Graphene Oxide and Pluronic Copolymer AggregatesâPossible Route

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Graphene Oxide and Pluronic Copolymer Aggregates – Possible Route to Modulate the Adsorption of Fluorophores and Imaging of Live Cells Niloy Kundu, Arpita Roy, Debasis Banik, Jagannath Kuchlyan, and Nilmoni Sarkar J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.5b05251 • Publication Date (Web): 12 Oct 2015 Downloaded from http://pubs.acs.org on October 13, 2015

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The Journal of Physical Chemistry

Graphene Oxide and Pluronic Copolymer Aggregates – Possible Route to Modulate the Adsorption of Fluorophores and Imaging of Live Cells Niloy Kundu, Arpita Roy, Debasis Banik, Jagannath Kuchlyan and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India E-mail: [email protected] Fax: 91-3222-255303 Abstract In recent years, amphiphilic triblock copolymers have attracted increasing attention due to their tunable properties and biocompatible nature and the degree of hydrophobicity of these block copolymers can be modulated by varying the hydrophobic poly (propylene oxide) (PPO) blocks and hydrophilic poly (ethylene oxide) (PEO) moieties. Beside these, compare to the conventional micelles block copolymer aggregates are more heterogeneous. For this reason, we have chosen two different fluorophores with different hydrophobicity so that we can monitor the different regions into the aggregates. We have showed the effect of theses Pluronic block copolymer aggregates on the adsorption of two fluorophores on graphene oxide (GO) surface. The PPO segment of the block copolymer strongly interact with the hydrophobic basal plane of GO. Thus, in presence of these aggregates the interaction between the GO and fluorophores is restricted depending on their location into the aggregates. The adsorption of the fluorophores is also depended on the hydrophobicity of the aggregates. In most of the cases, the adsorption phenomena follow the traditional Langmuir isotherm. Further, Fluorescence Correlation Spectroscopy (FCS) study successfully provides insight into the molecular diffusion of these fluorophores adsorbed on GO surface. In water, almost equal amount of fluorophores are adsorbed irrespective of their nature. However, in pluronic aggregates, the amount of adsorbed fluorophores decreases significantly depending on their position and hydrophobicity. In addition, our FCS result indicates that the molecular diffusion of these fluorophores in presence of GO and triblock copolymer deviate from the normal Fickian diffusion and show anomalous superdiffusion. Finally, we have also demonstrated that fluorophore loaded block copolymer and GO can be used as an effective tool for the live cell imaging. In presence of pluronic aggregates, fluorophores can be distributed in most of the cell surface and cellular uptake of GO is also

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increased. Furthermore, due to the biocompatible nature of these pluronics, GO-P123 can be served as drug delivery vehicle. 1. Introduction: In the year 1859, Oxford chemist Benzamine Brodie first produced graphite oxide which produces two dimension atomically thin graphene oxide (GO) after exfoliation.1 The extraordinary physiochemical properties such as high specific surface area2-3, electronic transport capabilities4-6, high mechanical strength7 and strong thermal and electrical conductive properties8,9 make it a promising candidate in several applications such as super capacitors10, transparent conductors11, nonlinear optics12, sensor13 and drug delivery14 etc. For this reason, it has gained significant attention in the field of chemistry, physics, material science and nanotechnology etc over the last few years.15-17 The two dimensional network of GO consists of various concentration of sp2 and sp3 carbon atoms. So, the chemical and optical properties of GO can be modified by careful tuning of sp2 hybridized carbon atoms. The honeycomb lattice structure of graphene oxide has chemically reactive oxygen containing groups which include carboxylic acid groups at the edges of GO and epoxy and hydroxyl groups on the basal planes. The presence of these reactive functional groups on graphene oxide surface create an opportunity to covalently modify the drug molecules which can be used for the targeted drug delivery in cell, while the presence of localized electron at nanosheet surface stabilize the drug molecule through the − interaction.18-19

However, the limited aqueous solubility of graphene precludes their use in the above mentioned area. Graphene oxide (GO) or other chemically converted derivatives form stable suspension in water albeit aggregate in biological media or in presence of electrolyte.20 Stable aqueous graphene solution can be obtained by using super acids21 or amphiphilic surfactants22,23. But it requires chemical functionalization of graphene and that prevent their use in biological processes. Again, graphene oxide solution is extremely unstable in presence of electrolytes as electrolytes screen the surface charge on the graphene oxide sheets and they form an aggregate.24 Amphiphilic block copolymers can be used as a steric stabilizer. It noncovalently binds to the GO surfaces and minimizes the aggregate inducing nanosheet-nanosheet interactions. Recently, Hersam and coworkers have shown that high concentration of graphene can be dispersed in water using nonionic block copolymers and the dispersion efficiency of graphene primarily


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depends on the length of the hydrophilic and hydrophobic domain of the block copolymer.25 Nonionic triblock copolymer (TBP) composed of two poly (ethylene oxide) (PEO) and one poly (propylene oxide) (PPO) segments. These PEO-PPO-PEO Pluronic TBP are commercially available with different compositions of hydrophilic PEO and hydrophobic PPO blocks. Due to their environment friendly nontoxic nature, chemical stability and structural diversity they are extensively used to prepare various biomimicking organized assemblies such as micelles, mixed micelles26 and vesicles27 depending upon various conditions like temperature, concentration etc. The central hydrophobic PPO block of the triblock copolymer strongly interact with the residual aromatic regions on the graphene oxide basal plane and two sterically stabilizing hydrophilic PEO blocks interact with the water molecules.28 Thus, nonionic block copolymers have several advantages over the other dispersing agents due to their nontoxic and biocompatible nature. It is well established that biomolecules or fluorophores are readily adsorbed on the GO surface due to the presence of both ionic and aromatic moieties and the fluorescence of the fluorophores29-32 or biomolecules33,34 are quenched in presence of GO. Fluorescence resonance energy transfer (FRET), nonradiative dipole-dipole interaction or the electron transfer are the main reason for such quenching.31-32 However, such adsorption can be restricted in presence of pluronic copolymer aggregates. Thus, in this manuscript, our main aim is to understand the restricted adsorption of different fluorophores on GO surfaces in presence of pluronic aggregates. Pluronic block copolymer aggregates are much more heterogeneous compare to normal micelles and Bhattacharyya et al have found the excitation wavelength dependency in solvation dynamics in P123 micelle which is due to the wide distribution of probe molecules into the P123 micelles.35 For this reason, we have taken both hydrophilic (Coumarin-343) as well as hydrophobic (Coumarin-480) fluorophores. Coumarin-343 (C-343) is sufficiently hydrophilic and located at the water-micellar interface (water-corona region) and Coumarin 480 (C-480) is located reasonably closer to the micellar surface (core-corona region) (Scheme 1).36 Here, the reason of choosing two different dyes having different locations in P123 micelle is to understand how the adsorption efficiencies of GO depend on the accessibility of the dye molecules and how the different regions of P123 micelles are affected in presence of GO. Beside the steady state fluorescence measurements, we have studied the diffusion properties of the fluorophores in presence of GO in Pluronic aggregates using the well known method, Fluorescence Correlation Spectroscopy (FCS). FCS study successfully provides insight into the organization and dynamic



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properties of the self-assemblies. In recent literature, FCS has been extensively used to study the diffusion coefficient of a molecule in simple solutions as well as in various organized assemblies such as micelles, vesicles and microemulsions at the single molecular level.37-42 The primary interest of FCS is the spontaneous intensity fluctuation which is due to the minute deviation of the small system from thermal equilibrium. Recently, Leng et al have shown the adsorption of fluorophores on metal oxide nanoparticles.43 Thus, FCS can also be employed to study the adsorption equilibrium at solid-liquid interface and this technique does not involve any separation step of adsorbed and non-adsorbed species. Pluronic aggregates also enhance the stability of GO in biological media24 and thus the cellular uptake of GO is increased in presence of pluronics. We have demonstrated that Pluronic aggregate stabilize GO can be used as potential biomarker for the live cell imaging and due to the biocompatible nature of the pluronics they can also be served as drug delivery vehicle. 2. Experimental Section: 2.1. Materials and Sample Preparation. Laser grade coumarin-480 (C-480), coumarin-343 (C343) and rhodamine 6G (R6G) ClO4 were purchased from Exciton. P123 and F127, triblock copolymers were obtained from Sigma Aldrich and were used without any further purification. Graphene oxide (GO) was synthesized from the graphite powder by modified Hammer’s method.44,45 Solid graphene oxide was dispersed completely in Milli-Q water to prepare 1mg/ml GO solution. 5 wt % P123 solution (which is much higher than its CMC value) was prepared by mixing (stirring with magnetic stirrer) appropriate amount of copolymer with 50 ml Milli-Q water in a sealed container at room temperature and solution was kept overnight for stabilization. Dye concentration was maintained as 2 µM throughout the steady states experiments. For the proper encapsulation of the dye in the micellar solution, the dye loaded solution was kept for few hours. Aqueous dispersion of GO (1mg/ml) was added gradually to the micellar solution and the solutions were vortexed to ensure the homogeneous distribution of both components. The chemical structures of the fluorophores are shown in Scheme 1 and for the FCS experiments dye concentration was maintained as 10 nm. 2.2. Structural Characterization. Transmission Electron Microscopy (TEM) measurement of GO, P123 micelles and GO-P123 mixed assemblies were carried out with analytical TEM (FEI. TECNAIG220S-TWIN) instruments operating at 200 KV. TEM samples were prepared by


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blotting a carbon coated (50 nm carbon film) Cu grid (300 mesh, electron microscopy science) with a drop of GO solution and GO in P123 solution and P123 micellar solution. The samples were allowed to dry for overnight. Atomic Force Microscopy (AFM) was performed on Agilent 5500 in tapping mode. A drop of freshly prepared GO solution was placed on a newly cleaved mica surface and sample was spin coated and air dried overnight before imaging. Fourier Transform Infrared Spectroscopy (FTIR) of the solid GO was recorded with Perkin-Elmer instrument (Spectrum RX, serial no 7313) by KBr pellet method and the zeta potential ( ) of the

GO solution was determined by Malvern Nano ZS instrument with 4mW He-Ne laser ( =

632 ).

2.3. Steady State and Time Resolved Fluorescence Measurements.

The absorbance and

steady state fluorescence spectra were collected with the use of Shimadzu (model no UV-2450) and Hitachi (model no F-7000) spectrofluorimeter respectively. All the samples were excited at 408 nm. Time resolved emission decays were collected with the help of time correlated single photon counting (TCSPC) set up at the excitation wavelength of 408 nm. Signals were collected at a magic angle (54.70) using Hamamatsu microchannel plate photomultiplier tube (3809U) and the instrument response function (IRF) of our system was 90 PS and the decays were analyzed using IBH DAS-6 decay analysis software. 2.4. Fluorescence Correlation Spectroscopy (FCS) and Microscopy. FCS measurements of the fluorophores in presence of different concentration of GO in water and pluronic aggregates were carried out using DCS 120 Confocal Laser Scanning Microscope (CLSM) system (Becker& Hickl DCS-120) with inverted microscope of Zeiss. The detailed description of instrumentation is described earlier.46 The autocorrelation function which describes the temporal fluctuation of the fluorescence intensity can be defined as,

=

(1)

where is the fluorescence fluctuation at time t and + is the fluctuation after a

delay and < > is the average fluorescence intensity.



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3D diffusion model have been used in order to fit the correlation curves. For K fraction of dyes which are diffused within the system with distinct diffusion coefficients, the correlation function is defined as,47

= ∑1"2 !" #

$' & ( %

)&

* +

, . & )& - . %

' 0

/

(2)

In the above equation, N denotes the number of fluorophores within the focal volume, !" is the

fractional weighting factor for the ith contribution to the autocorrelation curve and " is the diffusion time of the fluorescent species within the observation volume and is the delay or lag time. 3 denotes the structure parameter of the excitation volume and it is defined as (l/r), where l

is the longitudinal radii and r is the transverse radii. Transverse radii (r) can be determined

through the fitting of an autocorrelation curve of a fluorescent species with known diffusion coefficient. We have used R6G in water for this purpose and the diffusion coefficient 4 is 426

µm2s-1.48 and the autocorrelation curve of R6G in water (spectra not shown) is fitted with the

following equation in order to determine the global parameter r and 3.

= 1 +

678 : 67 1 + ; 98 : 9

(3)

Here, 4 is the diffusion coefficient of the fluorescent species. In the fitting analysis, r and 3 are

kept as linked global parameter. 3 = 5 is obtained after fitting the correlation curves of R6G in

water and observation volume (?@AA is obtained from the following equation, ?@AA = B/0 D B 3

(4)

The final value of D is obtained as 365 nm and ?@AA is 1.35 fl. All the FCS experiments were

performed at 200C and the diffusion coefficient can be obtained from the following equation,

4 =

9

6

(5)

Usually, diffusion is characterized by the ensemble averaged mean square displacements (MSD) i.e = 6Dt, where D is normal time dependent Stoke-Einstein diffusion constant and this

type of diffusion is termed as Fickian diffusion.49 However, in our cases, some of the FCS traces


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cannot be fit by the commonly used equation describing the transport of molecules through the focal volume via normal diffusion (i.e., α =1 in equation 2).Thus, an equation which is related to the anomalous diffusion is used to fit the FCS traces. < D 0 > = 6Γ F

(6)

The value of G in equation 6 and equation 2 denotes the extent of deviation from normal diffusion (α =1). For G >1, the process is termed as superdiffusive and for G

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