to caveolae mediated endocytosis as previously reported.20,22 Drug delivery inside the cellular compartment has been confirmed from the imaging experiment. When the cells were excited in the green light, red emission from the drug molecules were observed (Figure 8e). Yellow emission from the merged fluorescence images confirm that the nanocomposites carry the drug molecules inside the cells (Figure 8f). This imaging experiment also provide information about the perinuclear targeting of the drug molecules. For detailed study of the redox responsive drug release, drug loaded nanocomposite treated cultured cells were mixed with 10 µM GSH solution and incubated for 10 hours (Figure 9). Red emission from released drug molecules have been observed to be spread throughout the cells with more concentration near the perinuclear region (Figure 9e). 15 ACS Paragon Plus Environment


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After merging with the fluorescence images of nanocomposites, less yellow spots have been observed (Figure 9f) as compared to initial study where no GSH have been used. This study confirms that in presence of GSH more drug molecules are released from the nanocomposites and spread over the cellular compartments with higher concentration targeting the nucleus. For control experiments, we have used CHO cells as folate negative cell line (SI, Figure S13). No cellular internalization of the folate modified composites have been observed. To confirm specific targeting of cancer cells, KB cells were treated with composite nanoparticles without any folate conjugation. No cellular internalization was observed. In another control experiment, KB cells were pre-treated with free folic acid (1 µM) which eventually blocks the folate receptors on KB cells and ends up with negligible cellular internalization (SI, Figure S13). To assess the cytotoxicity of the drug loaded samples, we have conducted MTT assay as a standard cytotoxicity evaluation tool (Figure 10). For this purpose, KB cells were treated with different concentrations of drug loaded sample and incubated for 24 hours. Bare nanoparticle (without drug) show minimum toxicity effect and almost 90% cell viability has been observed. Drug loaded sample has shown ~40-60% cell viability depending on drug concentration in presence of external GSH (10 µM). From the cytotoxicity results it can be confirmed that the multifunctional nanocomposites effectively carry drug molecules inside the cells leads to higher apoptosis. For better understanding the effect of redox responsive drug release we have incubated the KB cells and drug loaded nanocomposites for 24 hours without any external GSH solution. We have observed that ~20-25% less cell death in this case which strongly confirms the redox responsive drug release phenomenon (SI, Figure S14) in our sample. We have also carried out control experiments to conclude the cytotoxicity results with CHO cells in presence of external GSH. Discussion: In this work our target was to develop a multifunctional bioengineered magneticfluorescent nanocomposite for targeted, responsive drug delivery and multimodal imaging applications. We have chosen Gd2O3 nanoparticles as magnetic component. The major advantage of this oxide is its paramagnetic nature and strong T1 contrast in MRI which has been well documented and widely accepted. Moreover, Gd2O3 has low toxicity, high chemical stability and high neutron capture cross-section for neutron therapy.79,80 Although, various synthetic methods are available (sol-gel, co precipitation, microwave, hydrothermal etc.),79-85 shape and size controlled synthesis techniques are rare for this nanoparticles.26, 82-85 16 ACS Paragon Plus Environment

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Moreover, surface functionalization of this nanoparticle is less explored which impedes it's wide-spread applications in bio-medical field. Here, we have adopted a high temperature colloidal synthesis route, since the shape and size of the nanoparticles can be easily tuned in this method by changing the reaction parameters. We are particularly interested in the preparation of small rod shaped nanoparticles since rods have added advantages in cellular internalization due to its anisotropic shape.

26, 83, 86, 87

It has been reported by other research

groups that rod shaped nanoparticles aligned with the blood flow inside the body and have shown better internalization efficiency. For rod shaped nanoparticle synthesis, we have used metal precursor salt Gd-oleate since it decomposes at high temperature along with long chain fatty acid, oleic acid as capping ligand. At 300oC the salt decomposes which can be observed by the colour change of the solution. We have optimized the reaction for one hour since shorter or longer time unable to provide rod shaped nanoparticles. We have observed some small near-spherical shape nanoparticles for 30 minutes reaction time. For longer reaction time we have observed much smaller rods, which is due to rupture of the long rods (SI, Figure S15). These nanorods are hydrophobic in nature as it is capped by long chain fatty acid. For biological applications we need hydrophilic nanorods with good colloidal stability and proper bioengineered surface. We have adopted a well developed one-pot polyacrylate coating. The advantages of this coating approaches are-(1) small hydrodynamic diameter, (2) excellent colloidal stability, (3) controlled surface functional groups and (4) robust coating even under harsh environments. For controlled surface functional groups we have used varying ratios of amine and acid monomer. In this work, the ratio between amine and acid monomer used is 1:9. This step lowers the number of surface amine groups during coating to avoid unwanted cytotoxicity. However, on the other hand it also provides sufficient surface amine groups for further conjugation. This ratio also maintains zwitterionic surface charge which plays crucial role during cellular internalization to avoid non-specific interactions. Various magnetic-fluorescent nanocomposites have been reported and a large amount of research have been conducted in last two decades. To avoid the photobleaching of dye molecules, various fluorescent nanoparticles have been developed as an alternative which includes semiconductor quantum dots (CdSe/ZnS, CdS etc.), doped quantum dots, carbon nanoparticles, silicon nanoparticles, plasmonic nanocluster, graphene quantum dots, organic nanoparticles etc. However, they suffer from severe toxicity (quantum dots), photo blinking, poor quantum yield, unrestrained shape and size, uncontrolled surface properties, large 17 ACS Paragon Plus Environment


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Stoke's shift and broad emission spectrum. Recently we have developed Eu3+ doped Gd2O3 nanoparticles which can overcome these drawbacks although strong phonon coupling reduces the quantum yield of the doped nanoparticles.26 In this work we have chosen fluorescein dye molecules due to its advantages like high quantum yield, sharp emission, small Stoke's shift and low toxicity. To minimize the fluorescence quenching issue we have used fluorescein dye as a monomer during polymer coating. During random coating, fluorescein molecules distributed in the robust polymer backbone which eventually place the dye molecules at various distances from the core nanoparticle. Lifetime measurement confirms that dye molecules have 2 different average lifetimes in the composite as compared to single lifetime of its own.50 These different lifetimes can be justified from the fact that, the dye molecules closer to the core get quenched by non-radiative electron transfer and lowered the fluorescence lifetime. Whereas, the dye molecules closer to the surface suffers minimum nonradiative transfer and lifetime retains almost intact. This nanocomposite have also shown stability against photobleaching. We have investigated photobleaching property in solution upon continuous exposure to UV light for 3 hours (SI, Figure S16). The composite have shown excellent resistance to photobleaching and even after a month almost ~80% fluorescence remain intact (in solution). We believe this is due to the distribution of the fluorescence molecules in the polymer backbone. We have tested the potential of these composite nanorod as multimodal imaging materials as well as drug delivery vehicle simultaneously. For drug delivery purpose we have developed a redox responsive drug release vehicle by modifying the drug molecules and conjugating it with the nanocomposite surface. The disulfide bond present between nanocomposite and drug molecules gets cleaved by excess glutathione in tumor cells by thiol exchange method and subsequently loaded drugs get released. This is particularly important since the GSH content in normal cell is very less whereas in tumor cells it increases 100 folds.88-91 This reducing environment triggers the drug release in a sustained manner. Drug loading has been confirmed by UV-visible and photoluminescence spectroscopy. Both the fluorescein molecules on the composite nanorod and daunorubicin drug molecules absorbs in the same region which has been confirmed from the broad hump of drug loaded composite in UV-visible spectroscopy. The loading efficiency has been calculated from the difference in absorbance value at 490 nm of the composite sample before and after drug loading. High loading efficiency of ~82.4% have been found along with encapsulation efficiency of ~30 µg/mg of the sample. Drug release study in solution has been performed in the absence and 18 ACS Paragon Plus Environment

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presence of added GSH (1 mM and 5 mM). In the absence of GSH, only 15% drug release was encountered which is evident from the design of the delivery vehicle as the disulfide linkages are strong enough to cleave. Under reducing environment in presence of GSH, disulfide linkages cleave via thiol exchange method. If we increase the thiol content more and more drug will be released as evident from the release profile. Drug release phenomenon have also been confirmed inside cellular compartments by fluorescence imaging. Here we have taken the advantage of dual colour emission from the drug loaded nanocomposites-green emission from the composite itself upon excitation in blue region and red emission from the drug molecules upon excitation in green region. Cell nucleus was stained by Hoechst to understand the intracellular release and localization of the drug molecules. Careful observation of the merged images (Figure 8) confirmed that both the nanocomposite and drug molecules co-localize near the nucleus of the cell as evidenced from the yellow emission. To understand redox responsive release of drugs, cells were treated with GSH via culture media. Here we have noticed comparatively more red emission near the nucleus as well as throughout the cell (Figure 9). This is because of the release of more drug molecules under reducing environment. This data has also been supported by the cytotoxicity measurements. Furthermore, proton relaxation study confirms its potential as MRI contrast agent as expected due to the presence of seven unpaired electrons in Gd3+. The major advantage of this magnetic-fluorescent nanocomposites are-small hydrodynamic size, unique shape, superior colloidal stability, intact fluorescence properties, controlled surface functionality, high drug loading efficiency, responsive and targeted delivery along with multimodal imaging capability. Conclusion: In conclusion, we have developed Gd2O3-fluorescein based multifunctional magneticfluorescent composite nanorod for redox responsive drug delivery and multi modal imaging. We have synthesized small Gd2O3 nanorods via colloidal synthetic route and further surface engineered to provide optimum surface functional groups for drug loading via disulfide linkage. We have found ~82.4% drug loading on the composite nanorod and also observed redox responsive drug release kinetics in solution as well as in cellular compartments. The major advantages of this magnetic-fluorescent drug delivery vehicles are small hydrodynamic size, excellent colloidal stability, stability of fluorescence, high loading efficiency, responsive



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drug release and multimodal imaging capability which we believe can overcome the existing drawbacks in drug delivery applications. Acknowledgement: AS acknowledges Department of Science and Technology (DST), India for providing research grant (DST INSPIRE Faculty Research Grant, Project No. GAP 0364). Authors would like to thank Dr. N. R. Jana, Indian Association for the Cultivation of Science, for his kind assistance in cellular imaging experiments and Dr. P. Deb, Tezpur University for his kind assistance in proton relaxivity measurements. Supporting Information: More DLS graphs, control cellular images, drug loading and releasing calculations, more TEM images etc. are available in supporting information. Reference: 1.

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Monodisperse

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Page 29 of 40

(a) Oleic acid (OA) Gd-Oleate

3000C, ODE 1 hour Gd2O3 nanorod

(b)

Amine monomer Acid monomer PEG monomer

O

NH 2

O HN H2N

H2N

Fluorescein monomer Polyacrylate coating

N

O N H

N

N H

OH

O O N O O

N

NH 2 NH 2

Folic acid-NHS

Gd2O3 nanorod

NH2

Cystamine

SH O

O

OH

SS-

NH2

N H

O

SS-

High glutathione inside tumor cells Oleic acid

Fluorescein monomer

Drug molecule

Folic acid

S -S -

HO

O H N

SS-

Epichlorohydrin

EDC Coupling

S-S-NH2

O

S-S-

DAUN

SS

H2N

O

S-S-

O

SS-

Cl

OH

S-S-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Polymer coating

Scheme 1: (a) Schematic representation of Gd2O3 nanorod synthesis. (b) Schematic representation of surface functionalization, composite preparation, drug loading and responsive drug release of Gd2O3-Fl composite nanorod based drug delivery vehicle.


10000

(a)

Page 30 of 40

(b)

(c)

8000

0

10

20

30

440

2000

40

50

60

642

4000

622

222

6000

400

Intensity (a.u.)

70

2θ (Degree) (d)

2000

C

Gd Cu

Gd 25 atom% O 75 atom%

(e) 1500

(f)

Gd Gd

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

321


Gd

Gd

1000 4

Gd

6

Cu

8

10

Energy (keV) 500

Gd

(222)

Cu O Gd Cu Gd

(440)

Gd Gd

0 0

2

4

Gd Cu Gd Gd

6

Gd

Cu

8

10

Energy (keV)

Figure 1: (a) XRD pattern of as synthesized Gd2O3 nanorods which showing distinct crystal planes of cubic Gd2O3. TEM images (b-c) of the as synthesized nanorods in different magnification exhibits monodisperse nature of the nanorods. (d) HRTEM (e) SAED pattern and (f) elemental analysis of the as synthesized nanorods confirmed the formation of phase pure Gd2O3 nanorods.


1.0

Absorbance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a) 0.8 0.6 0.4 0.2 0.0

300

400

500

600

Moment, M (emu/gm)

Page 31 of 40

6

(b) 4

-4

χv=3.95 X 10

2 -4

χv=2.63 X 10

0 -2 -4

Hydrophobic Hydrophilic

-6 -10000 -5000

Wavelength (nm)

0

5000

10000

Field, H (Oe)

Figure 2: (a) UV-visible spectra of as synthesized Gd2O3 nanorods in chloroform which exhibits a single peak at ~275 nm due to f-f electronic transition of Gd3+. (b) Magnetic measurement of both as synthesized and hydrophilic Gd2O3 nanorods exhibit paramagnetism.



(a)

(c)

(b)

1600

(e)

(d) Gd 10 atom% O 90 atom%

1200

Cu Gd

Number (%)

C

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

800 O

400

Cu Cu O Cu Gd Gd

Cu Gd Gd Gd

0 0

Page 32 of 40

2

4

6

8

10

0

20

Energy (keV)

40

60

80

100

Size, d (nm)

Figure 3: (a-c) TEM images of hydrophilic Gd2O3-Fl composites in different magnification exhibit that the nanorods retain their shape and size after composite preparation. (d) Elemental analysis confirms the presence of Gd with higher atomic percent of O. (e) DLS size distribution of the hydrophilic composite nanorods in pH 7.4 which is showing small hydrodynamic diameter (relative standard deviation is less than 0.7% and PDI value is 0.45).


Page 33 of 40

5

(a)

1.2

2.5x10

Only fluorescein Hydrophilic Gd2O3-Fl composite

PL intensity (a.u.)

1.4

Absorbance (a.u.)

1.0 0.8 0.6 0.4 0.2 0.0 300

400

500

600

700

800

(b)

5

2.0x10

5

1.5x10

5

1.0x10

4

5.0x10

0.0 450

550

650

750

Wavelength (nm)

Wavelength (nm) 5000

PL intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Decay Fit

(c)

4000 3000 τ1= 1.56 ns (12.82%) τ2= 4.09 ns (87.18%)

2000 1000 0

-8

1.0x10

-8

2.0x10

-8

3.0x10

Time (s) Figure 4: (a) UV-visible spectra of Gd2O3-Fl composite nanorods which exhibits a peak ~480 nm due to the absorbance of fluorescein molecules. UV-Vis spectra of free fluorescein molecules has also been recorded. (b) Fluorescence spectra of the composite nanorods showing strong emission at ~520 nm upon excitation at 450 nm due to the fluorescein molecules. (c) Lifetime measurement of Gd2O3-Fl composite nanorods showing biexponential fitting with two different average lifetime.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

% T (a.u.)


Page 34 of 40

1719 cm-1 556 cm-1 857 cm-1

3431 cm-1

1105 cm-1

1570 cm-1 2926 cm-1

1253 cm-1 1491 cm-1

400

1300

2200

3100

Wavenumber (cm-1)

4000

Figure 5: FTIR spectra of surface functionalized magnetic-fluorescent Gd2O3-Fl composite nanorods. The spectra shows all the representative peaks of the surface functional groups.


Page 35 of 40

(a)

After 3 days

(b)

Number (%)

Number (%)

Initial

0

20

40

60

80

0

100

20

After 7 days

(c)

40

60

80

100

Size, d (nm)

Size, d (nm)

After 30 days

(d)

Number (%)

Number (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

20

40

60

80

100

0

20

Size, d (nm)

40

60

80

100

120

Size, d (nm)

Figure 6: Colloidal stability study of Gd2O3-Fl composite nanorods in pH 7.4 (phosphate buffer) at different time intervals, initial (a), after 3 days (b), 7 days (c) and 30 days (d). The DLS size distribution exhibits excellent colloidal stability of the nanorods. All the measurements exhibit relative standard deviation between 0.3% to 0.7%.PDI value has been found between 0.2-0.4.



(a)

0.8

Hydrophilic Gd2O3-Fl composite Drug loaded Gd2O3-Fl composite Only drug

0.6 0.4 0.2 0.0 350

450

550

650

5

PL intensity (a.u.)

1.0

750

3.0x10

Hydrophilic Gd2O3-Fl composite

(b)

Drug loaded Gd2O3-Fl composite

5

2.5x10

Only drug

5

2.0x10

5

1.5x10

5

1.0x10

4

5.0x10

0.0 450

550

650

750

Wavelength (nm)

Wavelength (nm) 100

5 mM GSH

(c)

(%) Release

Absorbance (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 40

80 1 mM GSH

60 40

0 mM GSH 20 0 0

5

10

15

20

25

Time (h)

Figure 7: (a) UV-visible and (b) fluorescence spectra to confirm drug attachment on Gd2O3Fl nanorod composite. (c) Drug release profile with time in absence and in presence of glutathione. It is evident from the drug release profile that drug release from this system is redox-responsive. All the release data has been recorded five times and relative standard deviation has been found less than 1%.


Page 37 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a)

(b)

(c)

(d)

(e)

(f)

Figure 8: Cellular imaging study of KB cells with drug loaded Gd2O3-Fl composite nanorods in folate free RPMI media after 2 hours incubation. (a) bright field image, (b) images of the same region under UV excitation. Blue emissions are coming from the nucleus due to Hoechst staining. (c) images under blue excitation. Green emissions are coming from the fluorescein molecules of the composites. (d) merged image of b and c, (e) images under green excitation. Red emissions are coming from the Daunorubicin drug molecules. and (f) merged image of b, c and e confirms cellular internalization of the drug loaded samples. Yellow colour represents co-localization of composite (green emission) and drug molecules (red emission). (Scale bar 100 µm).



Page 38 of 40

(a)

(b)

(c)

(d)

(e)

(f)

Figure 9: Cellular imaging study of KB cells with drug loaded Gd2O3-Fl composite nanorods in folate free RPMI media in presence of 10 µM GSH after 10 hours incubation. (a) bright field image, (b) images of the same region under UV excitation. Blue emissions are coming from the nucleus due to Hoechst staining. (c) images under blue excitation. Green emissions are coming from the fluorescein molecules of the composites. (d) merged image of b and c, (e) images under green excitation. Red emissions are coming from the Daunorubicin drug molecules. and (f) merged image of b, c and e confirms thiol responsive drug release inside the cellular compartments. Yellow colour represents co-localization of composite (green emission) and drug molecules (red emission). (Scale bar 50 µm)


Page 39 of 40

120

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100 80 60 40 20 0

1

2

3

4

1

2

3

4

1

2

3

4

Figure 10: Cytotoxicity study of the Gd2O3-Fl nanorod composites before and after drug loading. Cytotoxicity studies confirm minimum toxicity of the bare nanoparticles whereas in presence of drug molecules KB cells are least viable in presence of external GSH (10 µM). It also confirms minimum effect of drug loaded samples on normal cell line (CHO) even in the presence of external GSH (10 µM). Varying drug concentrations under study are 125 µg/ml sample with 3.75 µg/ml daunorubicin (1), 250 µg/ml sample with 7.5 µg/ml daunorubicin (2), 375 µg/ml sample with 11.25 µg/ml daunorubicin (3) and 500 µg/ml sample with 15 µg/ml daunorubicin (4).



S S S

S

SH

SH S S

GSH

SH

SH SH

SH

S

Drug Fluorescein 100

Drug Release 80

% Release

S S

S

TOC

S

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 40

With GSH

60 40

Without GSH 20 0 0

6

12

18

24

Time (hours)


Surface Functionalized Multifunctional Gd2O3-Fluorescein Composite

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