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PEGylated Carbon Nanocapsule: A Universal Reactor and Carrier for In Vivo Delivery of Hydrophobic and Hydrophilic Nanoparticles Amritha Rammohan, Gargi Mishra, Binapani Mahaling, Lokesh Tayal, Ahana Mukhopadhyay, Sanjay Gambhir, Ashutosh Sharma, and Sri Sivakumar ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08885 • Publication Date (Web): 08 Dec 2015 Downloaded from http://pubs.acs.org on December 15, 2015

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ACS Applied Materials & Interfaces

PEGylated Carbon Nanocapsule: A Universal Reactor and Carrier for In Vivo Delivery of Hydrophobic and Hydrophilic Nanoparticles Amritha Rammohan1#, Gargi Mishra1#, Binapani Mahaling1, Lokesh Tayal1, Ahana Mukhopadhyay1, Sanjay Gambhir2*, Ashutosh Sharma1* and Sri Sivakumar(1, 3)* #

Authors have contributed equally to this work

1

Department of Chemical Engineering, Centre for Environmental Science & Engineering,

Thematic Unit of Excellence in Soft Nanofabrication, Indian Institute of Technology, Kanpur, UP-208016, India 2

Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, UP, India

3

Materials Science Programme, Indian Institute of Technology, Kanpur, UP-208016, India

E mail: [email protected], [email protected], [email protected]

ACS Paragon Plus Environment

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Abstract We have developed PEGylated mesoporous carbon nanocapsule as a universal nanoreactor and carrier for the delivery of highly crystalline hydrophobic/hydrophilic nanoparticles (NPs) which shows superior biocompatibility, dispersion in body fluids, good biodistribution and NPs independent cellular uptake mechanism. The hydrophobic/hydrophilic NPs without surface modification were synthesized in-situ inside the cavities of mesoporous carbon capsules (200850 nm). Stable and inert nature of carbon capsules in a wide range of reaction conditions like high temperature and harsh solvents, make it suitable for being used as nano/micro-reactors for the syntheses of a variety of NPs for bio-imaging applications, such as: NaYF4:Eu3+(5%), LaVO4:Eu3+(10%), GdVO4:Eu3+(10%), Y2O3:Eu3+(5%), GdF3:Tb3+(10%), Mo, Pt, Pd, Au and Ag. Multiple types of NPs (Y2O3:Eu3+(5%) (hydrophobic) and GdF3:Tb3+(10%) (hydrophilic)) were co-loaded inside the carbon capsules to create a multimodal agent for magneto-fluorescence imaging. Our in vivo study clearly suggests that carbon capsules have biodistribution in many organs including liver, heart, spleen, lungs, blood pool and muscles.

KEYWORDS: theranostics, nano/microreactor, biodistribution, multimodal-imaging, carbon capsules, Ln3+ doped nanoparticles


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1. Introduction Nanoparticles (NPs) as bioimaging agents have greatly advanced the speed, sensitivity, precision and resolution of bioimaging approaches.1 The efficient delivery of NPs to desired intracellular sites crossing all the physiological barriers is important to exploit their imaging potential. However, efficient delivery of NPs (hydrophobic and hydrophilic) faces several challenges: (1) Hydrophobic NPs due to their poor dispersibility in water show agglomeration in aqueous biological media, adsorption of proteins and early clearance from the body by the macrophage phagocytic systems despite having superior imaging properties.2 (2) Hydrophilic NPs overcome the above mentioned shortcomings however; they have inferior imaging properties (e.g. fluorescence and magnetism) and hindered intracellular delivery because of their decreased affinity to lipid membranes.3-10 (3) Furthermore, coating the surface of hydrophobic NPs with hydrophilic biocompatible materials (core-shell structure) such as silica, polymers and biomolecules (e.g. proteins, lipids etc.) may quench and alter the imaging properties (e.g. fluorescence and magnetic properties) due to the interface formation between the core and shell material.11-25 (4) Both hydrophobic and hydrophilic NPs have to meet certain additional challenges such as biocompatibility, efficient biodistribution and intracellular delivery. (5) Different NPs follow different cellular uptake mechanisms which leads to the their unique and different localizations within the cell.26 NPs reach specific intracellular sites depending upon their cellular uptake pathway which is determined by their surface interactions with cell membrane and it makes it essential to investigate the behavior of each particle separately.27 Thus, it is desirable to tailor a platform which enables internalization of a variety of NPs and their combinations by a nanoparticle-independent, predictable mode. Hence, it is substantial and attractive to develop a universal platform to deliver crystalline hydrophobic/hydrophilic NPs


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while preserving the desired properties of dispersion without agglomeration, reducing biomolecular adsorption, possessing superior biocompatibility, nanoparticle independent uptake mechanism of nanoparticles cargo and efficient biodistribution. We show that these often conflicting requirements can be met by in-situ encapsulation of NPs inside a biocompatible porous nano/micro capsule with an antifouling coating. Specifically, we demonstrate a versatile approach to deliver hydrophobic as well as hydrophilic functional

NPs

such

as

NaYF4:Eu3+(5%),

LaVO4:Eu3+(10%),

GdVO4:Eu3+(10%),

Y2O3:Eu3+(5%), GdF3:Tb3+(5%), Pt, Pd, Au and Ag by their in-situ syntheses in the amorphous carbon nano/microcapsules as nano/microreactors and NPs carriers. These nanocarriers are rendered hydrophilic by polyethylene glycol (PEG) grafting on its surface, which improves dispersion in biological media, biocompatibility, stealth, and reduces biomolecular adsorption. The developed delivery platform has the following advantages: (1) tunable size; (2) functionalized surface (e.g. anti-IgG antibody and PEG grafted); (3) stability under harsh conditions (thermo stable, tolerates wide pH range and stable in various solvents) in contrast to existing encapsulation approaches (e.g., liposomes, polymeric microcapsules, polymeric hydrogel capsules,28 polyelectrolyte based layer-by-layer assembly capsules, microemulsions,29 DNA capsules and dendrimers);30 (4) a wide range of in-situ synthesized encapsulated NPs (e.g. NaYF4:Eu3+(5%), LaVO4:Eu3+(10%), GdVO4:Eu3+(10%), Y2O3:Eu3+(5%), GdF3:Tb3+(5%), Mo, Pt, Pd, Au and Ag) and (5) simultaneous encapsulation of hydrophobic/hydrophilic imaging agents (e.g. GdF3:Tb3+ and Y2O3:Eu3+ NPs) bestowing a true multimodal capability in contrast to other platforms such as heterodimers, core-shell approaches, etc. which are limited to one or two modules. Further, this approach requires merely the modification of carbon capsules surface for biological applications which is independent of the encapsulated NPs, which is in contrast to the


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existing approaches (e.g. ligand exchange reactions, surface-modification with biomolecules, core-shell approach, etc.).31-32 Carbon capsules also minimize direct nanoparticle/bio interfaces in blood which in turn reduces bio-physicochemical interactions and colloidal forces experienced due to suspended proteins, vesicles, cells and membranes. This can minimize the biomacromolecular coating on the surface of NPs which can also reduce the loss in the imaging properties in biological media. It is to be noted that bare carbon capsules have been checked for in vivo biocompatibility and found to be suitable for intravenous delivery.33 Although encapsulation vehicles such as silica and TiO2 microcapsules also possess some of the above advantages (e.g. stability under harsh conditions), they have not been explored for bioimaging application so far.34-35 Apart from the above material advantages, PEGylated carbon capsules co-loaded with Y2O3:Eu3+(5%) and GdF3:Tb3+(5%) addresses the issue of the targeting single intracellular site with dual functionality (fluorescence and magnetism) with desirable biocompatibility and in vivo biodistribution in majority of organs (heart, bone marrow, lungs, liver, spleen etc.). Our preliminary in vitro degradation studies show that PEGylated carbon capsules are biodegradable. Thus, the developed delivery platform is a promising system for safe delivery of contrast agents for multimodal imaging for in vitro and in vivo applications. 2. Experimental section 2.1 Materials Tetraethyl orthosilicate (TEOS), n-octadecyltrimethoxysilane (C-18 TMS), europium chloride hexahydrate (EuCl3.6H2O), lanthanum nitrate hydrate (La(NO3)3.xH2O), europium nitrate pentahydrate (Eu(NO3)3.5H2O), gadolinium nitrate hexahydrate (Gd(NO3)3.6H2O), terbium nitrate pentahydrate (Tb(NO3)3.5H2O), yttrium acetate hydrate (CH3COO)3Y.xH2O, triethyl amine (TEA), oleylamine,

bis-amino polyethylene glycol (6000 kDa), stannous chloride


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(SnCl2), fluorescein isothiocyanate-phalloidin (FITC-phalloidin), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), trypsin-EDTA, Dulbecco’s-modified eagle’s medium (DMEM),

2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride

(DAPI),

penicillin-

streptomycin antibiotic, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), filipin III, chlorpromazine, cytochalasin, rottlerin, genistein, anti-mouse IgG-FITC (antibody produced

in

rabbit),

N-hydroxysuccinimide

(NHS),

N-(3-Dimethylaminopropyl)-N′-

ethylcarbodiimide hydrochloride (EDC), myeloperoxidase enzyme, phosphate buffer saline (PBS) powder packets, gelatin (from cold water fish skin) and Gd standard were purchased from Sigma Aldrich. Sulphuric acid, nitric acid, ethanol and dimethyl sulphoxide (DMSO) were obtained from Merck Chemicals, India. Hydrofluoric acid (HF), nitric acid (HNO3) and hydrogen Peroxide 30% (H2O2) were obtained from S.D. Fine Chem. Ltd. and ammonium fluoride (NH4F) and oleic acid was obtained from Loba Chemie, India. Sodium chloride, sodium hydroxide (NaOH pellets), methanol, acetone, chloroform, cyclohexane, hydrogen peroxide (40% v/v) and aqueous ammonia (25% v/v) were purchased from Fisher Scientific. BCA protein assay kit was purchased from Thermo Scientific. All the above chemicals were used as received. HeLa, HepG2, A498 and L929 cell lines were purchased from National Centre for Cell Science, Pune, India. MM418 cell line was gifted by Dr. Andrew Jackson, Division of translational Oncology, Nottingham University. 99mTc was obtained from Isorad Ltd. 2.2. Preparation of carbon capsules Carbon capsules were prepared by adapting a silica template based synthesis protocol.36-37 In a flask, absolute ethanol (35 ml), de-ionized water (5 ml) and aqueous ammonia (25%, w/v) (1.5 ml) were mixed followed by the addition of TEOS (4.5 ml). A mixture of TEOS (4 ml) and C-18 TMS (2 ml) were added drop-wise to this solution and this solution was incubated for 45 min.


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After 45 min, the solvent was removed at 60 °C under vacuum using rotary evaporator. The obtained white powder was dried overnight at 100 °C. 1 g of this powder was treated with H2O2 (30%, w/v in water) at room temperature for 36 h. The product was re-dispersed in a mixture of 9 ml of H2O and 1 ml of conc. H2SO4 and heated in air at 100 °C for 5 h and after which the temperature was raised to 160 °C for 15 h. The dark solids obtained were pyrolyzed in a high temperature muffle furnace (Eurotherm) under N2 atmosphere at 800 °C with a ramp rate of 3 °C/min for 1 h. The carbonized sample was etched by hydrofluoric acid (HF) (48%, w/v) and washed five times in water to remove excess HF. The above protocol leads to the formation of capsules of ~200 nm size. Silica templates of different sizes can be made by changing the precursor’s concentrations; various precursor concentrations utilized for the size tuning has been reported in supporting information (Table S1).

2.3 Synthesis of NPs-loaded carbon capsules 2.3.1 GdF3:Tb3+(10%)-loaded carbon capsules The synthesis protocol for the synthesis of GdF3:Tb3+(5%) is adapted from a report.38 Citric acid (0.3 M) was prepared and the pH was adjusted to 6 by the adding few drops of ammonium hydroxide solution. Carbon capsules (10 mg) were dispersed in citric acid solution (900 µl, 0.3 M) and it was heated up to 70 °C. Methanolic solution of gadolinium nitrate hexahydrate salt (500 µl, 0.52 M) and terbium nitrate pentahydrate (500 µl, 52 mM) were added to above reaction mixture and stirred for 15 min. NaF (100 µl, 0.75 M) was added to the above mixture at 70 °C and kept under stirring for 2 h. The reaction mixture was cooled to room temperature and the NPs-loaded carbon capsules were separated by centrifugation (6500 rpm) followed by repeated washings with water to remove the excess un-encapsulated NPs and reactants.


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2.3.2 Y2O3-:Eu3+(5%) loaded carbon capsules The synthesis protocol for the synthesis of Y2O3:Eu3+(5%) is adapted from a report.39 Carbon capsules (25 mg) were dispersed in oleylamine (10 ml) under sonication for 10 min and the mixture was degassed at 70 °C for 10 min. Yttrium acetate (0.15 M) and europium nitrate (7.5 mM) were added to the above mixture and heated at 310 °C for 30 min under reflux conditions. The reaction mixture was cooled to room temperature and the NPs-loaded carbon capsules were separated by centrifugation (6500 rpm). The NPs-loaded carbon capsules were washed by hexane to remove excess un-encapsulated NPs and reactants. 2.3.3 Y2O3:Eu3+(5%) and GdF3:Tb3+(10%) co-loaded carbon capsules To perform co-loading of NPs inside carbon nanocapsules, synthesis protocol mentioned in the experimental section 2.3.2 was followed. After this first step, the prepared Y2O3 NPs-loaded carbon capsules were dispersed in aqueous solution of citric acid (10 ml, 0.3M) (pH 6, is adjusted by adding NH4OH) and heated at 75 °C. Aqueous solution of lanthanum nitrate (500 µl, 0.1 M), terbium nitrate (500 µl, 10 mM) were added into the above mixture followed by the addition of aqueous solution of sodium fluoride (4 ml, 0.5 M) and heated at 75 °C for 2 h. The reaction mixture was cooled and centrifuged at 6500 rpm for 3 min. The precipitate was washed with water to remove all un-encapsulated NPs. The above precipitate was again dispersed in aqueous citric acid solution (5 ml, 0.3 M at pH 6) at 75 °C followed by drop wise addition of aqueous solution of gadolinium nitrate (2 ml, 0.2 mmol) and stirred for 5 min. The above reaction mixture was cooled to room temperature and the NPs-loaded carbon capsules were separated by centrifugation (6500 rpm). Carbon capsules loaded with both yttrium oxide and gadolinium fluoride NPs washed with ethanol and water to remove the un-encapsulated NPs and reactants and dried.


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Synthesis protocols of carbon capsules loaded with NaYF4:Eu3+(5%), LaVO4:Eu3+(10%) and GdVO4:Eu3+(10%), Mo, Pt, Pd, Au and Ag are mentioned in supplementary information 2.4 PEG modification of carbon capsules Carbon capsules (5 mg) were suspended in a mixture of H2SO4 (7.5 ml, 0.1 M) and HNO3 (2.5 ml, 0.1 M) for 30 min in order to obtain –COOH groups on carbon surface. The carbon capsules were centrifuged (6500 rpm) and washed several times with water. Carbon capsules were further treated with thionyl chloride (0.2 ml) in DMF (4 ml) at 120 °C for 2 h under stirring condition. Bis-amino PEG (6000 kDa) (5 mg) was added to the above mixture solution at 120 °C and allowed to stirred for 24 h. The above reaction mixture was cooled to room temperature and PEG functionalized carbon capsules were separated by centrifugation (6500 rpm). These functionalized carbon capsules were washed in ethanol and dried at room temperature. After PEGylation, the carbon capsules encapsulated NPs and free NPs were compared via FTIR to check any change in the surface ligands of the NPs.

2.5 Characterization of carbon capsules and nanoparticle-loaded carbon capsules The carbon capsules and NPs-loaded carbon capsules were characterized by Scanning Electron Microscopy (SEM) (SUPRA 40 VP Gemini, Zeiss, Germany) at an accelerating voltage of 10 kV, Transmission Electron Microscopy (TEM) (FEI Technai G2 U-Twin) at 200 kV and X-Ray Diffraction (XRD) (Thermo Electron diffractometer) operating in the θ–2θ Bragg configuration using Cu Kα at 40 kV radiation (λ=1.5406 Å) from 5° to 80° at 2o min-1 scan rate to understand the size, morphology and crystalline nature of the capsules, respectively. For the SEM analysis sample (500 µg) was dispersed in water (1 ml) and sonicated for 5 min, 10 µl of sample was spread on a cleaned silicon substrate and dried in vacuum desiccator for 30 min. For TEM


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analysis, sample (100 µg) was dispersed in water (1 ml) and sonicated for 5 min and sample (10 µl) was loaded on a carbon coated TEM grid (mesh size 200) and grids were dried for 24 h in a vacuum desiccator. For XRD characterization, the dried powder samples (1 mg) were taken and analyzed. Additionally, samples containing Eu3+, Tb3+ ions were characterized by fluorescence spectroscopy. For fluorescence analysis, powder samples (100 µg) were dispersed in ethanol and sonicated for 10 min and analyzed using fluorescence spectrometer (Edinburgh instruments FLSP 920 (185-2600 nm)) equipped with double monochromator, 450 W Xenon lamp excitation source, and Peltier element cooled Hamamatsu R928-P PMT detector. The luminescence lifetime was estimated by recording the decay curve using a 100 W micro flash lamp (µF920H) as the excitation source. Emission scans were done from laser source with VIS/NIR detector using filter at 515 nm. Tb3+ doped NPs were excited at 488 nm, Eu3+ doped NPs were excited at 464 nm. MultiSkan UV-Vis spectrometer was used for photospectrometric analysis, Olympus Ix81 motorized inverted microscope and Zeiss LSM 710 confocal microscope were used NPs and cells imaging. 2.6 In vitro degradation of Y2O3:Eu3+(5%) and GdF3:Tb3+(10%) co-loaded PEGylated carbon capsules PEGylated carbon capsules (1 mg/ml) were incubated with myeloperoxidase enzyme (50 U/ml) in PBS (300 µl) under stirring conditions in the presence of 10 mM of H2O2 at 37 °C for 40 h. The reaction mixture was supplemented with 25 U/ml of enzyme after every 24 h period. Control experiments were performed with 10 mM of H2O2 and myeloperoxidase enzyme (50 U/ml) separately to investigate the effect of peroxidase enzyme activity on the PEGylated carbon


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capsules. Samples were analyzed using UV-Vis absorption spectroscopy at different time intervals to find out the % degradation.

2.7 Cytotoxicity assay, uptake studies and inhibitor studies Cells were cultured in DMEM medium containing FBS (10%, v/v) and penicillin/streptomycin (1%, v/v) at 37 °C in an atmosphere containing 5% CO2. For MTT cytotoxicity assay, 104 cells were seeded in each well of the 96 well plates and incubated for 6 h in a CO2 incubator. After 6 h, carbon capsules dispersed in complete media were added to each well with different concentrations (50, 100 and 200 µg/ml). A negative control was taken which contained cells only. After 18 h, cells were washed with PBS, trypsinized and centrifuged at 2000 rpm for two min; the supernatant was discarded to remove residual carbon capsules and its aggregates to avoid false reading which might come because of scattering by carbon capsules. Cells were added to 96 well plates again and MTT dye (500 µL, 0.5 mg/ml) dissolved in basal media was added to each well. After 4 h, media was gently removed from the wells and 200 µl of dimethyl sulphoxide (DMSO) was added to each well. The absorbance of the purple solution was measured after 30 min using a multi-plate reader in MultiSkan UV-Vis spectrometer at 570 nm. All readings were taken in triplicate. For study of cellular uptake of the carbon capsules, cells (104) were seeded in 24 well plates and culture media was added to it. After 4 h, NPs loaded carbon capsules (100 µg/ml) were added to each well. After 18 h, culture media was removed and the actin cytoskeleton were stained with FITC-phalloidin (50 µg/ml). Nuclei were stained using DAPI (10 µg/ml) for 10 min. The staining protocols were used as provided by the suppliers.


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For inhibitor studies, 104 HeLa and MM418 cells were seeded in each well of 24 well plates. After adhesion of cells to the substrate, different concentrations of inhibitors dissolved in serum free DMEM media were added to each well and incubated for 1 h. Concentrations of different inhibitors used were as follows: filipin III: (5 µg/ml), chlorpromazine (10 µg/ml), cytochalasin (10 µg/ml), rottlerin (25 µg/ml), genistein (100 µM).40 After 1 h, the media containing inhibitors was removed and fresh culture media was added to each well. RITC labeled Y2O3/GdF3 coloaded PEGylated carbon capsules (100 µg/ml) were added in each of these wells and incubated for 4 h. Cells of each well were fixed with formaldehyde (1 ml, 4%), stained with FITCphalloidin (100 µl, 50 µg/ml) and imaged using confocal fluorescence microscopy. Control experiments were done to confirm that the required inhibition was achieved at the used concentrations of inhibitors, when possible. It was checked by the inhibition of transport of transferrin and cholera toxin by chlorpromazine, genistein and filipin-III, respectively. Cells were exposed to chlorpromazine (10 µg/ml), genistein (100 µM) and filipin III (5 µg/ml ) for 1 h followed by incubation with FITC labeled transferrin (100 µg/ml) for 20 min and FITC labeled cholera toxin (100 µg/ml) for 30 min. Cells were fixed with formaldehyde (1 ml, 4%), and imaged using confocal fluorescence and bright field microscopy to count the number of cells with internalized transferrin and cholera toxin. 20 different frames were chosen to count the number of cells showing capsules uptake and a number average was taken. All the image analyses were done in triplicates for each sample. All the cell culture experiments were repeated three times independently to check the reproducibility of the results.

2.8 Antibody attachment and Antibody loading analysis on PEGylated carbon capsules Anti-mouse IgG-FITC (whole molecule) was attached to the carbon capsules surfaces using EDC-NHS coupling.41 PEGylated carbon capsules (2 mg) were ultra-sonicated in water (5 ml)


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and suspended in MES buffer (1 ml, 500 mM) (pH 6.1) followed by addition of NHS (2.3 ml, 50 mg/ml) and EDC (1.2 ml, 10 mg/ml) at room temperature and incubated for 30 min. This reaction mixture was centrifuged at 6500 rpm and washed in excess MES buffer (50 mM) and dried. Carbon capsules were re-suspended in MES buffer (9 ml, 50 mM) containing Ig-G antibody (10 µg/ml) (pH 6.1) and stirred at 150 rpm for 1 h. Finally, the antibody modified capsules were centrifuged out at 6500 rpm and washed thrice with MES buffer (50 mM) and dried. The amount of antibody attached to the surface of each capsules was calculated by BCA assay using BCA protein assay kit (Thermo Scientific) and the protocol mentioned by the manufacturer was used. A standard curve of antibody concentration vs absorbance at 562 nm was plotted to find out antibody concentration of unknown samples. 2.9 Radiolabeling and In vivo PET based Biodistribution studies PEGylated carbon capsules were labeled with

99m

Tc using coordination bonding. PEGylated

carbon capsules were dispersed in water (0.15 ml) and filtered through 30 µm filter followed by addition of

99m

Tc in presence of SnCl2 (80 µl/ml) for 30 min at room temperature. The binding

was confirmed by thin layer chromatography. All animal experiments methods and protocols were approved (Institutional Animal Ethics Committee approval number (CDRI/IAEC/2012/17) and conducted according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA 34/199). Male Sprague Dawley (SD) rats (200 ± 20 g) were utilised for the studies. Rats were obtained from the National Laboratory Animal Centre, CSIR-CDRI and were kept in a 12 h light–dark cycle, with controlled temperature (22–24 °C) and humidity (50-60%) and free access to standard rodent food and water. For biodistribution, male SD rats (n=5) were given


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anesthesia using ketamine and xylazine in the ratio (1:1). Samples with initial radioactivity of 0.493 mCi were taken for intravenous injection. Rats were injected with 500 µl of 99mTc labeled PEGylated carbon capsules and PET scan was done in prone and supine positions. Whole body scans were taken at 2X zoom by Infenia Hawkeye-4 after 30 min. 2.10 In vivo Pharmaco-kinetics studies For biodistribution and pharmacokinetics study, 8 weeks old C57BL/6J 15 mice were obtained from Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur’s (IITK) animal house. Animals were kept under 12 h light/dark conditions at 25 °C. Animals were fed with normal diet. The experiment was approved by institute ethical committee, IITK. 100 µL of PBS to control animals and 100 µL of PBS containing 40 µg nanoparticles to experimental animals was administered intravenously. Animals were sacrificed at 1 h, 6 h and 24 h time points using asphyxiation. Blood was collected from the heart immediately after sacrificing the animal and kept in ice followed by storing in -80 °C for further studies. The tissue processing and ICPMS study was performed by modified method as described previously.42 For ICPMS studies 35 mg of blood was dried in oven at 80 °C for 24 h in 30 ml glass vials. The samples were then treated with 6 ml of HNO3 and H2O2 (3:1) at 120 °C for 24 h. The samples were then diluted with 25 times in MQ water. Gadolinium was quantified from a standard curve plotted at a range of 0.05 to 5ppm in 2% HNO3 using ICPMS (ICPMS (Agilent 7900).

3. Results and Discussion 3.1 Fabrication of NPs loaded carbon capsules and surface modification Mesoporous hollow carbon capsules in the size range of 200-850 nm were prepared by a template-based synthesis adapted from an earlier approach.37 A capsule of diameter ~650 nm


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possesses the shell thickness of ~140 nm. SEM images of carbon capsules of various sizes (180– 850 nm) are shown in Figure 1 which shows that the size of the carbon capsules can be tailored for desired applications. We have been able to synthesize carbon capsules of sizes ranging from 180-850 nm by varying the concentration of silica precursor, solvent concentration and catalyst (ammonia) (Table S1).

Figure 1: Scanning electron micrographs of carbon capsules of various sizes. (a) 180-200 nm (b) 550-570 nm (c) 600-620 nm (d) 580-600 nm (e) 360-380 nm (f) 450-470 nm (g) 280-300 nm (h) 200-220 nm (i) 850-870 nm The as-prepared carbon capsules were found to be dispersible in a variety of solvents such as ethanol, methanol, oleic acid, water, acetone, cyclohexane, n-hexane, chloroform, etc. which allows synthesis of a wide variety of NPs in the hollow core. Schematic representation for fabrication of nanoparticle-loaded carbon capsules is shown in scheme 1.


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Scheme 1: Synthesis of nanoparticle-loaded PEGylated carbon capsules

The hollow core mesoporous shell carbon capsules were incubated with the NPs precursors along with solvents at required reaction conditions for loading different NPs. The precursors diffused into the hollow cavity of carbon capsules through the pores and the NPs were formed inside the core of the capsules. The synthesized NPs remain trapped inside the capsules because they were larger than the shell’s average pore size (3 nm ± 1 nm; Figure S1). NPs-loaded carbon capsules were separated from the NPs formed outside the capsules by centrifugation followed by repeated washing steps. We have synthesized a variety of hydrophobic (NaYF4:Eu3+, LaVO4:Eu3+, GdVO4:Eu3+, Y2O3:Eu3+, Pt, and Pd) as well as hydrophilic (Au, Ag and GdF3:Tb3+) NPs inside carbon nanocapsules illustrating their generic and versatile nature to function as nanoreactors (Figure 2, TEM images). In order to load multi-functional NPs having two different imaging functionalities (magnetic and fluorescence imaging), Y2O3:Eu3+ NPs-loaded carbon capsules were synthesized in the first step and then incubated with precursor solution of GdF3:Tb3+ at required conditions for co-loading as shown in the scheme 1.


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In order to increase stealth and biocompatibility, NPs-loaded carbon capsules were surface modified with polyethylene glycol-bis-amine (MW ~6000) as shown in the scheme (Figure S2) which was confirmed by FTIR peaks at 1593 cm-1 for amide bond and at 1390 and 1450 cm-1 for ethylene groups present on PEG molecules (Figure S3). PEGylation of the surface leads to a better dispersion of capsules in water (Figure S4). Since, PEGylation was done after encapsulating NPs inside the capsules, encapsulated NPs were checked for possible changes in their surface functional groups caused by PEGylation which could adversely affect the imaging properties of NPs. Surface functional groups on NPs surfaces were analyzed by FTIR and it showed that bare Y2O3:Eu3+ NPs and encapsulated Y2O3:Eu3+ NPs in PEGylated carbon capsules possess similar peaks corresponding to oleic acid (1663 and 2978 cm-1). It clearly suggests that the ligands on the NPs surfaces were not exchanged during PEGylation (Figure S5). The PEG packing density on carbon capsules surface was calculated by analyzing the percentage of C, H, and N using CHN/O analyzer (Perkin-Elmer 2400 Series CHNS/O Analyzer). PEG packing density was found to be 5.5 x 109 molecules per capsule (size of capsules used for calculation was ~350 nm). The free amino groups imparted by bis-amino-PEG molecules were utilized to bind FITC-tagged IgG antibodies on the surface of carbon capsules. The hydrodynamic diameter of unmodified PEGylated carbon capsules and antibody modified PEGylated carbon capsules were measured to be 224 ± 20 nm and 247 ± 25 nm, respectively. Antibody quantification on the carbon capsules shows that each capsule can bind to ~4400 antibodies approximately (calibration curve, Figure S6). The rough estimate of area occupied by antibodies on carbon capsules surface is ~6.16 x 10-22 m2. Furthermore, we have been able to synthesize carbon capsules of sizes ranging from 180-850 nm by varying the concentration of silica precursor, solvent concentration and catalyst (ammonia) (Table S1; Figure 1 (a-i)). By altering the sizes of carbon capsules,


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payload of encapsulated NPs inside the capsules can be controlled to deliver the desired amount of NPs inside the cells. Also, different sizes of carbon capsules would be taken up by different uptake pathways and would reach different destination inside the cells. This ability of carbon capsules to target various intracellular sites inside the cells can be exploited for intracellular imaging applications.

3.2 Electron microscopic characterization of nanoparticle-loaded carbon capsules The size and surface morphology of NPs-loaded carbon capsules have been characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 2a-k shows the TEM image of bare carbon capsules, NaYF4:Eu3+(5%), LaVO4:Eu3+(10%), GdVO4:Eu3+(10%), Y2O3:Eu3+(5%), Pt, Pd, Ag, GdF3:Tb3+(10%), GdF3:Tb3+(10%) prepared by cation

exchange

method,

and

co-loaded

hydrophobic

Y2O3:Eu3+(5%)/hydrophilic

GdF3:Tb3+(10%) nanoparticle-loaded carbon capsules, respectively and Figure S7 shows the TEM images of Au and Mo NPs loaded carbon capsules. Prior to TEM imaging, the NP-loaded carbon capsules were dispersed in water to mimic their biological environment. Contrast in the TEM images clearly suggests the formation of nearly mono-dispersed NPs without significant agglomeration in large number inside the mono-dispersed carbon capsules. There is a probability of agglomeration of NPs inside the carbon capsules when dispersed in water, however, this is not seen in TEM images and it has not affected the imaging properties which is supported by our luminescence lifetime data. The lifetime of Tb3+ ion in NaYF4:Tb3+ loaded carbon capsule in water and ethanol is found to be 3.3 ms showing no noticeable agglomeration has taken place. In the TEM image of the co-loaded carbon capsules (Figure 2k), the larger darker spots are from Y2O3:Eu3+ and the smaller and lighter spots belong to the GdF3:Tb3+ NPs. This clearly shows that two different NPs are encapsulated in the same carbon capsule. The uniform elemental


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distribution of Gd and Y in the EDAX elemental mapping (Figure 2l) proves the co-loading of the two different NPs. EDAX elemental mapping of Mo, Co, Ag, Pd, and Au NPs loaded carbon capsules are reported in Figure S8 showing presence of various NPs in carbon capsules. Above results suggest that this versatile approach of co-loading can be utilized to prepare other combinations of therapeutic and diagnostic NPs also.

Figure 2: Electron Microscopic characterization of NPs-loaded carbon capsules. TEM image of (a) bare carbon capsules, (b) NaYF4:Eu3+(5%)-loaded carbon capsules, (c) LaVO4:Eu3+(10%)-loaded carbon capsules, (d) GdVO4:Eu3+(10%)-loaded carbon capsules, (e) Y2O3:Eu3+(5%)-loaded carbon capsules, (f) Pt-loaded carbon capsules, (g) Pd-loaded carbon capsules, (h) Ag-loaded carbon capsules (i) GdF3:Tb3+(10%)-loaded carbon capsules, (j) cation exchange synthesized GdF3:Tb3+(5%)-loaded carbon capsules and (k) Y2O3:Eu3+(5%) and cation exchange synthesized GdF3:Tb3+(10%) co-loaded carbon capsules. (Inset in each Figure shows


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the respective NPs-loaded carbon capsules at higher magnification). (l) Elemental mapping of Y and Gd in the Y2O3:Eu3+(5%) and GdF3:Tb3+(10%) co-loaded carbon capsules.

In order to investigate the surface morphology of NPs-loaded carbon capsules SEM images were taken (Figure S9) which suggest that the morphologies of bare capsules and NPs-loaded capsules are similar. It also shows that there are very few NPs present on the surface of the capsules. TEM and SEM images prove that in-situ synthesis of nanoparticle inside the carbon capsule even at high temperatures (~300 ºC) has minimal effect on the morphology carbon capsules contrary to existing techniques for encapsulation such as micro-emulsions, liposomes, dendrimers etc. 3.3 X-ray diffraction characterization of nanoparticle-loaded carbon capsules XRD characterization was performed to establish the crystalline phase of the encapsulated NPs inside the carbon capsules. Figure 3 and Figure S10 show the XRD pattern of NPs-loaded carbon capsules and bare carbon capsules, respectively. It is clearly evident from the XRD data that all the NPs synthesized inside the carbon capsules are crystalline in nature.


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Figure 3: XRD characterization of NPs-loaded carbon capsules. XRD data for (a) NaYF4:Eu3+(5%)-loaded carbon capsules, (b) Au-loaded carbon capsules, (c) GdVO4:Eu3+(10%)-loaded carbon capsules, (d) Y2O3-loaded carbon capsules, (e) Pt-loaded carbon capsules, (f) Pd-loaded carbon capsules, (g) Ag-loaded carbon capsules, (h) GdF3:Tb3+(10%)-loaded carbon capsules, (i) Y2O3:Eu3+(5%) and cation exchange synthesized GdF3:Tb3+(10%) co-loaded carbon capsules (Reference peaks of materials are shown in the respective graphs).

We note that the peak at 2θ ~22º in all the samples is due to graphitic carbon, which is also present in the XRD data of bare carbon capsules. NaYF4:Eu3+-loaded carbon capsules were found to have peaks matching with cubic crystalline α-NaYF4 phase, GdVO4:Eu3+-loaded carbon capsules contained peaks of tetragonal zircon crystalline phase. Y2O3-loaded, Au-loaded, Ptloaded, Pd-loaded and Ag-loaded carbon capsules possess XRD peaks matching with the cubic


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phase of respective materials. The XRD data of GdF3-loaded carbon capsules prepared by synthesis method explained in experimental section clearly demonstrates that GdF3 particles were formed in hexagonal phase. The Y2O3:Eu3+ and GdF3:Tb3+ co-loaded sample shows peaks arising from cubic phase of Y2O3 and that arising from the orthogonal phase of GdF3. XRD data of LaVO4 loaded carbon capsules suggest the formation zircon phase of LaVO4 NPs along with some impurities. The XRD data shows that in situ synthesis of NPs inside the carbon capsules has no effect on crystallinity and phase of NPs as compared to ex-situ processes. 3.4 Fluorescence characterization Fluorescence of the lanthanide-based NPs was measured to illustrate the luminescence properties of NPs synthesized inside carbon capsules (Figure S11). Figure S11 (a and b) illustrates the emission spectra of LaVO4:Eu3+(10%) and NaYF4:Eu3+(5%) respectively, excited with 395 nm light. The emission peaks at 590 and 612 nm which are observed in both spectra correspond to 5

D07F1 and 5D07F2 transitions of Eu3+ ions, respectively. Figure S11 (c and d) shows the

emission spectra of GdF3:Tb3+(5%) and Y2O3:Eu3+(5%) loaded sample which clearly show the characteristic peaks for Tb3+ (Figure S11 c) and Eu3+ ions (Figure S11 d) in the respective emission spectrum. Hence, it is shown clearly that the NPs retain their desirable imaging properties when synthesized and encapsulated within the carbon capsules, for e.g. green emission is shown by GdF3:Tb3+ NPs loaded inside carbon capsules. 3.5 In vitro degradation of Y2O3:Eu3+(5%) and GdF3:Tb3+(10%) co-loaded PEGylated carbon capsules In order to understand the biodegradation of nanoparticle-loaded PEGylated carbon capsules we performed in vitro degradation experiment in presence of myeloperoxidase enzyme (released by


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leukocytes in the body) and hydrogen peroxide at 37 °C. Figure S12 demonstrates the degradation behavior of carbon capsules and suggests that carbon capsules are degradable in contrast to experiments without myeloperoxidase. However, the degradation rate is slow (~16% in 40 h). We note that similar results were observed in the case of carbon nanotubes and carbon nanohorns.43 3.6 In vitro cytotoxicity assay to analyze biocompatibility Prior to any bioimaging application, it is important to analyze the in vitro cytotoxicity of the carbon capsules. For this purpose, in vitro cytotoxicity of Y2O3/GdF3 co-loaded PEGylated carbon capsules (180 nm) (50-200 µg/ml) was tested against various cancer cell lines which include HeLa, A498, L929,HepG2 and MM418 cell lines. Figure 4 (a-d) and figure S13 shows that PEGylated carbon capsules have good biocompatibility for all the cancer lines tested for cytotoxicity. Further, the cytotoxicity of Y2O3/GdF3 co-loaded PEGylated carbon capsules is minimal even up to 200 µg/ml capsules concentration which shows more than 80% of cell proliferation in all the four cell lines making it suitable for use in biological applications.


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Figure 4: Cytotoxicity analysis. MTT assay of PEGylated carbon capsules loaded with GdF3:Tb3+ and Y2O3:Eu3+(5%) (180 nm) on (a) HeLa cells, (b) A498 cells, (c) HepG2 and (d) L929 cells

3.7 Cellular Internalization of carbon capsules and elucidation of uptake pathway To evaluate the imaging potential of NPs loaded carbon capsules in biological system, it is required to understand the nature of intracellular uptake of the imaging vehicle. For this purpose, internalization of PEGylated NPs loaded carbon capsules was studied in HeLa, A498, L929, HepG2 and MM418 cells. RITC dye was tagged to PEG for probing it under the confocal laser scanning microscope (CLSM). The CLSM images (Figure 5) clearly show the internalization of significant number of RITC-labeled PEG modified Y2O3:Eu3+ and GdF3:Tb3+ coloaded carbon capsules by A498, L929, HepG2 and HeLa cells (Figure 5a-d). In addition, Figure 5e shows the CLSM image of MM418 with bright green emission which arises from Tb3+ ions suggesting their


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imaging capability. It is noted that PEG modified Y2O3:Eu3+ and GdF3:Tb3+ coloaded carbon capsules are not labeled with RITC in this particular case and blue emission arises from nuclei stained with DAPI.

Figure 5: Cell uptake analysis CLSM images of cellular uptake of RITC labeled PEGylated carbon capsules loaded with GdF3:Tb3+ and Y2O3:Eu3 by (a) A498, (b) L929, (c) HepG2 cells, Panel A represents the fluorescent images of RITC-PEGylated carbon capsules loaded with GdF3 and Y2O3 NPs, Panel B shows DIC images of the cells, and Panel C represents the merged


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images of A & B to show internalization of capsules inside the cells (A1, B1, C1 correspond to A498 cells, A2, B2, C2 correspond to L929 cells and A3, B3, C3 cells correspond to HepG2 cells), (d) CLSM images of cellular uptake of RITC labeled PEGylated carbon capsules loaded with GdF3:Tb3+ and Y2O3:Eu3+(5%) by HeLa cells ( green fluorescence is from FITC-phalloidin and red from RITC labeled capsules and (e) Merged images of cellular uptake of unlabeled PEGylated carbon capsules loaded with GdF3:Tb3+ (green fluorescent particles) and light microscopy image of MM418 cell, nuclei is stained with DAPI (blue). It is important to understand the uptake pathway of NPs as it ultimately decides their sub-cellular fate and destination. In order to understand the uptake mechanism, HeLa cells were treated with different inhibitors (filipin-III, chlorpromazine, cytochalasin-D (cyto D), rottlerin and genistein), which block different uptake pathways selectively. Filipin-III and genistein are known to stop caveolae-mediated endocytosis. Filipin-III does it by binding to 3β-hydroxysterol, which constitutes a major component of glycolipid microdomains and caveolae and genistein does it by disruption of the actin filament and by utilizing recruitment of dynamin II whereas chlorpromazine

selectively

inhibits

clathrin-mediated

endocytosis.45

Rottlerin

blocks

macropinocytosis, while cytochalasin-D blocks actin polymerization which inhibits both phagocytosis and macropinocytosis.46

Understanding of uptake mechanism of the Y2O3/GdF3-coloaded PEGylated carbon capsules can help in tailoring the capsule internalization at subcellular level. Uptake mechanism has been studied with MM418 and HeLa cells. Cells were counted and cultured for 6 h and blocked with inhibitors at various concentrations and thereafter Y2O3/GdF3-coloaded FITC-tagged PEGylated carbon capsules were added to the cells. A control experiment was also done to check the cytotoxicity of inhibitors on MM418 and HeLa cells and found that concentration of inhibitors used does not show significant cytotoxicity with both MM418 and HeLa cells (Figure S14A and


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B) After 4 h, cells were fixed for microscopic observation. Counting under the microscope was done to quantify the number of cells possessing internalized capsules. Figure 6a and S15 reveal that filipin III inhibited HeLa cells show minimal capsule uptake which suggests that the capsules were taken by caveolae/raft mediated endocytosis mechanism.47 Figure 6b also shows that filipin III and genistein inhibited MM418 cells show minimal capsule uptake which suggests that the capsules were taken by caveolae/raft mediated endocytosis mechanism.47 Transport of molecules such as transferrin and cholera toxins B are known to enter cells selectively via clathrin and caveolae mediated endocytosis, respectively. We performed the inhibition studies with them as positive controls (with MM418) to confirm that the inhibition of uptake mechanisms occurred at the used concentrations of inhibitors (Figure S16). As expected, transport of transferrin and cholera toxin was inhibited by chlorpromazine and genistein. In contrast, filipin –III does not show significant inhibition in the uptake of cholera toxin suggesting that cholera toxin is not uptaken by caveolae-mediated pathway in MM418. Caveolae/raft mediated endocytosis occurs by caveolae coated vesicles escaping the lysosomal degradation and the monomeric vesicles formed in this pathway are transported to Golgi apparatus. Filipin is known to interact with units of cholesterol in the cell’s plasma membrane and inhibits caveolae invagination.48 Genistein is a tyrosine kinase inhibitor which inhibits the process of internalization of invagination.49 Caveolae/raft mediated endocytosis occurs by caveolae coated vesicles escaping the lysosomal degradation and the monomeric vesicles formed in this pathway are transported to Golgi apparatus.50


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Figure 6: Cell uptake mechanism analysis of capsules with different inhibitors, a) HeLa cells and b) MM418 cells Above results clearly suggests that PEGylated carbon capsules containing two different NPs are internalized via this uptake mechanism. This is because the uptake pathway depends only on the surface of carbon capsules and not on the encapsulated NPs. This is crucial for the delivery of variety of NPs, since it enables NPs-independent intracellular internalization by a single and thus, predictable mode.

3.8 Biodistribution analysis Successful translation of any theranostic vehicle from laboratory scale to clinical level requires a study of their biodistribution, pharamacokinetics, blood residence time and clearance from the body. Thus, the promising in vitro properties of NPs-loaded PEGylated carbon capsules were further investigated for their in vivo biodistribution in rats. For this purpose, biodistribution of Y2O3/GdF3 co-loaded PEGylated carbon capsules was studied inside the male SD rats using radionuclide imaging on a gamma camera. PEGylated carbon capsules of 350 nm and 180 nm were labeled with

99m

Tc (half-life 6 h). Rats were anesthetized and intravenously injected with


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samples (50 µg/ml) through penile vein. Whole body scans were done after 30 min post injection on a dual head gamma camera. 350 nm sized capsules were found to be mainly localized in liver, spleen, lungs and urinary bladder (Figure 7 c & d).

Figure 7: Bio-distribution analysis. (a, c & e) Biodistribution of free 99mTc (2 X), 99mTc labeled 350 and 180 nm sized capsules respectively post injection 30 min by PET (organ wise percentage); (b, d and f) Biodistribution images of free 99mTc (2X image), 99mTc labeled 350 and 180 nm sized capsules (1X image) respectively, post injection 30 min by PET With the reduction in size to 180 nm, the distribution becomes more widespread with the capsules now also seen in the heart blood pool, bone marrow with some affinity to muscular tissue as well (Figure 7 e & f) whereas in control rats it a majorly distributed in liver and gastric


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area (Figure 7 a & b). It can be seen that the biodistribution of free

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99m

Tc is different from the

NPs-loaded PEGylated carbon capsules suggesting that the 99mTc is bound with carbon capsules and have not leached from the carbon capsules. Thus, it is evident that hepatic system, spleen, lungs and urinary bladder can be visualized through bigger and smaller carbon capsules; whereas the smaller 180 nm capsules are also present in blood pool (plasma protein binding) and muscle. A slight accumulation of radioactive isotope seen in urinary bladder may be attributed to a small amount of free 99mTc present during circulation. No adverse effect was seen on the rats after the injection of the vehicle inside them, and they were alive and survived well after the injection. 3.9 In vivo pharmaco-kinetics studies and clearance Preliminary in vivo studies were performed to investigate the distribution of Y2O3/GdF3 coloaded PEGylated carbon capsules in blood and their clearance from the body. Blood samples were taken out at different time points from the mice and concentration of Gd3+ ions present in the capsule were measured using ICPMS analysis. Results (Figure 8) clearly confirm that significant concentration of carbon capsules were present in the blood pool up to 24 h which supports their long circulation ability in the blood. This may be due to the presence of PEGylated surface which prevents phagocytosis and clearance by the reticulo-endothelial system.51-52 It is interesting to note that significant amount of Gd3+ ion were present in urine samples suggesting their clearance via urine which is further supported by the PET-based biodistribution analysis. We also observed the clearance through faeces.


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Figure 8: a) Pharmaco-kinetic distribution of NPs loaded PEGylated carbon capsules in blood pool; Clearance kinetics of PEGylated carbon capsules via b) urine and c) faeces. 4. Conclusions We have developed PEGylated mesoporous carbon nanocapsules as a universal multimodal nano-vehicle to deliver unmodified crystalline hydrophobic and hydrophilic NPs with desired properties of dispersion and transport in biological media without agglomeration, reduced biomolecular adsorption, biocompatibility, good biodistribution, nanoparticle-independent uptake mechanism and antibody functionalization allowing potential targeting abilities. Delivery of different NPs cargo to the cells using PEGylated carbon capsules was achieved via a single uptake pathway which was governed by the capsule surface functionalization rather than the NP surfaces. NP-loaded PEGylated carbon capsules (~180 nm) showed good in vitro biocompatibility while the biodistribution study in rats demonstrated the ability to image a wide range of organs including hepatic, spleen, lungs and urinary bladder, bone marrow, blood pool (plasma protein binding) and muscle. The smaller particles were found to circulate longer in the blood enabling enhanced imaging possibilities. In addition to diagnostics, these results also strongly argue for the future potential of the multi-modal delivery platform developed in advanced disease treatments such as stimuli-responsive targeted drug delivery, hyperthermic


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agent-based therapy, etc. by an appropriate choice of the encapsulated NPs and capsule surface functionalization.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The supporting information contains chemistry of PEGylated carbon capsules with its digital image, detailed material characterization (BET of carbon capsules, FTIR, SEM and EDAX images of NPs loaded carbon capsules, TEM images of NPs loaded carbon capsules, fluorescence spectra of NPs loaded carbon capsules), antibody calibration curve and supporting in vitro experiments. This material is available free of charge via the Internet.

Acknowledgements This work was conducted in Indian Institute of Technology, Kanpur (India). S.S and A.S acknowledge DST Nanomission for funding. We acknowledge Sanjay Gandhi Post Graduate Institute, Lucknow (India) for in vivo experiments. We acknowledge T. Radhakrishnan and Mr. Muvva D. Prasad, University of Hyderabad for providing TEM imaging facilities. We acknowledge Mrs. Monika Thakur (IIT Kanpur), Akansha Shukla and Dr. Manish Dixit (SGPGI) for their help in imaging and animal experiments respectively. We acknowledge and thank Dr. Naibedya Chattopadhyay and Dr. Deepshikha Tewari of Central Drug Research Institute, India for providing rats and animal facilities. We also acknowledge Dr. Andrew Jackson and Miss Poonam Bilimoria (Nottingham University, Oncology research, Nottingham, UK) for providing help in conducting experiments for uptake analysis for reviewer’s comment. We Acknowledge Dr. Bandyopadhyay, IITK for providing mice for biodistribution study.


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PEGylated Carbon Nanocapsule: A Universal ... - ACS Publications

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