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Photoluminescent Hybrids of Cellulose Nanocrystals and Carbon Quantum Dots as Cytocompatible Probes for in vitro Bio-imaging Jiaqi Guo, Dongfei Liu, Ilari Filpponen, Leena-Sisko Johansson, Jani-Markus Malho, Sakeena Quraishi, Falk Liebner, Hélder A. Santos, and Orlando J. Rojas Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 22 May 2017 Downloaded from http://pubs.acs.org on May 23, 2017
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Photoluminescent hybrids of cellulose nanocrystals and carbon Quantum dots as cytocompatible probes for in vitro bio-imaging Jiaqi Guo,1 Dongfei Liu,2 Ilari Filpponen,1,3* Leena-Sisko Johansson,1 Jani-Markus Malho,4 Sakeena Quraishi,5 Falk Liebner,5 Hélder A. Santos2 and Orlando J. Rojas1,4,6 1
Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, FI-00076 Aalto, Finland 2
Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland
3
Alabama Center for Paper and Bioresource Engineering, Department of Chemical Engineering, Auburn University, Auburn, AL 36849-5127, United States
4
5
Department of Applied Physics, School of Science, Aalto University, FI-00076 Aalto, Finland
Division of Chemistry of Renewable Resources, Department of Chemistry, University of Natural
Resources and Life Sciences Vienna, Konrad-Lorenz-Straße 24, 3432 Tulln an der Donau, Austria 6
Departments of Forest Biomaterials and Chemical and Bimolecular Engineering, North Carolina State University, Raleigh, NC 27695, United States
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KEYWORDS: cellulose nanocrystals (CNC); carbon quantum dots (CQD); carbodiimide coupling; bio-based hybrids; cytocompatibility
ABSTRACT
We present an approach to construct biocompatible and photoluminescent hybrid materials comprised of carbon quantum dots (CQD) and TEMPO-oxidized cellulose nanocrystals (TO-CNC). First, the amino-functionalized carbon quantum dots (NH2-CQD) were synthesized using a simple microwave method and the TO-CNCs were prepared by hydrochloric acid (HCl) hydrolysis followed by TEMPO-mediated oxidation. The conjugation of NH2-CQD and TO-CNC was conducted via carbodiimide-assisted coupling chemistry. The synthesized TO-CNC@CQD hybrid nanomaterials were characterized using X-ray photoelectron spectroscopy, cryo-transmittance electron microscopy, confocal microscopy and fluorescence spectroscopy. Finally, the interactions of TO-CNC@CQD hybrids with HeLa and RAW 264.7 macrophage cells were investigated in vitro. Cell viability tests suggest the surface conjugation with NH2-CQD not only improved the cytocompatibility of TO-CNC, but also enhanced their cellular association and internalization on both HeLa and RAW 264.7 cells after 4 and 24 h incubation.
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INTRODUCTION Nanomaterials have pervaded almost everywhere in industry – e.g., medicine, plastics, electronics, and aerospace.1,2 Their exceptional properties, such as high surface area, strength and hardness at high temperature, good wear, erosion and corrosion resistance, make them feasible for numerous applications.3 As a novel form of cellulose, nanocellulose has accounted a growing interest as a renewable building block for the design of advanced functional bio-materials.4 Nanocellulose, in particular, cellulose nanocrystals (CNC) can be isolated by means of top-down methods from variable botanical sources, such as wood, straw and cotton.5 Typically, CNCs are manufactured by hydrolysis of cellulosic fibers in concentrated sulfuric acid, yielding highly crystalline particles (up to 90%) with the dimensions of 100−300 nm in length and 5−10 nm in width.6 Owing to its superior properties including good mechanical strength, low density, high aspect ratio and the ability to form hydrogen bonds, CNC has been widely used to create novel biobased nanomaterials, such as polymer nanocomposites, mechanically adaptive materials and mesoporous photonic solids.7-10 Moreover, with regard to biological systems, numerous advantages of CNC, such as ignorable ability to induce inflammatory responses and low toxicological risk, have been demonstrated.11,12 The results have revealed that CNC exhibits low cytotoxicity to the animal and human cells when compared to those of other nanomaterials, such as multiwalled carbon nanotubes (MWCT) and certain metallic nanoparticles.13-15 Moreover, unlike microcrystalline cellulose (MCC), CNC has not been shown to trigger an inflammatory response in human monocyte-derived macrophages.16 In addition, it should be mentioned that CNC presents the ability to penetrate HEK-293 cells (derived from human embryonic kidney cells) and Sf9 insect cells (derived from the pupal ovarian tissue of Spodoptera frugiperda), and this penetration can be tuned by adjusting the surface charge of CNC.15 The interaction of nanomaterials with biological systems, such as cells, is determined by several factors such as the shape, size, material composition, stiffness, surface charge, and surface moieties
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of nanomaterials.17 Nanomaterials with larger aspect ratios and sharper angular are taken up in larger amounts and faster rates. Therefore, the needle-like morphology of TO-CNC possesses a great potential for biological applications. Another possible advantage of utilizing the rod-like shape of CNCs is that the spherical particles tend to have shorter blood circulation times, while anisotropic analogues can align with blood flow leading to longer circulation times. The abovementioned low toxicity, ignorable inflammatory response and the ability to penetrate cells make CNC a promising candidate to be utilized in different fields such as bone tissue engineering, drug delivery and carriers in biological systems.12,18-20 Nanocellulose-based imaging probes have been developed with fluorescent dyes, e.g. fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RBITC), coumarin derivative, Lucifer yellow derivative, and their interactions with cells have been investigated.18,21-25 Carbon quantum dots (CQD) – discovered in 2004 during purification of single-walled carbon nanotubes – constitute a comparatively new subclass within the big family of zero-, one- and twodimensional fluorescent, carbon-based nanomaterials.26-28 Obtained either by top-down or bottomup approaches, carbon quantum dots have spatial dimensions typically within the single-digit nanometer range similar as nanodiamonds, carbon nanotubes (CNT), fullerene nanoparticles, graphene nanoribbons, graphene oxide, graphene quantum dots and polymer dots. However, different from inorganic semiconductor quantum dots, both fundamentals of the observed fluorescence and opportunities to tailor the photoluminescence properties of CQD are still a matter of debate.29 Compared to traditional metal-based quantum dots (QD), CQDs have distinct advantages, such as low toxicity, renewability, biocompatibility, low cost, and better chemical resistance. These features render CQD as promising nanomaterial for a wide field of applications, such as biosensing, biomedical imaging, biomarker development, energy conversion and anticounterfeiting. Moreover, surface functionalization of CQD is not only used to improve quantum yield and water dispersibility, but also to tune the emission wavelengths30 or enhance the interaction
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of CQD with other (nano)materials.31,32 Due to their excellent optical properties, CQDs have mainly been investigated as bioimaging agents.33 Albeit earlier investigations have described the covalent attachment of CQD on to carboxymethylated CNF and a conjugation of QD to CNC,34,35 there are no reports on the use of CNC as a carrier for covalently attached CQD. In this work, we present a facile approach towards novel photoluminescent TO-CNC@CQD hybrid materials and, for the first time, explore their interaction with HeLa and RAW 264.7 macrophage cells where the hybrid function is used as a bio-imaging probe. Fluorescent NH2-CQD, TO-CNC and their conjugated TO-CNC@CQD hybrids were characterized using techniques such as atomic force microscopy (AFM), transmittance electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), confocal microscopy and fluorescence spectroscopy. Furthermore, the conjugation of NH2-CQD with TO-CNC model surface was demonstrated in situ by means of quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). We foresee that the synthesized non-toxic and photoluminescent hybrids may find use in bio-imaging, biotoxicity studies, sensing devices and optical components.
MATERIALS and METHODS Materials. All chemicals and materials in this work were used of analytical grade without further purification. Main used chemicals, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2,2,6,6-tetramethyl-1-piperidinyloxyradical (TEMPO) and 2,2-(ethylenedioxy)-bis-(ethylamine) were purchased from Sigma-Aldrich. Milli-Q (MQ) water (resistivity 18.2 MΩ) was used for all aqueous solutions preparation. Synthesis of cellulose nanocrystals (CNC). CNCs used in this work were prepared via acid hydrolysis of ashless filter paper according to the method detailed by Araki et al.36 5.0 g of grinded filter paper (30 mesh, Whatman 541 filter paper) were mixed with 150 mL of HCl (4 N) solution, and the hydrolysis reaction was allowed to proceed ~4 h at 80 °C. The resultant mixture was
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precipitated in 2500 mL MQ-water overnight, and then washed by centrifugation at 8000 rpm until pH above 4. The high content CNC suspension (ca. 50 g/L) was collected after dialysis for one week (MWCO 6000−8000Da). TEMPO-mediated oxidation of CNC (TO-CNC). TO-CNCs were obtained via TEMPOmediated oxidation according to the method outlined elsewhere.37,38 14.6 mL of the as prepared CNC suspension (44.5 g/L) was dispersed in 35.4 mL H2O (total volume 50 ml) containing 10 mg of 2,2,6,6-tetramethyl-1-piperidinyloxyradical (TEMPO, 0.065 mmol) and 200 mg of sodium bromide (1.9 mmol). Prior to the oxidation, the suspension was allowed to stir for 30 min. 4.9 mL of 13% NaClO (8.6 mmol) solution was then slowly added over 20 min under stirring and the pH was kept around 10 by adding 0.5 M of NaOH (produced COOH-groups decrease the pH). The reaction was considered complete when the pH remained stable. 5 mL of methanol was then added to quench the oxidation reaction. The mixture was dialyzed against deionized (D.I.) water for one week after adjusting the pH to 7. The translucent suspension was stored at 4 °C until further use. The dry content of TO-CNC was evaluated (ca. 6.6 g/L). Synthesis of amino-functionalized carbon quantum dots (NH2-CQD). The NH2-CQDs were synthesized as described elsewhere.34,39 Briefly, 5 mL of glycerol and 2 mL of 10 mM of sodium phosphate solution were mixed with 1 mL of 2,2-(ethylenedioxy)-bis-(ethylamine) in a small beaker covered with aluminum foil. Then microwave-assisted pyrolysis was carried out for 10 min at 750 W. After cooling down to room temperature, the high viscous syrup was diluted and dialyzed against distilled water (MWCO 1000 Da) for 3 days. The light yellow-brown aqueous suspension was collected and stored at 4 °C for further usage. The dry content of NH2-CQD suspension was measured to be ca. 4.5 g/L. Synthesis of fluorescent TO-CNC@CQD hybrid nanomaterials and TO-CNC covalently equipped with a fluorescent reactive dye. Both amino-functionalized CQDs and the reactive dye Alexa FluorTM 488 were grafted onto respective TO-CNC samples by common EDC/NHS coupling
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chemistry as detailed in previous reports.40,41 Briefly, an aqueous dispersion of TO-CNC (40 mL, 6.6 g/L) was activated with 0.38 g of EDC and 0.46 g of NHS under vigorous stirring at pH 5 for 15 min. Subsequently, 5.5 mL of a 4.4 g/L aqueous dispersion of NH2-CQD (pH 9.2, phosphate buffer) was added. The reaction was left to proceed overnight under gentle mixing at room temperature. The resulting suspension was washed and dialyzed (MWCO 12000−14000 Da) against distilled water and stored for further usage. The dry content of TO-CNC@CQD hybrid materials was measured to be ca. 4 g/L. For the labeling of TO-CNC with the reactive dye Alexa Fluor® 488, 1 mL of TO-CNC (6.6 g/L) was activated with 9.5 mg of EDC and 11.5 mg of NHS under stirring at pH 7 for 30 min, and subsequently, 50 µg of the cadaverine derivative (AF488; Thermo Fisher, USA) was added. The reaction was left to proceed overnight under stirring (400 rpm) at room temperature. The resulting suspension (AF488-CNC) was washed and dialyzed (MWCO 12000−14000 Da) against D.I. water. The final concentration of TO-CNC/AF488 was fixed to 2 g/L. Infrared spectroscopy. FTIR characterization was performed with a Thermo Scientific Nicolet Avatar 360 FTIR spectrometer in absorbance mode using the KBr pellet technique. Spectra were acquired for a total of 32 scans in the range of 400−4000 cm–1 with a resolution of 4 cm–1. Fluorescence spectroscopy. Photoluminescence (PL) emission measurements of liquid samples were carried out using a LS 45 Fluorescence Spectrometer (PerkinElmer, Finland). The excitation wavelength was set up at 365 nm. The emission was recorded from 365 to 650 nm. Zeta (ζ)-potential measurements. The ζ-potential of 0.25 g/L aqueous dispersions of NH2-CQDs was measured using a Nano ZS90 Instrument, and the pH was adjusted by adding a small amount of 0.5 M of NaOH or HCl. Quantum yield measurements. The quantum yield, Φ, of the synthesized NH2-CQD was calculated using quinine sulfate (Φst = 0.54) as a reference standard. From the given NH2-CQD solution, five different concentrations were prepared. The absorbance of these concentrations lied in
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the range of 0.02−0.1. The integrated fluorescence intensity was evaluated by calculating the area under the emission curve using a Hitachi-F7000 fluorescence spectrometer. The Φ of the NH2-CQD was calculated according to the following equation: Φs = Φst (Ms/Mst) (ηs2/ηst2) where Y was the quantum yield, M was the measured integrated emission intensity, and η was the optical density. The subscript “st” refers to standard with known quantum yield and “s” for the sample. ηs (H2O) and ηst (0.1 M H2SO4) are the refractive indices of the solvents which are both approx. 1.33.(See supporting information) Confocal laser scanning microscopy. Leica TCS SP2 confocal laser scanning microscopy (Leica microsystems CMS GmbH, Manheim, Germany) was applied to study the fluorescence of TO-CNC@CQD hybrids. The fluorescence images were taken from dry samples, which were dropped onto cleaned silica wafers. Images (750 × 750 µm2) were obtained at excitation and detection wavelengths of 488 and 500−530 nm, respectively. The intensity images were scanned using an averaging mode and constant imaging conditions (laser powers of 500 and 600 V during surface imaging). Atomic force microscopy. TO-CNC deposited on a silica wafer were characterized in air at room temperature (23 ºC) via a Multimode AFM with a Nanoscope V controller operated at tapping mode (Bruker Corparation, USA). Transmission electron microscopy (TEM). Bright field TEM images were acquired using a FEI Tecnai 12 TEM operating at 120 kV. Cryo-TEM imaging was performed using Jeol 3200FSC cryoTEM, which was operated at 300 kV in bright field mode. The imaging was carried out using an Omega-type Zero-loss energy filter. Images were acquired with Gatan Ultrascan 4000 CCD camera. Vitrified samples were prepared using a Fei Vitrobot by pipetting 3 µL of a sample dispersion on to holey carbon copper grids under 100% humidity. The samples were cryo-transferred to the cryoTEM.
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X-ray photoelectron spectroscopy (XPS). The surface chemical composition of synthesized TO-CNC, NH2-CQD and TO-CNC@CQD hybrids was investigated using a Kratos Analytical AXIS Ultra electron spectrometer equipped with a monochromatic Al Kα X-ray source at 100 W and a neutralizer. The XPS experiments were performed on the dry thin-films, in which the suspensions were precipitated on cleaned silica wafer. 100% cellulose ash free filter paper was employed as an in-situ reference in the measurements.42 In order to obtain reliable results, at least three spot areas of each sample were scanned with the size of 400×800 µm. TO-CNCs model film preparation. In order to employ surface-sensitive techniques, e.g. SPR and QCM, it is necessary to prepare thin, uniform cellulose model surfaces. In this work, TO-CNC model films were prepared according to the procedure outlined by Aulin et al.43 Gold-coated wafer (SPR measurement) and quartz sensors (QCM measurement) were firstly cleaned in an UV/ozone oven for 15 min. Prior to grafting of TO-CNC, the substrates were coated with 1 g/L of polyethylene imine (PEI) to enhance the interaction of TO-CNC with the respective substrates. The concentration of TO-CNC for spinning coating was 1.5 g/L. Finally, the coated substrates were cured at 80 ºC for 10 min. Quartz crystal microbalance with dissipation monitoring (QCM-D). The interactions of NH2CQD with TO-CNC were studied using a Q-Sence E4 Device (Västra Frölunda, Sweden) and a constant liquid flow of 100 µL/min. The covalent interaction of NH2-CQD with the cellulose sensor surface can be assessed based on the shift of frequency. In the experiments, QCM sensors were coated with TO-CNC using a spin coating technique. The measurements were not started unless the frequency stabilization with MQ-water/buffer had been completed. In order to activate the TO-CNC sensor surface, EDC/NHS solution was introduced (pH 5) and the NHS-CNC surface was formed. After extensive rinsing, the activated TO-CNC, i.e., NHS-CNC, surface was exposed to the respective NH2-CQD suspension (30 min, pH 9.2, 20 mM). When the plateau of NH2-CQD conjugation with the TO-CNC sensor surface was reached, the latter was rinsed with buffer and
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MQ-water, respectively. In order to understand the roles of electrostatic and covalent interactions between TO-CNC and NH2-CQD, a control experiment was carried out following the above procedure, except without EDC/NHS activation. Surface Plasmon Resonance (SPR). SPR experiments were performed with a SPR Navi 200 (Oy Bionavis Ltd., Tampere, Finland) using exactly the same experimental conditions as in QCM-D measurements. Due to the conjugation of mobile molecules on to the stationary model surface, the optical resonance at the interface between the solid surface and the surrounding medium becomes shifted and can be monitored as a change in the SPR angle. Moreover, while compared with QCMD measurements, SPR has a negligible coupled water/buffer effect which allows the determination of adsorbed dry mass.44 Cell culture. The interaction of cells with the synthesized TO-CNC@CQD hybrid materials were studied for HeLa (human cervical cancer cells, passage #27–32) and RAW 264.7 macrophage (murine leukemic monocyte macrophages, passage #20–23) cells (both from the American Type Culture Collection, USA). The cell lines were selected due to their ability to mimic the different cell-types of the body and, therefore, relevance in biological applications. Specifically, the selected RAW 264.7 was to mimic the mononuclear phagocyte system, which is a part of the immune system. HeLa cells derive from human cervical cancer cells, which have been widely used to develop the cancer models. It is worth mentioning here that by studying the interaction of CQDsCNCs and HeLa cells, it is possible to evaluate the potential of CQDs-CNCs in cancer therapy. Both cell types were cultured in Dulbecco’s modified Eagle’s medium with 4.5 g/L glucose, supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% L-glutamine, penicillin (100 IU/mL), and streptomycin (100 mg/mL) (all from Hyclone, GE Healthcare, USA). The cell cultures were maintained in a standard incubator (BB 16 gas, Heraeus Instruments GmbH, Germany) at 37 °C with an atmosphere of 5% CO2 and 95% relative humidity. Prior to each test,
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the cells were harvested with 0.25% (v/v) trypsin/ethylene diamine tetraacetic acid/phosphate buffer saline (PBS). Cell viability tests. The cytocompatibility of NH2-CQD, TO-CNC and TO-CNC@CQD hybrid materials towards HeLa and RAW 264.7 macrophage cells was evaluated. The negative control was 1× Hank's balanced salt solution (HBSS, pH 7.4). HeLa or RAW 264.7 macrophage cells (100 µL, 2×105 cells/mL) were seeded in 96-well plates and left to attach for 24 h. After cell attachment, the medium was removed and cells were washed three times with 1× HBSS (pH 7.4). Then each formulation in 1× HBSS (100 µL) was added into each well and incubated for 4 or 24 h. After removing the samples and washing for three times with 1× HBSS, the CellTiter-GloTM reagent (Promega, USA) was used to quantify ATP as an indicator of metabolically active cells present. The plate was then incubated at room temperature for 20 min to stabilize the luminescent signal. The luminescence was recorded on a Varioskan flash fluorometer (Themofisher Scientific, USA). Confocal microscope images. For the confocal microscope images, suspensions of HeLa or RAW 264.7 macrophage cells (400 µL, 1×105 cells/mL) were seeded in Lab-Tek Chamber Slides (Thermo Fisher Scientific, USA). After 24 h of cell attachment, the wells were rinsed three times with 1× HBSS (pH 7.4) and then the NH2-CQD, AF488-CNC and TO-CNC@CQD hybrid nanomaterials (50 µg/mL) were added. After 4 or 24 h of incubation, the liquids were removed and the wells were washed three times with 1× HBSS (pH 7.4). CellMaskTM (Thermo Fisher, USA) with a concentration of 2 µg/mL was added for cell plasma membrane staining, according to the manufacturer’s specifications. Then the cells were fixed with paraformaldehyde (PFA, 4.0% w/v) in 1× phosphate buffer saline (PBS) for 20 min at room temperature. Confocal images were taken with a Leica TCS SP5II (Leica Microsystems, Germany) inverted confocal fluorescence microscope, equipped with argon (488 nm) and UV (diode 405 nm) lasers, and using a HCX PL APO 63×/1,20 W CORR/0.17 CS (water) with a water immersion micro dispenser.
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Flow cytometry studies. The extent of cell uptake for the prepared nanomaterials was analyzed using flow cytometry. HeLa or RAW 264.7 macrophage cell suspension (1×106 cells/mL, 2.0 mL) was seeded in 6-well plates. Following by 24 h of cell attachment to the wells, the cells were firstly rinsed three times with 1× HBSS (pH 7.4), and then the NH2-CQD, AF488-CNC and TOCNC@CQD hybrid materials (50 µg/mL) were incubated with the cells for 4 or 24 h. After washing with 1× HBSS (pH 7.4), the cells were harvested, and then fixed with PFA (4.0% w/v) in 1× PBS for 20 min at room temperature. Exactly 10000 events were collected on a LSR II flow cytometer (BD Biosciences, USA) with a laser excitation wavelength of 488 nm using FACSDiva software. Statistical analyses. Results are expressed as the mean ± standard deviation (s.d.) for at least three independent experiments. Data were analyzed using one-way analysis of variance (ANOVA) followed by a Bonferroni’s post-hoc test for multiple comparisons (Origin 8.6; OriginLab Corp., USA).
RESULTS AND DISCUSSION Preparation of TEMPO-oxidized cellulose nanocrystals (TO-CNC). Typically, CNCs are produced by hydrolysis with either hydrochloric (HCl) or sulfuric (H2SO4) acid.6 The principal difference between these two methods is the formation of negatively charged sulfate half ester groups during H2SO4 hydrolysis.45 These groups provide electrostatic repulsion between the CNCs, thus making their dispersion more stable than that of the HCl-hydrolyzed CNCs, which tends to readily aggregate. However, sulfate groups are rather labile and therefore their presence has a detrimental effect to the thermal properties of CNC. Moreover, they can also contribute to unwanted side reactions, such as nucleophilic substitution and elimination. In the present study, CNCs were first produced by HCl hydrolysis and then oxidized using TEMPO-mediated oxidation to produce the TO-CNC. It is well-known that only the primary hydroxyl groups on the surface of native CNC become oxidized during the TEMPO-oxidation.45 TEMPO-oxidized CNCs were characterized by
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FTIR and a new band at 1730 cm-1 attributed to the C=O stretching was assigned (see Figure S1). The degree of oxidation (DO) was estimated by the intensity ratio between the band at 1730 cm-1 (C=O) and 1050 cm-1 (band from cellulose backbone). The DO value was found to be approximately 0.24, which is in agreement with our previous report.37 The morphology of TO-CNC was investigated using AFM and TEM (see Figure S2). As expected, CNCs were rod-like particles having dimensions of ca. 5−10 nm (width) and ca. 200 nm (length). These findings indicate that the introduction of carboxyl groups did not significantly affect size and shape of CNCs. Preparation of amine-bearing carbon quantum dots (NH2-CQD). For the synthesis of the photoluminescent NH2-CQD we used a previously reported method.34,39 The synthesized NH2-CQD exhibited an excellent water-solubility and the emission of green luminescence under the UV light (excitation wavelength of 365 nm) is demonstrated in Figure 1d. The charge density of the NH2CQD was determined by polyelectrolyte titration34 and it was found to be + 500 µmol/g at pH 4.5. It is worth mentioning that the obtained charge density results of NH2-CQD followed the trend of previously reported study and no positive charge at pH above 8.5 was observed.34 The pH dependent charge of NH2-CQD was further confirmed by ζ-potential measurements, as shown in Figure 1c. FTIR technique was applied to explore the structural features of NH2-CQD (Figure 1a). The signals were attributed to amide I (1645 cm-1), amide II (1570cm-1) and amide III stretching bands (1472cm-1), respectively. Moreover, the stretching bands at 3380 cm-1 and 1113 cm-1 were attributed to stretching of –OH, N-H and C-O(H), respectively.46 The dimensions of synthesized NH2-CQD were determined by TEM which indicated round particles with an average size of 10 nm (Figure 1b).
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Figure 1. NH2-CQD characterization: (a) FTIR confirms the amino-functionalization of NH2-CQD; (b) TEM image showing the morphology of NH2-CQDs (scale bar: 100 nm). (c) Mean ζ-potential values of NH2-CQDs as a function of different pH values. (d) Photographs of TO-CNC (top) and TO-CNC@CQD (bottom) suspensions under visible and UV light (365 nm).
Attachment of NH2-CQD on TO-CNC model thin films. The interactions between NH2-CQD and TO-CNC were monitored by surface sensitive QCM-D and SPR techniques. As a simple and high-resolution mass sensing technique,47 QCM has been extensively employed to study polymer and protein adsorption,48-51 quantum dot adsorption,52 covalent interaction53 and gas-phase reactions.54 Moreover, QCM-D is sensitive towards conjugation of mobile molecules onto stationary model surfaces and provides information regarding the degree of hydration of (coupled water). By combination of QCM-D with SPR, better understanding of hydration and conjugation can be achieved.44 In this study, we employed QCM-D to demonstrate the EDC/NHS-assisted
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covalent coupling between the carboxyl groups of TO-CNC and the amine groups of NH2-CQD. It is important to note that under acidic and even neutral conditions, the surface amine groups of NH2CQD are positively charged (RNH3+ groups), whereas under alkaline conditions these groups are deprotonated and converted to neutral NH2 groups.34 Therefore, the QCM-D experiments were conducted at pH 9.2 (buffer) to minimize non-specific interactions, i.e., electrostatic attraction between positively charged NH2-CQD and negatively charged TO-CNC. Figure 2a shows the QCM-D sensograms of the NH2-CQD attachments onto the TO-CNC surface with and without EDC/NHS activation. After introducing of NH2-CQD into the system, the negative frequency change is observed in both the EDC/NHS activated and nonactivated reference TO-CNC model surfaces, i.e., the amount of adsorbed mass is increased (Figure 2a). This increase in the adsorbed mass might be attributed to the water uptake (swelling) of TOCNC at alkaline conditions and/or the EDC/NHS assisted coupling reaction of the NH2-CQD and TO-CNC.46 However, it should be noted here that the frequency of EDC/NHS activated surface (red profile) is slightly more decreased than that of the reference surface (black profile). This indicates that a higher amount of NH2-CQDs can be attached to the surface of TO-CNC when EDC/NHS activation is employed. However, the amount of attached NH2-CQD was found to be lower than those reported for the adsorption of cationic polyelectrolytes onto cellulose model films.55 The results presented thus far point out that NH2-CQD can be attached onto the TO-CNC model surface, while the contributions of the coupled water and physically bound NH2-CQD to the mass gain could not be exactly measured. Therefore, the same set of experiments was carried out using SPR, which allows monitoring of the adsorbed dry mass only. Figure 2b shows that the SPR angle increased (increased adsorption) when NH2-CQDs were introduced onto the EDC/NHS activated TO-CNC model surface (red profile), while no significant change was observed in the case of the nonactivated reference surface (black profile). Moreover, it is important to note here that the reference curve returned to the baseline level after rinsing at pH 9.2 whereas in the case of the EDC/NHS
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activation the NH2-CQD still remained at the TO-CNC surface. This is taken as an indication of EDC/NHS assisted coupling of the carboxyl groups of TO-CNC and the amine surface groups of the NH2-CQD. The conjugated amount of CQD was calculated from the QCM-D and SPR data (see Supporting Information) and the amounts were found to be 47.2 ng/cm2 and 4.9 ng/cm2, respectively. It should be mentioned here that the significant difference between the CQD amounts can be explained by the bound water which can only be observed during the QCM-D measurements.
Figure 2. Impact of EDC/NHS activation of TO-CNC model films on the covalent attachment of NH2-CQDs as a function of elapsed coupling time as monitored by (a) QCM-D and (b) SPR. The ionic strength was kept constant (20 mM). Red curve denotes the EDC/NHS activated TO-CNC model surface and black curve indicates the non-activated reference TO-CNC model surface. The EDC/NHS activation step is not included in the sensograms. Surface elemental composition of TO-CNC@CQD hybrids. X-ray photoelectron spectroscopy (XPS) was employed to analyze the surface chemical composition of the synthesized hybrid materials. This technique has been utilized for exploring the surface chemistry of various bio-based substrates.56 Wide scan spectra of TO-CNC, NH2-CQD and TO-CNC@CQD are shown in Figure
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3a−c, which illustrate the high resolution regions of N 1s and C 1s, respectively. The surface elemental compositions were calculated from the peak intensities of XPS spectra and are shown in Table 1. No significant differences between the TO-CNC and cellulose standard were observed. Moreover, the O/C ratio of TO-CNC (O/C = 0.6) was found to be similar with that of cellulose standard (Table 1). As expected, the N signal of NH2-CQD was significantly higher than that of TO-CNC sample (Figure 3b). Table 1 indicates that the NH2-CQD presents a higher N content (7.1 ± 0.1%), a higher C content (72.2 ± 0.4%) and lower O/C ratio (0.26) when compared to that of the TO-CNC. Interestingly, the N content was found to decrease to 2.6 ± 0.1% after conjugating the NH2-CQD with TO-CNC (to form TO-CNC@CQD hybrids). Based on this, the loading efficiency of NH2-CQD onto the TO-CNC was calculated to be ~36%. Moreover, the O/C ratio reduced from 0.6 to 0.5, which can be taken as a further indication of the successful coupling reaction.
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Figure 3. XPS spectra of TO-CNC (red), NH2-CQD (blue) and TO-CNC@CQD hybrid (green). The samples were deposited on silica wafer and air-dried. (a) A wide spectrum. (b) and (c) High resolution regions of N 1s and C 1s. Table 1. and C
Table 1. Surface chemical composition of TO-CNC, NH2-CQD and TO-CNC@CQD hybrids. The data was obtained from the peak intensities of XPS spectra shown in Figure 3. Surface composition (%) O 1s
N 1s
O/C
TO-CNC
35.7±1.1 62.1±1.1 0.55±0.1
0.6
NH2-CQD
19.0±0.4 72.2±0.4
7.1±0.1
0.26
TO-CNC@CQD
32.5±0.7 64.4±0.5
2.6±0.1
0.5
0
0.67
Filter Paper* *
C 1s
40.1
59.8
Whatman filter paper as a reference. Fluorescence properties of TO-CNC@CQD hybrid materials. Figure 1d shows the
photographs of TO-CNC and TO-CNC@CQD suspensions which both appear as translucent and homogeneous, while a color change (yellow-brownish) is observed after the conjugation of NH2CQD onto the TO-CNC. The yellow-brownish color originates from the ordinary color of NH2CQD. The excitation of TO-CNC@CQD with UV light (365 nm) resulted in a green fluorescence, whereas in the case of the pristine TO-CNC no emission was observed. In addition, the cryo-TEM image of TO-CNC@CQD in Figure 4b reveals the presence of CQDs only on the surface of CNCs. Fluorescence properties of TO-CNC and TO-CNC@CQD were further investigated by confocal fluorescence microscopy (Figure 4a). As expected, the TO-CNC@CQD hybrids exhibited a strong green fluorescence while excited at 488 nm. Moreover, fluorescence spectroscopy was employed to investigate the wet state properties of TO-CNC, NH2-CQD and TO-CNC@CQD (Figure 4c). The spectra revealed a strong fluorescent signal at 450 nm for the NH2-CQD and TO-CNC@CQD,
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which further indicates that the photoluminescent bio-based hybrids were successfully synthesized. Moreover, the photoluminescence quantum yield of NH2-CQD was measured (as a function of pH) by using quinine sulfate as a reference and 360 nm as the excitation wavelength. As expected, the highest quantum yield (16.8%) was obtained at lower pH after which it gradually decreased to 9% upon increasing the pH (see supporting information).
Figure 4. (a) Fluorescence characterization of TO-CNC (lower) and TO-CNC@CQD (upper) by confocal microscopy. Scale bar: 80 µm. (b) TEM image of the TO-CNC@CQD. Red arrows
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indicate the CQDs on the surface of TO-CNC. Scale bar: 50 nm. (c) Fluorescence spectra of dispersions of TO-CNC, NH2-CQD and TO-CNC@CQD.
Cell viability tests. The nature and extent of interactions between cells and nanomaterials are affected by both the cell type and features of the respective nanomaterials, such as composition, size, shape, crystallinity, surface charge and surface ligands.57-59 The cytocompatibility of TO-CNC, NH2-CQD and TO-CNC@CQD with different cancer cell lines (HeLa cells, RAW 264.7 macrophages) were evaluated using an ATP-based luminescent assay (Figure 5). The cell lines were selected due to their importance as suitable models mimicking the different cell-types of the body (epithelial cells and macrophages). For NH2-CQD within the tested concentration range (1−1000 µg/mL), no significant decrease on the cell viability for HeLa and RAW 264.7 macrophage cells were observed after 4 h incubation (Figure 5a and c). When we increased the incubation time to 24 h, both cell lines showed a clear NH2-CQD concentration-dependent cell viability, especially RAW 264.7 macrophage cells. Specifically, at a NH2-CQD concentration of 1000 µg/mL, the viability of HeLa and RAW 264.7 macrophage cells dropped to ca. 80% and ca. 40%, respectively, after 24 h incubation. Among all nanomaterials evaluated, TO-CNC turned out to have the most pronounced concentration-dependent cell anti-proliferation effect on both cell lines studied after 4 and 24 h incubation.60 For example, the RAW 264.7 macrophage viability decreased to ca. 50% after 4 h incubation with TO-CNC (1000 µg/mL) and ca. 40% more after extending the incubation time to 24 h. NH2-CQDs are positively charged (in neutral and acidic conditions) spheres, whereas TO-CNCs show needle-like morphology with negatively charged surface (at pH above 4). The stronger cell anti-proliferation effect of TO-CNC suggested that the morphology induced cell toxicity was stronger than that induced by the surface charge. In comparison to bare TO-CNC, the conjugation of NH2-CQD onto the surface of TO-CNC promoted an increase in cell viabilities in both cell lines studied after 4 and 24 h incubation. The less pronounced cell anti-proliferation effect for TO-
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CNC@CQD hybrid nanomaterials could be attributed to the surface cover of TO-CNC by the relatively cytocompatible NH2-CQD.
Figure 5. Cytocompatibility of different nanomaterials with HeLa (a and b) and RAW 264.7 macrophage cells (c and d) after 4 h (a and c) and 24 h (b and d) incubation as a function of the nanomaterial concentration. The 1× HBSS (pH 7.4) solution and Trition X-100 were used as the negative and positive controls, respectively. Errors bars represent the mean ± s.d. (n = 4). The viability of the cells incubated with NH2-CQD and TO-CNC@CQD hybrids were both compared with those incubated with TO-CNC; the levels of significance were set at probabilities of *p < 0.05, **p < 0.01, and ***p < 0.001. We evaluated the cellular interactions of the nanomaterials (AF488-CNC, NH2-CQD and TOCNC@CQD hybrids) with HeLa (Figure 6) and RAW 264.7 macrophages (Figure 7) cells by confocal fluorescence microscopy after 4 and 24 h incubation. In Figure 6 and Figure 7, all the cells showed a normal morphology under the concentrations studied (50 µg/mL), indicating a good cell viability and supporting the cell viability studies in Figure 5. In Figure 6a and Figure 7a, more NH2CQD nanoparticles were associated and internalized with HeLa and RAW264.7 macrophage cells
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than AF488-CNC after 4 h incubation. The surface charge of the nanomaterials is known to play a crucial role in cell association and internalization, where positively charged particles are associated and internalized in a greater extent than those negatively charged ones.57 Moreover, the shape and size of nanomaterials have also been found to greatly affect their cellular uptake.61 Therefore, the positively charged surface and smaller particle size of NH2-CQD can be assumed to translate into their more pronounced cellular association and internalization. Surface conjugation of NH2-CQD was supposed to improve the cellular interaction of TO-CNC which was confirmed even after short incubation time (4 h, Figure 6a and Figure 7a). When the incubated time increased to 24 h (Figure 6b and Figure 7b), an enhancement of cellular association and internalization was only detected for NH2-CQDs.
Figure 6. Confocal fluorescence micrographs of HeLa cells incubated at 37 ºC with NH2-CQDs, AF488-CNCs and TO-CNC@CQD hybrids for 4 h (a) and 24 h (b). The plasma cell membranes are stained with CellMask in red and the nanomaterials are in green.
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Figure 7. Confocal fluorescence micrographs of RAW 264.7 macrophage cells incubated at 37 ºC with NH2-CQDs, AF488-CNCs and TO-CNC@CQD hybrids for 4 h (a) and 24 h (b). The plasma cell membranes are stained with CellMask in red and the nanomaterials are in green. The extent of cell internalization after 4 and 24 h of incubation was also assessed using flow cytometry (Figure 8). As shown in Figure 8a and Figure 8b, the NH2-CQD associated and internalized to a larger extent in HeLa cells than the AF488-CNC and TO-CNC@CQD hybrid nanomaterials regardless of the incubation time. The mean fluorescence intensity (MFI) for HeLa cells was ca. 1172 for NH2-CQD after 4 h incubation; this value increased to ca. 1209 after 24 h incubation. In contrast, the MFI value for AF488-CNC was ca. 148 and 130 after 4 and 24 h incubation with HeLa cells. The greater extent of cellular internalization of NH2-CQD can be ascribed to their positively charged surface and small particle size. Surface conjugation of NH2CQD with TO-CNC had a similar impeding effect on internalization, however, to a somewhat lesser extent as evident from the MFI values (ca. 270 and 287 for 4 and 24 h incubation with Hela cells).
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The increased MFI values for TO-CNC@CQD hybrids (while compared to those of AF488-CNC) can be attributed to the successful surface conjugation of the TO-CNC with positively charged NH2CQD to enhance the cellular interactions of TO-CNC. RAW 264.7 cells are a macrophage-like cell line, and their association and internalization of nanomaterials is believed to be more pronounced.62 Specifically, the MFI values for NH2-CQD, AF488-CNC and TO-CNC@CQD hybrids were ca. 1553, 192 and 371 for 4 h incubation with the RAW 264.7 macrophage cells. After 24 h incubation, the MFI values increased to ca. 2855 for NH2-CQD, ca. 351 for AF488-CNC, and ca. 586 for T)CNC@CQD hybrids confirming the cell internalization trend obtained for HeLa cells. Overall, the confocal fluorescence microscope experiments are corroborated with the flow cytometry results.
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Figure 8. Flow cytometry histograms (left column) of NH2-CQD, AF488-CNC and TOCNC@CQD hybrids (100 µg/mL) incubated with HeLa (a and b) and RAW 264.7 macrophage cells (c and d) for 4 h (a and c) and 24 h (b and d). For the flow cytometry analysis, the mean fluorescence intensities (MFI, right column) of each nanomaterials were calculated. Errors bars represent the mean ± s.d. (n = 3). The MFI values are all compared with the control cells (#); NH2-
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CQD and TO-CNC@CQD@ hybrids are both compared with AF488-CNCs (*); the levels of significance are set at probabilities of #p < 0.05, ##p < 0.01, and *** and ### p < 0.001.
CONCLUSIONS We have prepared photoluminescent cellulose nanocrystals functionalized with biocompatible and non-toxic carbon quantum dots via carbodiimide-assisted coupling chemistry. The successful preparation was confirmed by X-ray photoelectron spectroscopy, cryo-transmittance electron microscopy, confocal microscopy, and fluorescence spectroscopy. In vitro studies showed that the TO-CNCs present stronger cell anti-proliferation effect when compared to that of the NH2-CQDs which suggested the significant role of the morphology induced cell toxicity. Moreover, the cytocompatibility of TO-CNC towards HeLa and RAW 267.4 macrophage cells was improved by surface conjugation with NH2-CQDs. The cellular association and internalization of TOCNC@CQD hybrids were more pronounced for both cell types tested compared to the CQD-free TO-CNC counterparts. The results obtained in this work demonstrate the potential of TOCNC@CQD hybrid nanomaterials for bio-imaging applications.
ASSOCIATED CONTENT The following files are available free of charge. Supporting information. FTIR spectra of cellulose nanocrystals (CNC) and TEMPO-oxidized cellulose nanocrystals (TO-CNC); AFM and TEM characterization of TO-CNC; The details of the adsorbed mass calculations based on the QCM-D and SPR measurements.
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AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected];
ACKNOWLEDGEMENT This research was supported by the Academy of Finland through its Centers of Excellence Programme (2014-2019) and in part by the Austrian Federal Ministry for Agriculture, Forestry, Environment and Water Management through the WoodWisdom Net+ project AeroWood (S. Quraishi, F. Liebner). Dr. Joseph Campbell is greatly acknowledged for performing the XPS analysis.
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