Green Synthesis of Zwitterion-Functionalized Nano-Octahedral Ceria

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Green Synthesis of Zwitterion-Functionalised Nanooctahedral Ceria for Enhanced Intracellular Delivery and Cancer Therapy Rashid Mehmood, Sajjad Seifi Mofarah, Aditya Rawal, Florence Tomasetig, Xiaochun Wang, Jialin Yang, Pramod Koshy, and Charles C. Sorrell ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06726 • Publication Date (Web): 19 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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ACS Sustainable Chemistry & Engineering

Green Synthesis of Zwitterion-Functionalised Nanooctahedral Ceria for Enhanced Intracellular Delivery and Cancer Therapy Rashid Mehmood1,2*, Sajjad Seifi Mofarah1, Aditya Rawal3, Florence Tomasetig4, Xiaochun Wang2, Jia-Lin Yang2, Pramod Koshy1, Charles Christopher Sorrell1 1

2

3

4

School of Materials Science and Engineering, UNSW Sydney, High Street, Building E10, Kensington NSW 2052, Australia Adult Cancer Program, Lowy Cancer Research Centre, UNSW Sydney, Corner Botany & High Streets, Kensington NSW 2052, Australia NMR Facility, Mark Wainwright Analytical Centre, UNSW Sydney, High Street, Building F10, Kensington NSW 2052, Australia BMIF Facility, Biomedical Imaging Facility, UNSW Sydney, Botany Street, Building E26, Kensington NSW 2052, Australia

* Corresponding Author: [email protected]

Abstract The present work reports a new method for the green chemical synthesis of biomaterials using an integrated, room-temperature, aqueous, chemical technique involving surfactantfree precipitation of nanoceria, surface silanisation, and functionalisation with zwitterionic agents by metal-free O-PET-ATRP. The synthesis mechanism for each of these steps is presented. The present work is the first to report the use of water, rather than organic solvent, as medium for O-PET-ATRP. The nanoparticles were characterised by FTIR, laser Raman microspectroscopy, XPS, TGA, TEM, and NMR. The functionalisation resulted in retention of nanoparticle shape, hindrance of plasma protein adsorption, maintenance of small hydrodynamic size, and establishment of a near-electroneutral surface. The latter demonstrates that the zwitterion formed a continuous passivating layer on the nanoceria surfaces.

These outcomes resulted in higher uptake of functionalised nanoceria and

enhanced redox performance. Nanoceria provided cytoprotection to normal cells while cytotoxicity was observed in fibrosarcoma cells. Nanoparticles generated pH-controlled redox responses in fibrosarcoma cells where, at physiological conditions of pH 7.4, antioxidant activities were observed while prooxidant behaviour was generated at tumour microenvironment conditions of pH 6.4. These effects were accentuated at both pH values for functionalised nanoceria, which is a direct result of the functionalisation.

Keywords: cerium oxide, aqueous synthesis, organocatalysis, redox, fibrosarcoma 1 ACS Paragon Plus Environment

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Introduction Green chemical syntheses have become increasingly important owing to the need to develop sustainable materials and processes. In pursuit of these methodologies and inspired by natural photosynthesis mechanism, efforts have been made to utilise chlorophyll as a catalyst for materials syntheses.1,2 The use of the environmentally-benign green solvent water as a reaction medium also follows along this line.3-7 Further, photoinduced organocatalysis is being explored for micromolecular to macromolecular syntheses,8-12 where, instead of heat, non-invasive visible light is used to initiate the chemical reaction, thereby facilitating the use of heat-sensitive materials and functional groups. These processes also have the advantage that the organocatalytic synthesis mechanism can avoid the use of a metal, the retention of which creates concern for environmental and biological impacts. Consequently, considerable efforts have been undertaken to use this mechanism in organic and macromolecular synthesis, the most important of which incorporates the well-known atom-transfer radical polymerisation (ATRP).13-16

ATRP was established and developed using metal catalysts, particularly copper-based ligated complexes.17

However, the increasing use of macromolecules in microelectronics and

biomaterials has highlighted the probability of retained metal-contaminated end-products.18 Although recent work has reported attempts to lower the residual catalyst load,19-21 its complete removal is still desired22 and so metal-free organocatalysed photoinduced electron transfer ATRP (O-PET-ATRP) offers a potential solution.23,24 ATRP has been implemented successfully to synthesise antifouling and hydrophilic zwitterionic polymers for drug delivery, where both positive and negative charges in the structure yield a net zeta potential of zero.25,26 The antifouling character of these zwitterionic agents is critical to nanoparticlebased medicines because it can prevent adsorption of nonspecific proteins and so prolong the blood circulation time.25,26 The effective neutralisation of the surface charge hinders the adsorption of polar proteins and facilitates retention of the small hydrodynamic size of nanoparticles in vitro and/or in vivo environments, which enhance their delivery to the target. One such nanoparticle is redox-active ceria (cerium oxide, CeO2-x, nanoceria) because it can mimic the biological enzyme reactions that regulate reactive oxygen species (ROS) through redox chemistry.27-40 2 ACS Paragon Plus Environment

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The redox mechanism of nanoceria as well as the ROS regulation mechanism are dependent on intrinsic Ce3+ defects and the formation of charge-compensating oxygen vacancies,38,41-43 where continuously reversible Ce3+ ↔ Ce4+ switching occurs owing to the low reduction potential of the Ce3+/Ce4+ pair.44 This establishes a dynamic equilibrium of transient charge compensation between the two valences, where the presence of an oxygen vacancy allows the oxidation of ROS in biological media while simultaneous oxygen occupancy by small neutral molecules, such as oxygen and water, causes transient oxygen vacancy annihilation. These are followed by Ce4+ → Ce3+ reduction owing to the low reduction potential and the coordination instability, resulting in oxygen vacancy re-formation. Importantly, nanoceria has been found to generate pH-dependent redox response. Once in the cell microenvironment, it can induce cytotoxic effects by producing ROS at weak acidic pH values while providing cytoprotection to healthy cells by scavenging ROS at weak basic pH conditions.31,38,39 These dual actions make it an ideal material to employ in cancer therapy, where it has been used to modulate tumour-stroma interactions in skin cancer cells. The cytotoxic and cytoprotective effects have been observed in cancer and normal cells, respectively.32 These therapeutic effects of nanoceria in cancer cells are enhanced by functionalising its surface with biocompatible agents, such as polyethylene glycol (PEG), dextran, hyaluronic acid, heparin, etc. Although PEG has been utilised to functionalise a range of biomaterials, including nanoceria, its use is associated with several disadvantages, including immunogenicity, cytoplasmic vacuolisation, heterogeneity, reduction in binding affinities, and reduction in bioactivity, which limit its use for biomedical applications.35-37

Therefore, the present work reports the green synthesis of pristine truncated nanooctahedra of ceria and their functionalisation with zwitterionic sulfobetaine (SB) by a green chemical technique for the purpose of enhancing delivery through optimised cell uptake, induced antifouling, and reduced nonspecific protein adsorption. A strategy of surfactant-free aqueous chemistry and room-temperature precipitation was used to synthesise the pristine nanoparticles, which was followed by surface modification with silane and functionalisation with zwitterionic sulfobetaine by using the O-PET-ATRP technique. While previous studies have used O-PET-ATRP in organic solvents,23,24 the present work presents a seminal strategy for a green chemical approach using water as solvent; this is advantageous since these organic 3 ACS Paragon Plus Environment


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compounds are not environmentally benign. Fibrosarcoma cells were used to analyse the in vitro redox response of nanoparticles. The data reported include synthesis and surface modification techniques, structural and surface characterisations, protein adsorption, redox effects and associated biomedical responses, and cellular uptake.

Materials and methods Materials Sulfobetaine methacrylate (SBMA), fluorescein-o-methacrylate (FMA), triethylamine (TEA), cerium (III) nitrate hexahydrate (Ce(NO3)3·6H2O, 99.999 wt% trace metal basis), aqueous ammonia solution (25 wt%), 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA), CCK-8 (cell counting kit 8), ethanol (99.5%), human plasma, fluorescein (FS), and cerium standard (100 mg/mL Ce in HNO3) were purchased from Sigma-Aldrich, Australia.

Dulbecco’s

phosphate buffered saline (DPBS, Gibco Life Technologies, USA), and trypsin-EDTA (ethylenediaminetetra acetic acid, Gibco Life Technologies, USA) were purchased from Thermo Fisher Scientific, Australia. Hydrochloric acid (32 vol%) and nitric acid (76 vol%) were purchased from RCI Labscan Limited, Australia. Sodium hydroxide (98 wt%) was purchased from Chem-Supply, Australia; sodium cacodylate was purchased from Proscitech, USA; and blue light diode (LED, 4.8 W, 𝜆 max = 450 nm) light strips were purchased from RS Components, Australia. Mitosox red dye was purchased from Thermo Fisher Scientific, Australia. Glutaraldehyde (25%, pH 7.4) and osmium tetroxide (4%) were purchased from Electron Microscopy Sciences, USA; LX112 resin embedding kit was purchased from Ladd Research, USA; fibrosarcoma (HT1080) cell lines were purchased from American Type of Cell Culture, USA: RPMI 1640 medium (Roswell Park Memorial Institute-1640, USA) was purchased from Invitrogen; and 3-(trimethoxysilylpropyl)-2-bromo-2-methylpropionate (BMPS) was purchased from Gelest Incorporation, USA.

Nanoparticle synthesis by surfactant-free precipitation Synthesis of ceria truncated nanooctahedra (NO) weas carried out by a wet-chemical and surfactant-free precipitation method using aqueous ammonia solution. Briefly, a 0.15 M solution of cerium nitrate in deionised water was magnetically stirred for 30 min at room temperature. Aqueous ammonia solution was added dropwise using a syringe and driver

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(Graseby MS 26, UK) while monitoring the pH of the solution until it increased to ~8.0. The turbid suspension was stirred for 24 h at room temperature. The suspension was centrifuged at 3000 rpm for 10 min and the supernatant removed. The resulting thick slurry was dried at 85oC for 24 h. The dried solid was crushed with a stainless steel spatula to fine powder and stored in an enclosed plastic vial.

Surface modification of nanoparticles by silanisation Surface silanisation of the nanoceria was done using a silane (BMPS) coupling agent. NO particles were weighed (10 mg) in a round-bottom flask, which was sealed with a rubber septum. An inert atmosphere was created within the flask by displacive introduction of argon gas by purging with an argon-filled balloon for 30 min. This was followed by the addition of 10 mL of deionised water using a disposable syringe to achieve a concentration of 1 mg/mL of NO. 0.25 mL BMPS and 0.1 mL of aqueous ammonia solution then were added dropwise to the flask using a syringe and driver (Graseby MS 26, UK). The flask was sonicated for 20 minutes and then magnetically stirred at room temperature for 24 h.

The resultant

suspension of silane-modified NO particles (SNO) was centrifuged at 3000 rpm for 5 min and washed with deionised water three times to remove unreacted silane, after which drying was done in a vacuum desiccator for 24 h.

Surface functionalisation of nanoparticles by metal-free O-PET-ATRP The O-PET-ATRP reaction was done in an inert atmosphere at room temperature.18 Briefly, 50 mg of SNO particles were placed in a glass vial (100 mm H x 25 mm Ø) containing a magnetic stirring bar. Subsequently, monomers and organic photoredox catalyst were added in a molar ratio of 300/100/0.1 (SBMA/FMA/FLS). The vial was sealed with a rubber septum and an inert atmosphere in the flask was achieved by purging with an argon-filled balloon for 30 min. The organic electron donor (TEA) was introduced using a disposable syringe at a specific molar ratio according to the experimental design. 10 mL of deionised water were added to the vial using a disposable syringe in order to form a suspension. The vial was sonicated for 20 minutes and then was transferred to the centre of a cylindrical array of a strip of blue LEDs (75 mm H x 125mm Ø) located in the dark, after which the suspension was magnetically stirred at room temperature for 24 h. The resultant suspension of functionalised truncated nanooctahedra (FNO) was dried in a vacuum desiccator for 24 h for further use. 5 ACS Paragon Plus Environment


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Characterisation The functional groups of NO, SNO, and FNO were identified by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR; Spotlight 400 FT-IR, Perkin Elmer, UK, wavelength range 400-4000 cm-1) and laser Raman microspectroscopy (Raman; Renishaw, UK, inVia Raman microscope, 35 mW helium-neon laser, 514 nm, 20X magnification, 1.5 μm beam diameter, 200-800 cm-1). The organic and inorganic surface chemical composition of the particles was determined by X-ray photoelectron spectroscopy (XPS; Thermo Fisher Scientific, USA, ESCALAB 250Xi spectrometer, 13.8 kV, 8.7 mA, 500 μm beam diameter, monochromated AlKα X-rays, 1487 eV).

The natures of the decompositions of the

functionalisation agents on the nanoparticle surfaces were examined by thermogravimetric analysis (TGA; TA instruments, USA, Q5000, 20o-1000oC, 10oC/min heating rate). The average particle sizes and standard deviations (SD) were determined using transmission electron microscopy (TEM; Philips CM200, 200 kV, Netherlands). The particle sizes were confirmed by dynamic light scattering (DLS); the zeta potential also was determined using the same instrument (Zetasizer Nano ZS, Malvern Instruments, UK, 4 mW He-Ne laser, 633 nm). For this work, ceria NO and FNO particles were suspended in 3 mL of RPMI biological medium or deionised water at a concentration of 20 μg/mL using 10 mL individual glass tubes. The suspensions were sonicated for 5 min preceding measurement.

The localised carbon bonding at different positions of the functional groups was assessed using solid-state nuclear magnetic resonance (NMR; Bruker, USA, Biospin Avance III 300 MHz spectrometer, wide-bore 7 T superconducting magnet, 75 MHz frequency for 13C nucleus and 300 MHz for 1H nucleus). Approximately 80 mg of each sample was packed into 4 mm H zirconia rotors with Kel-F® caps and magic-angle spun at 12 kHz. The 13C NMR spectra were acquired using the 1H-13C Multi-CP technique using a 1 ms Hartman-Hahn cross-polarisation contact pulse ramped from 70% to 100 % for polarisation transfer. The 13C 90° pulse length was 4 μs. The Spinal-64 1H decoupling scheme with a 75 kHz decoupling field strength was used during acquisition. Recycle delays of 1.5 s were used to ensure full relaxation of the 1H nuclei and 8 k transients were signal-averaged for sufficient signal-to-noise ratio. The


13C

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chemical shifts were referenced to tetramethylsilane (TMS) using glycine as a secondary reference.

Effects of pH on Ce3+ ↔ Ce4+ switching Nanoparticles (FNO, 500 mg) were suspended in an aqueous solution at pH 7.4 or pH 6.4 for 24 h. The suspensions then were centrifuged at 3000 rpm, the supernatants were removed, and the resultant thick slurries were dried in a vacuum desiccator at room temperature for 24 h. The solid aggregates were crushed with a stainless steel spatula and each resultant powder was analysed by high-resolution XPS (Thermo Fisher Scientific, USA, ESCALAB 250Xi spectrometer, 13.8 kV, 8.7 mA, 500 μm beam diameter, monochromated AlKα X-rays, 1487 eV).

Protein adsorption The adsorption of human plasma proteins was evaluated first by suspending ceria NO and FNO particles in glass tubes containing 2 mL of plasma solution (0.1 mg/mL concentration) to achieve a particulate concentration of 200 μg/mL. The suspensions were transferred to an incubator (Forma 4140TS, Thermo Fisher Scientific, Australia) held at 37°C in an atmosphere of 5 vol% CO2 in air for 1 h. Following incubation, the suspensions were centrifuged at 3000 rpm for 10 min, after which the supernatant protein concentration was evaluated using UVVis spectrophotometry (UV-Vis; PerkinElmer, USA, UV-Visible Spectrometer, single beam).

Stock suspension preparation for cell analysis Stock suspensions of NO and FNO particles were prepared at pH 7.4 and 6.4 using 1 M HCl or NaOH. The nanoparticles were weighed in a sterile test tube and then suspended in RPMI medium at a concentration of 100 or 200 μg/mL. The pH of the solutions was adjusted and monitored using a pH meter (FG2, Mettler-Toledo, Australia).

Cell culturing The fibrosarcoma cell line (HT1080) and normal fibroblast cell line (MRC-5) were dispersed in cell culture flasks in RPMI-1640 medium (supplemented with 2.0 mM L-glutamine, 10 vol% fetal bovine serum and 1% v/v penicillin/streptomycin) in a PC2 biosafety hood. The flasks



then were transferred into the incubator held at 37°C in an atmosphere of 5 vol% CO2 in air for cell growth and remained there until further use.

Cell viability The procedures for cell viability, ROS analysis, confocal microscopy imaging, electron microscopy imaging, and quantitative cellular uptake are described elsewhere.38,39 Briefly, for cell viability, RPMI medium (100 μL) was used to suspend 2 × 103 cells/well in 96-well culture plate. Cells were allowed to adhere for 24 h in the incubator, after which 100 μL of the NO or FNO stock suspension was added to the wells. The plate then was subjected to incubation intervals of 24 h, 48 h, or 72 h. After this, 10 μL of CCK-8 reagent were added to the wells and plate again was incubated for 3 h. The optical absorbance of CCK-8 was measured at 450 nm using a plate reader (Molecular Devices, USA, SpectraMax M3, Model MT05412).

These experiments were performed in triplicate; cell controls without

nanoparticles were treated identically.

ROS analysis The experimental procedure for ROS measurement was nearly identical to that used for cell viability except that a ROS-sensitive dye (DCFH-DA) was used to detect ROS instead of CKK-8 dye. Briefly, after cell incubation with nanoceria, each well was washed with 1 mL DPBS solution. The cells were detached from the bottom of the plate by adding 10 μL of trypsin and incubated for 5 min. Cells then were exposed to 90 μL of 10 μM dye solution, followed by incubation for 30 min. The dye was cleaved by cell action and the fluorescence intensity of its by-product (DCF) within the cell was measured at 485/535 nm using a fluorometer (Fluoroskan Ascent Microplate Fluorometer, Thermo Fisher Scientific, Australia). These experiments were performed in triplicate; cell controls without nanoparticles were treated identically. The identical experimental procedure was carried out using Mitosox dye to detect the mitochondrial superoxide radical.

Confocal microscopy imaging HT1080 cells (2 × 103 cells/well) were seeded in 500 μL RPMI medium for 24 h, followed by incubation with 100 μL stock suspensions of NO or FNO (200 μg/mL) for 4 h. The residual nanoparticles from the suspensions were removed by washing with 500 μL of DPBS three 8 ACS Paragon Plus Environment

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times. The cells were incubated with organelle stains, including lysotracker (50 nM for 30 min), plasma membrane stain (10 μg/mL for 30 min), and nucleus Hoechast stain (5 μg/mL for 30 min), respectively, all of which were purchased from Thermal Fisher Scientific, Australia. Following this, the cells were washed with 500 μL of DPBS three times. The live cells then were resuspended in 500 μL of DPBS and imaged by confocal laser scanning microscopy (Leica TCS SP8 DLS microscope, Germany) using a 63X/1.40 OIL CS2 objective in a sequential mode for each channel.

Electron microscopy imaging of cells The experimental procedure was nearly identical to that used for confocal microscopy imaging except that, after incubation, the cells were fixed in 1 mL of glutaraldehyde (2.5 % 0.1 M sodium cacodylate buffer, Sigma Aldrich, Australia) for 1 h at room temperature. The cells then were washed with sodium cacodylate buffer (1 mL of 0.1 M) and subjected to stepwise postfixing treatment in by microwave heating (Pelco BioWave, TED Pella, Inc., USA) as follows: 1) Microwave heating of 1 mL osmium tetroxide (1% in 0.1 M cacodylate buffer) 2 min, 2) Washing in 1 mL of 0.1 M cacodylate buffer, 3) Washing in 1 mL of deionised H2O (twice), 4) Washing in ethanol (30, 50, 70, 80, 90, and twice at 100 vol%), 5) Infiltration in the LX112 resin with increasing resin concentrations (33, 67, and twice at 100 vol% in 100% ethanol). The resin was hardened in a drying oven at 60oC for 24 h. Thin sections (60 nm) of resin were cut using an ultramicrotome (Leica EM UC6, Leica Microsystems, Germany). The sections then were placed on Formvar-coated 200 mesh copper grids (Ted Pella Inc., USA) and imaging was done by TEM (JEOL, TEM-1400, Japan) at an accelerating voltage of 120 kV.

Quantification of nanoceria cellular uptake The preparation procedure was nearly identical to that for the confocal microscopy except that, after washing with DPBS, 10 μL of trypsin were added to each well and incubated for 5 min. The cells were digested in 50 μL of concentrated nitric acid for 12 h, heated (90°C, 20 min) in a drying oven, and resultant suspensions were diluted with deionised water to 1 mL. The cerium concentration of the suspension (1 mL) was determined by inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer quadrupole, NexION, USA). The obtained values were recorded in μg/mL. These experiments were performed in triplicate.



Results and discussion The environmentally-friendly surfactant- and metal-free aqueous green synthesis methodologies at room temperature of the present work yielded well dispersed truncated nanooctahedra of ceria (NO; average TEM size of 50 grains = 10 ± 2 nm SD) by precipitation, silane-modified surfaces (SNO; average TEM size of 50 grains = 12 ± 2 nm SD) by silanisation, and zwitterion-functionalised surfaces (FNO; average TEM size of 50 grains = 14 ± 3 nm SD) by O-PET-ATRP, as shown in the stepwise synthesis scheme in Fig. 1. It is known that the nanoceria morphology plays a critical role in redox behaviour and cellular uptake.39,40 Thus, the present work reports the synthesis of discrete pristine truncated nanooctahedral particles that maintained their morphological integrity after silanisation as well as after functionalisation with sulfobetaine, as shown in the TEM images in Fig. 1. These procedures are advantageous because the commonly used high-temperature methods generally result in the formation of hard agglomerates, which reduces the surface area, and a reduction in the surface oxygen vacancy concentration, which reduces the redox performance.43,45


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Figure 1. Schematic representation of materials synthesis in aqueous medium at roomtemperature: (a) Pristine nanoceria synthesis by surfactant-free precipitation (NO), (b) nanoceria surface-modified by silanisation (SNO), (c) functionalised nanoceria by O-PET-ATRP synthesis (FNO), (i) TEM image of NO (higher magnification image in inset), (ii) TEM image of SNO (higher magnification image in inset, (iii) TEM image of FNO (higher magnification image in inset) 11 ACS Paragon Plus Environment


The redox behaviour of nanoceria has been reported to be associated with its surface or subsurface regions, where available redox-active sites in the form of oxygen vacancies provide the capacity for the switching of the Ce3+/Ce4+ couple.46 Hence, the avoidance of surfactants and organic solvents is critical because these carbon-chain molecules, when present during crystallisation, can cause structural alteration by hybridisation, intercalation, and/or embedding within the structure; they also may adsorb on the rough surfaces. These processes are capable of blocking the redox-active sites, which are the oxygen vacancies, and so represent potentially serious impediments to the redox performance.45 Consequently, the present work reports the precipitation of nanoceria without the use of a surfactant to fabricate pristine truncated nanooctahedra, the growth mechanism of which can be explained by dissolution and recrystallisation phenomena, during which dissolution in basic conditions results in Ce3+ ions’ forming Ce(OH)3 nuclei, which then grow to form CeO2-x crystals.40,42 Since the rates of dissolution and recrystallisation are low under room temperature conditions, these facilitate effective ionic packing and the formation of crystallographic facets of greater packing densities and larger interplanar spacing, the order of which is {111} > {100} > {110}.34,41,42 As shown in Fig. 2(a), the dissolution of the ceria precursor (Ce(NO3)3·6H2O) leads to the formation of cerium hydroxide nuclei, which grow to form nanooctahedra of exposed {111} facets. These nanooctahedra form {100} truncations for the abovementioned reasons as well as for surface area reduction.


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Figure 2. Synthesis mechanisms of nanoceria: (a) nanoceria synthesis: (i) nucleation of Ce(OH)3 from Ce(NO3)3·6H2O dissolved in H2O in basic solution (by NH4OH), (ii) growth of Ce(OH)3 nuclei to form nanooctahedra, (iii) truncation of nanooctahedra, (b) surface



silanisation: (i) hydrolysis of silane methoxy groups in the presence of aqueous ammonia, (ii) condensation of hydroxyl groups of nanoceria and silane, (c) surface functionalisation using O-PET-ATRP: (i) catalyst ([TEA]/[FS]) activation by blue light and radical initiation, (ii) chain propagation by addition of monomers (SBMA, FMA), (iii) termination by effect of bromine radical

After crystallisation, surface bonded hydroxyl groups can provide the link to attach functional ligands or molecules on the surfaces of nanoparticles. This highlights the advantage of the surface silanisation process, which involves Si bonding to the OH bonded to CeO 2 and hence does not involve oxygen vacancies.

Importantly, the attachment of other functional

molecules or agents on nanoparticles surfaces involves their direct linkage with silanes rather than occupation of the oxygen vacancies. The oxygen vacancies can be occupied temporarily by O-containing species, particularly the common ROS species superoxide (●O2 –), hydroxyl (●OH), and hydrogen peroxide (H2O2), but this occupation is dynamic.47 Since surface functionalisation of the materials by ATRP and in particular O-PET-ATRP also offers potential advantages in the blood circulation time of the materials and nonspecific protein adsorption by improvements in hydrophilicity, dispersibility, and adsorptivity, this approach was utilised in the present work.48,26 However, all previous work using the O-PET-ATRP method has involved organic solvents, 21-24 whereas the present work is the first to report the use of the green solvent water.

Consequently, all three stages of the aqueous, room-temperature synthesis process of the present work offer advantages over alternative techniques using organic solvents or surfactants. The mechanisms of surface silanisation and O-PET-ATRP are presented in Fig. 2(b) and Fig. 2(c), respectively. Surface silanisation, shown in Fig. 2(b), involves the hydrolysis of the silane methoxy groups and their condensation with the dangling hydroxyl groups on the nanoparticle surfaces. The silane terminal group then is used to functionalise the particle surfaces with sulfobetaine and fluorescein using O-PET-ATRP. The detailed mechanism of OPET-ATRP is described in Fig. 2(c).

The structures of NO, SNO, and FNO also were analysed by ATR-FTIR, Raman, XPS, and TGA, as shown in Fig. 3(a-f). 14 ACS Paragon Plus Environment

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Figure 3. Characterisation of NO, SNO, and FNO: (a) FTIR spectra, (b) laser Raman microspectra, (c) XPS spectra (survey), (d) XPS spectra (N1S, FNO), (e) XPS spectra (S2P, FNO), (f) TGA

The broad peak in the FTIR spectra at 3400 cm-1 for all three samples (NO, SNO, and FNO) is attributed to the symmetric stretching of the hydroxyl group.49 The transmittance intensity of this peak was the least for FNO, which suggests the modification of the surface OH groups during functionalisation. The presence of stretching vibrations for C=O, Si-O, and Si-C- at 1720 cm-1, 900 cm-1, and 1130 cm-1, respectively, in SNO and FNO differentiates these materials 15 ACS Paragon Plus Environment


from NO.49 The establishment of silane linkages and oligomers in SNO and FNO, respectively, is shown by the C-H stretching peak at 2900 cm-1 in Fig. 3(a) and the Raman spectra in Fig. 3(b). The correspondence of the peaks (aromatic C-C and C=C, C-N, C-S, S-O) for FTIR and Raman spectra for the general structure of FNO supports the NMR data for the localised structure of the functionalisation agents. These two data sets confirm the oligomerisation of SBMA and FMA monomers. The absence of peak shift in the main cerium oxide Raman peak at 465 cm-1 indicates that little or no structural alteration occurred. Further, the oxygenvacancy-related Raman peak28 at 600 cm-1 is consistent for all three materials, which demonstrates that the oxygen vacancy concentration remained relatively unaffected by these processes. Fig. 3(c) reveals the surface chemical analyses of the materials by high-resolution XPS, where the survey scan spectra show the peaks of all relevant elements. The quantified atomic percentages of the major elements calculated from these data are as follows:

Material

C1s (at%)

Ce3d (at%)

O1s (at%)

NO

17.65

19.10

63.18

SNO

23.57

14.43

57.87

FNO

34.31

10.12

46.91

The 17.65 at% C on the NO particles represents adventitious carbon. Since all three types of nanoparticles were subjected to the same exposure conditions, it could be assumed that 5.92 at% C derive from silanisation and 16.66 at% derive from oligomeric functionalisation. However, examination of the TGA data in Supplementary Information Figure 1 reveals that the weight losses for both functionalisation agents up to ~750°C are similar, which suggests the alternative interpretation that the deposition of adventitious carbon is enhanced by the oligomer (on 39 C units; molecular weight 903 g/mol) rather than the silane (on 7 C units; molecular weight 257 g/mol). Although the attachment of silane and sulfobetaine on oxide nanoparticles is known48, it is not possible to determine a degree of deposition since the pyrolysis segments of the data include not only pyrolysis of the functionalisation agents but also pyrolysis of adventitious carbon, pyrolysis of fluorescein, and calcination of Ce(OH)4 gel. However, the establishment of a near-electroneutral surface demonstrates that the zwitterion formed a continuous passivating layer on the FNO surfaces. These data may be contrasted with the TEM particle size data, where the respective NO, SNO, and FNO sizes were 16 ACS Paragon Plus Environment

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10, 12, and 14 nm, and the DLS particle size data, where the NO and FNO sizes were 11 and 14 nm, respectively.

Further, the presence of the Si2p peak at 101 eV and the Br3d peak at 69 eV indicates the attachment of the silane to the pristine NO surfaces.48 Similarly, the N1s peaks and the S2p peaks, shown in Fig. 3(d,e) reveal the attachment of the SBMA monomer, which thus confirms the oligomerisation48. The presence of two N1s peaks and two S2p peaks indicates their different chemical environments, where the N1s peak at higher binding energy (402.5 eV) is attributed to N in the quaternary ammonium cations (–N+(CH3)2–) and the N1s peak at lower binding energy (389.5 eV) is attributed to N in the carbon chain in secondary amine (–N–) form.50 The two S2p peaks at 167.9 eV and 169 eV are considered to be representation of the C–S and C–SO3 environments, respectively.50 Since the C–Br bond is unstable under X-ray radiation,48 its low intensity in SNO and absence in FNO are expected. The determination of an N/S ratio of 1/1 demonstrates the structural consistency of the monomer. These results are in good agreement with previous studies of silane and sulfobetaine coatings.48

The TGA data (Supplementary Information Figure 1) show that the pristine NO exhibited loss of adsorbed H2O at ≤85°C, followed by exponential weight loss up to ~500°C, which results from the calcination of the hydrated and passivating surface layer in the form of gelled Ce(OH)4.51 At higher temperatures, oxidation of the oxygen vacancies causes a slight weight gain. The data for SNO are similar and indicate (1) loss of adsorbed H2O at ≤85°C, (2) rapid and substantial weight loss from pyrolysis combined with the onset of calcination of the underlying gelled Ce(OH)4 up to ~500°C, and (3) weight loss owing to the decomposition of SOx, which dominates the weight gain from SiO2 formation from the Si in silane. The data for FNO also are similar in that they exhibit the first two regimes. However, at temperatures >300°C, the weight loss from calcination of Ce(OH)4 is overshadowed by the pyrolysis of residual sulfobetaine. At ~750°C, SiO2 formation from the Si in silane causes a noticeable weight gain.

Since the linear chain of SBMA and FMA is formed directly on silane-coated nanoparticle surfaces, the only feasible method to analyse the extent and degree of monomers linkage is 13C

NMR, which has been used in the present work. These NMR spectra and peak chemical

shifts indicating the localised carbon functional groups of the SNO and FNO are shown in Fig. 17 ACS Paragon Plus Environment


4(a) and (b), respectively. In Fig. 4(a), all of the expected 13C peaks of the initiator, shown in inset (i), can be assigned with the exception of peak f at 37 ppm. This exception may be linked to an initiator-initiator coupling reaction, where a silanol group attacks the bromine endgroup instead of coupling to the ceria, as suggested in Fig. 4, inset (ii). Comparison of the relative intensities of peak q to those of a, c, and j suggests that approximately half of the initiator sites were self-coupled.


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Figure 4.

13C

NMR spectra of SNO and FNO: (a) Silane initiator coating, (b) oligomer of

sulfobetaine methacrylate and fluorescein-o-methacrylate

Further, the 13C NMR spectra of the FNO in Fig. 4(b) indicate the successful coupling of the zwitterionic SBMA and the FMA monomers to the nanoceria-bound initiator, as represented by peak g (46 ppm). Peak h (52 ppm) is ascribed to the distinctive methyl groups at the quaternary nitrogen of sulfobetaine.53 The quaternary and carbonyl carbon sites of the fluorescein are indicated by the presence of peaks k (83 ppm) and o (166 ppm), respectively, as shown in Fig. 4, inset (iii).54 Comparisons of the relative intensities of peak a against peaks g, o, and q confirm the same degree of polymerisation is ~2, thus indicating that the polymerisation reaction is one of oligomerisation. The low extent of polymerisation can be attributed to a combination of the characteristics of the monomer and catalyst as well as the reaction conditions. It is likely that the self-coupling behaviour of silane may have facilitated the agglomeration of the nanoparticles in suspension, as demonstrated in the TEM images of Fig. 1, which then were taken up by the fibrosarcoma cells, as shown subsequently in Fig. 10. The latter can be seen in the TEM image of FNO in Fig. 1 and the cell-uptake images, described subsequently.

The biological environment is a complex medium containing charged biomolecules, which perform specific functions and circulate in the bloodstream. More than half of human blood is comprised of plasma, which has a high concentration of proteins that consist of positively and negatively charged amino acids that tend to adsorb on oppositely charged surfaces. When nanoparticles are introduced as a foreign material in the body, they tend to adsorb nonspecific proteins.52 However, zwitterionic functionalisation can be an essential adjunct to such nanoparticulate therapies through two main attributes. First, this imparts hydrophilicity to the nanoparticles, which enhances ease of circulation and distribution through the bloodstream. Second, it also imparts antifouling characteristics owing to the effective neutralisation of the surface charge, which hinders the adsorption of nonspecific proteins and thus prolongs the blood circulation time.26 The consequent reduction in protein surface adsorption effectively allows retention of a smaller hydrodynamic size in in vitro and in vivo environments, which enhances both the diffusivity and enhanced permeability and retention



(EPR) effect.26 Fig. 5(a-d) confirms these effects by contrasting the relevant characteristics of NO and FNO in biological medium.

Figure 5. DLS data for nanoceria in biological medium: Hydrodynamic size of (a) NO, (b) FNO; zeta potential of (c) NO, (d) FNO

Fig. 5(a,b) reveals the capacity of the zwitterionic sulfobetaine to retain a hydrodynamic particle size of FNO (21 nm) smaller than that of the protein-adsorbed nanoceria NO (35 nm) when in biological medium. Further, Supplementary Information Figure S2 confirms that there is only a slight difference in the hydrodynamic size of NO (11 nm) and FNO (14 nm) when in aqueous medium; similar sizes were documented by TEM: NO (10 ± 2 nm), FNO (14 ± 3 nm). These data suggest that the near-electroneutrality deriving from equal amounts of


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positive and negative charges of the passivating zwitterion on the surfaces of the FNO is responsible for the hindrance of protein adsorption.

The functional groups at the surface of the nanoparticles can form weak electrostatic interactions, which also facilitate the agglomeration of particles.55 This enhances the cellular uptake and redox behaviour of nanoceria in the fibrosarcoma cells, which are verified by the increased cellular uptake and redox behaviour of FNO compared to NO; these data are discussed subsequently. The negative zeta potential of the pristine NO is consistent with the gelled Ce(OH)4 surface layer and the nil zeta potential of FNO derived from the zwitterionic effect. The model for these phenomena and its quantification are shown in Fig. 6(a-c).



Figure 6. Plasma protein adsorption on nanoceria: (a) Model representing protein corona formation on NO, (b) model representing resistance of FNO to plasma proteins, (c) quantification data for protein adsorption on NO and FNO

The in vitro cell viability assays for NO and FNO at pH 7.4 and 6.4 at nanoparticle concentrations of 100 and 200 μg/mL with fibrosarcoma (HT1080) or normal fibroblast (MRC5) cells are shown in Fig. 7(a-d). The normal cells exposed to both NO and FNO were significantly viable at pH 7.4 while cytotoxicity was observed at acidic (pH 6.4) condition in fibrosarcoma cells, which are in agreement with earlier reports on nanoceria under similar acid-base conditions.31,38,39 Increased cytotoxic effects of FNO with increasing exposure time and, to a lesser extent, nanoparticle concentration were observed at pH 6.4.


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Figure 7. Cell viability at different nanoceria concentrations: (a) 100 μg/mL at pH 7.4, (b) 100 μg/mL at pH 6.4, (c) 200 μg/mL at pH 7.4, (d) 200 μg/mL at pH 6.4 The redox behaviour of nanoparticles was investigated using two ROS-sensitive dyes (DCFHDA and Mitosox). DCFH-DA is used for H2O2 and •OH while mitochondrial •O2– is detected using Mitosox. These data are presented in Fig’s 8 and 9, which show the in vitro redox analyses for NO and FNO at pH 7.4 and 6.4. Fig. 8(a,c) and Fig. 9(a,c) show the antioxidant effects (biological pH 7.4) for NO and FNO, where the decreases in fluorescence provide direct measurements of ROS scavenged by nanoparticles. Conversely, Fig. 8(b,d) and Fig. 9(b,d) show the prooxidant effects (weak acidic pH 6.4), where the increases in fluorescence provide direct measurements of the ROS produced by nanoparticles. The antioxidant effect is more significant than the prooxidant effect owing to the pH-facilitated decrease in the reduction potential. Further, the redox performances at both pH values of the FNO were greater than those of the NO, which resulted from the establishment of the protein corona that forms around the latter.

The particle size, zeta potential, protein adsorption, and redox

performance data are consistent in suggesting higher cellular uptake of FNO compared to that of NO. These data allow differentiation of the extent of redox by nanoparticles for •O2– versus H2O2 and •OH, where the former is greater than the latter. These observations suggest that higher uptake of nanoparticles into cells occurs for those in close proximity to mitochondria.



Figure 8. Redox performance (DCF dye intensity) of nanoceria in fibrosarcoma cells: (a) 100 μg/mL at pH 7.4, (b) 100 μg/mL at pH 6.4, (c) 200 μg/mL at pH 7.4, (d) 200 μg/mL at pH 6.4

The Ce3+ ↔ Ce4+ redox switching and charge-compensating oxygen vacancies provide the mechanism for ROS regulation at the cellular level.

Hence, these vacancies serve as

catalytically active sites for the oxidation of ROS and the counter-reduction of oxygen and water molecules. The kinetics of these reactions can be regulated by particulate parameters38 but, more particularly, the pH of the medium. Consequently, for FNO, the following highresolution XPS data for the powders processed at normal physiological pH 7.4 and tumour microenvironment pH 6.4 were obtained:

At pH 7.4

Ce3+ (37 at%) / Ce4+ (63 at%) 24 ACS Paragon Plus Environment

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At pH 6.4

Ce3+ (30 at%) / Ce4+ (70 at%)

These data demonstrate that Ce3+ ↔ Ce4+ oxidation or reduction is enhanced in weak basic or acidic pH conditions, respectively. These data support the view that the redox conditions reflect potential prooxidative and antioxidative behaviour of nanoceria in biological media at different pH levels. Consequently, the in vitro ROS analysis data in Fig. 8(c,d) and Fig. 9(c,d) show that the dye (DCFH-DA and Mitosox) intensities at pH 7.4 are greater than those at pH 6.4, suggesting that antioxidation dominates prooxidation. This is in agreement with the observation that lower reduction potentials occur at higher pH values.43,44 Consequently, since the pH fluctuates within cells in the range 4.7-8.056, then these changes facilitate the Ce3+ ↔ Ce4+ switching, which is known to occur readily over the pH range ~4.5-8.5.51 Thus, repeat switching reversibility can occur in essentially all cellular conditions.



Figure 9. Redox performance (Mitosox dye intensity) of nanoceria in fibrosarcoma cells: (a) 100 μg/mL at pH 7.4, (b) 100 μg/mL at pH 6.4, (c) 200 μg/mL at pH 7.4, (d) 200 μg/mL at pH 6.4

In order to assess the cellular uptake behaviour of the nanoparticles, light microscopy, electron microscopy, and quantitative mass spectrometry were done for NO and FNO. These data are given in Fig. 10. The light microscopy images are shown in Fig. 10(a, b), where green regions represent the population of nanoparticles while cell membrane, nucleus, and lysosomes are indicated by red, blue, and white regions, respectively. However, since light microscopy provides indirect evidence of nanoceria uptake, electron microscopy analyses were done to confirm uptake, the data of which are shown in Fig. 10(c,d). These qualitative data are quantified through ICP-MS analyses for Ce contents, as shown in Fig. 10(e).

It is known that the surface charge of a nanoparticle can play a critical role in its circulation in the bloodstream as well as its delivery into the cell.57,58 The relevant factors in the surface charge are largely a function of the type and extent of exposed crystallographic plane as these involve the surface stoichiometry, surface chemistry, nature of the terminating bonds, atomic packing densities, surface energies, adsorption energies, acidities, etc.38,41,42 Many of these can be assessed by standard approaches, including determination of particle size (related to surface area), zeta potential, protein adsorption, and redox performance. In the present work, these parameters are consistent in demonstrating the greater cellular uptake and intracellular delivery of FNO relative to NO, thereby suggesting a link between the surface charge and chemistry differences between the functionalised and pristine nanoparticles. This is confirmed clearly by the opposite surface charges of these, where the slightly positively charged FNO is more readily taken up by the negatively charged cell membrane. Most importantly, since the functionalisation does not change the basic shape of the nanoparticles, then this avoids the spheroidisation generally associated with surface modification,59 the associated reduction of the cell-particle contact angle, and the resultant reduction in cellular uptake.58


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Figure 10. Cellular uptake of nanoceria: (a) Confocal microscopy image of NO, (b) confocal microscopy image of FNO; note: Green = nanoceria, red = plasma membrane, blue = nucleus, white = lysosomes, (c) TEM image of NO, (d) TEM image of FNO, (e) ICP-MS cell uptake of NO and FNO

Conclusions Green, aqueous, room-temperature, chemical synthesis methods to fabricate redox-active ceria nanoparticles have been developed.

The integrated methods include aqueous

surfactant-free precipitation to fabricate pristine nanoparticles, surface modification by silanisation, and functionalisation with zwitterionic sulfobetaine using O-PET-ATRP. The main 27 ACS Paragon Plus Environment


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advantages of these methods are the use of an environmentally-friendly reaction conditions, an environmentally benign solvent, and the potential for improved redox performance through the retention of active sites owing to the absence of organic surfactant and/or solvent. The present work is the first to report the use of water as a solvent for O-PET-ATRP. The effectiveness of this integrated green approach to synthesise discrete and redox-active nanoparticles for fibrosarcoma therapy was confirmed by FTIR, laser Raman microspectroscopy, XPS, TGA, TEM, and NMR. The functionalisation was successful in hindering the adsorption of nonspecific proteins and prolonging the blood circulation time by effectively retaining a small hydrodynamic particle size through the establishment of continuous, passivating, zwitterionic, electrically neutral surfaces.

Critically, the

functionalisation also resulted in the retention of the original particle shape, which avoided spheroidisation. These features contributed to the achievement of higher uptake of FNO compared to that of pristine nanoceria and consequent enhanced redox performance. The redox behaviour at cellular levels was altered by the pH of the biological environment, where physiological environment conditions of pH 7.4 were cytoprotective and antioxidative toward normal and cancer cells but tumour microenvironment conditions of pH 6.4 were cytotoxic and prooxidative toward cancer cells.

Competing financial interests The authors declare no competing financial interests.

Acknowledgements The authors wish to acknowledge grant support from the Australian Research Council (DP170104130), scholarship support (R.M.) through an Australian Government Research Training Program (RTP) Scholarship, and the characterisation facilities of the Mark Wainwright Analytical Centre at UNSW Sydney.

Author contributions R.M. designed the project, undertook experimentation and data analysis, wrote the first draft of the manuscript, and worked on all subsequent drafts. S.S.M. assisted in laser Raman microspectroscopy and data analysis. A.R. assisted in the NMR data acquisition and analysis. F.T. assisted in confocal imaging. X.W and J.L.Y. provided the facilities for all biological studies. 28 ACS Paragon Plus Environment

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P.K. provided technical support. C.C.S. provided subsequent data analysis, worked on all subsequent drafts of the manuscript, and supervised the project.

Supporting Information The supporting Information (Thermogravimetric analysis and dynamic light scattering data) is available free of charge via the Internet at http://pubs.acs.org.

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For Table of Contents Use Only Synopsis The present work reports an environmentally-friendly, integrated, room-temperature aqueous chemical technique for the synthesis of biomaterials for cancer therapy.


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Green Synthesis of Zwitterion-Functionalized Nano-Octahedral Ceria

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