ZnO nanoparticle-protein interaction: Corona

ZnO nanoparticle-protein interaction: Corona formation with associated unfolding A. K. Bhunia, P. K. Samanta, S. Saha, and T. Kamilya Citation: Applied Physics Letters 103, 143701 (2013); doi: 10.1063/1.4824021 View online: http://dx.doi.org/10.1063/1.4824021 View Table of Contents: http://scitation.aip.org/content/aip/journal/apl/103/14?ver=pdfcov Published by the AIP Publishing

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APPLIED PHYSICS LETTERS 103, 143701 (2013)

ZnO nanoparticle-protein interaction: Corona formation with associated unfolding A. K. Bhunia,1 P. K. Samanta,2 S. Saha,1 and T. Kamilya3,a) 1

Department of Physics & Technophysics, Vidyasagar University, Paschim Medinipur 721102, India Department of Physics, Ghatal R.S. Mahavidyalaya, Paschim Medinipur 721212, India 3 Department of Physics, Narajole Raj College, Paschim Medinipur 721211, India 2

(Received 1 July 2013; accepted 12 September 2013; published online 2 October 2013) The interaction as well as the formation of bioconjugate of Bovine Serum Albumin (BSA) and Zinc Oxide nanoparticles (ZnO NPs) is investigated. The surface binding along with reorganization of BSA on the surface of ZnO NPs forms stable “hard corona.” The time constants for surface binding and reorganization are found to be 1.10 min and 70.68 min, respectively. The close proximity binding of BSA with ZnO NPs via tryptophan is responsible for bioconjugate formation. Fibrillar aggregated structure of BSA is observed due to conformational change of BSA in C 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4824021] interaction with ZnO NPs. V

Nanomaterials are at the foremost periphery of the swiftly developing field of commercial exploration of nanotechnology.1 The advent of nanotechnology has accelerated the evolution of materials with many exceptional sizedependent properties for use in several biological and medical applications.1–4 Owing to the high surface to volume ratio, nanoparticles (NPs) are highly reactive. However, the knowledge about the effects of NPs on biological systems and their potential toxicity is very limited yet. Thus, the interaction of NPs especially having luminescence property with proteins has emerged as a key parameter in nanomedicine5–8 and nanotoxicology9–13 in the recent research. However, there are only few NPs (Zinc Oxide (ZnO), silver, and gold) known to us which are less-toxic and biocompatible having excellent tuned luminescence property that can be used in several biomedical and pharmaceutical applications for mankind.14–16 ZnO is very well known multifunctional wide and direct band gap semiconductor having excellent size dependent tunable optical property which is of great interest in the NPs based drug delivery, bio-imaging, and biomedical research.14,15 However, before going into realizing these applications, it is very much essential to understand the way of interaction of bare ZnO NPs with blood plasma protein. The study of protein-NPs conjugation will provide us with the information of the phenomena occurring at the protein-NPs interface at the molecular level as well as the information about conformational changes of protein occurring at the protein-NPs interface. Whenever NPs come in physiological fluid systems they are surrounded by the protein molecules. The NPs then get associated with the protein molecules and a dynamic layer of proteins is formed on the surface of NPs. This conjugated system is known as “NPs-protein corona.”17,18 It is noteworthy that the biological impact of proteincoated NPs is mainly related to the “hard corona,”19 composed of an inner layer of selected proteins with the highest affinity having with lifetime of several hours in a slow exchange with a)

Author to whom correspondence should be addressed. Electronic mail: [email protected]

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the environment. The surface adsorption may lead to structural changes/unfolding of protein. The function of proteins depend on their three dimensional structures; the conformational changes induced by adsorption may significantly affect the function of proteins and still result in loss of biocompatibility, activity, as well as potential immunogenicity.20–22 Therefore, the concept of bio-safety and biocompatibility of ZnO NPs is a key issue in favor of application in human body. Only few studies on ZnO NPs-protein interaction as well as formation of bioconjugate have been reported.23 However, the interaction mechanism of the formation of ZnO NPs-protein corona and associated unfolding along with the development of mimic model remain unclear so far. In order to recognize the physical basis of the biological activity of ZnO NPs under conditions of environmental exposure in a better way, we have focused our aim to analyze the formation of ZnO NPs-protein corona and interaction of ZnO NPs with a model protein, Bovine serum albumin (BSA) by major spectroscopic along with microscopic techniques. BSA and Tryptophan (TRY) were purchased from Sigma Chemical Co. (U.S.A.). The samples were used as received without any further purification. The BSA solution with predetermined concentration of BSA (CBSA ¼ 0.01 mg/ml) was prepared by using triple distilled water, deionized with a Milli-Q water purification system from Millipore, U.S.A. The pH and the resistivity of freshly prepared water were 6.8 and 18.2 MX cm, respectively. ZnO NPs were synthesized using a simple wet chemical method as reported elsewhere24 with minor modification. The reagents used for fabricating ZnO NPs were of analytical grade (MERCK, 99.99% pure) and used as supplied. Under constant stirring of 0.5M zinc nitrate solution, NaOH solution (1M) was added drop-wise for 5 min and the stirring was continued further for 30 min. A white precipitate was deposited at the bottom of the flask. The precipitate was then filtered and washed 2–3 times with Millipore water for removal of any residual salts and dried in a furnace. The so prepared ZnO NPs were dispersed in Millipore water using ultrasonication for 15 min. The concentration of

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the ZnO was varied from 0.01 mg/ml to 1 mg/ml. BSA-ZnO NPs mixed solutions were prepared by mixing 0.01 mg/ml BSA with ZnO NPs, ranging from 0.01 to 1 mg/ml with proper ratio. The optical absorption spectra of the samples were recorded by using Shimadzu-Pharmaspec-1700 UV-VIS after ultrasonication of the samples in water. The fluorescence spectrum of the as prepared samples was obtained by using Hitachi-F7000-FL spectrophotometer. The slit width at the excitation and the emission of the spectrophotometer were adjusted to 10 nm and 5 nm, respectively. The circular dichroism (CD) spectra of the samples were recorded by JASCO J-815 CD Spectrometer. The Fourier Transform Infrared (FTIR) spectra of the samples were obtained by Perkin Elmer LS-55 in absorption mode. For microstructural study, a small drop of the water dispersed samples was placed on a thin carbon coated copper grid and kept for some time for drying. High Resolution Transmission Electron Microscopy (HRTEM) of the prepared samples was acquired using JEOL-JEM-200 operating at 200 kV. The selected area electron diffraction (SAED) pattern of the said NPs was also carried out in situ with HRTEM. Typical absorption spectra of the ZnO NPs, BSA, and ZnO NPs-BSA conjugated system are shown in Fig. S1 (see supplementary material).51 The TRY of BSA exhibits an absorption peak at 278 nm owing to the p-p* transition of aromatic amino acid residues.25–27 ZnO NPs exhibit an absorption peak at 368 nm due to excitonic transition at room temperature.28 A small red shift (2 nm) of the absorption peak of BSA is observed due to binding of BSA with ZnO NPs and the formation of the bio-conjugate.29 Additionally, the absorption peak of ZnO NPs is slightly blue shifted to 366 nm from 368 nm in the presence of BSA which is consistent with a change in the dielectric function on the surface of ZnO NPs upon adsorption of BSA.30 The shifting of absorption peaks of BSA and ZnO confirms that ZnO NPs interacts with BSA through TRY. From the absorption spectra (see Fig. S2 in supplementary material),51 it is found that the critical concentration is high enough to stabilize the all ZnO NPs and is set at 0.01 mg/ml. We have observed the time-dependent absorption spectra (Fig. 1) of

FIG. 1. Time variation of Absorption spectra BSA-ZnO NPs complex with CBSA ¼ 0.01 mg/ml and CZnO ¼ 0.01 mg/ml. Inset shows the plot of change of absorption intensity of ZnO NPs with time. Red line shows the fitting curve fitted by Eq. (1).

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ZnO NPs with BSA to detect the onset of corona formation and its stabilization. It is also found that the intensities of ZnO NPs and BSA both decrease with time. The kinetics of ZnO NPs-BSA corona formation is studied by fitting the change of absorption intensity of ZnO NPs with time by non-linear least square fitting based on Levenberg-Marquardt algorithm of Microcal Origin 7.5 with the following exponential association equation31 (inset of Fig. 1): t t It ¼ IO þ A1 1 exp þ A2 1 exp ; (1) t1 t2 where Io and It are the absorption intensities at time zero and t, respectively. The constants A1 and A2 are the relative contributions and t1 and t2 are corresponding time constants of two mechanisms (surface binding and reorganization of BSA), respectively, involving in ZnO NPs-BSA interaction. We have found that t1 ¼ 1.10 min for the binding of BSA with surface of ZnO NPs for the formation of corona and t2 ¼ 70.68 min reveals to reorganization of BSA due to binding with ZnO NPs. The higher value of A1 (0.033) than A2 (0.008) implies that the binding is more pronounced than unfolding. The exponential association confirms that the formation of “ZnO NPs-BSA” corona starts immediately after incorporation of ZnO NPs into BSA. The protein (BSA) needs a long time to shield the original surface of ZnO NPs and reorganization of BSA occurs.31–34 HRTEM images enable the visualization of the structures formed under experimental conditions. Almost spherical ZnO NPs are found to form with diameter 15–20 nm (Fig. 2(a)). The ZnO NPs are polycrystalline in nature with the existence of the (101), (102), and (110) planes (Fig. 2(b)). The BSA molecules are attached to the ZnO NPs and form BSA-ZnO NPs corona (shown by circle in Fig. 2(c) and branched into fibrillar aggregated structure as observed from

FIG. 2. HR-TEM images (a) pure ZnO NPs, (b) SAED pattern of pure ZnO, (c) BSA-ZnO NPs complex (inset shows the SAED pattern), and (d) enlarge portion of circle of Fig. (c). Inset shows the HRTEM image of pure BSA.


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Fig. 2(c). The corona is clearly observed in Fig. 2(d). The magnified image of Fig. 2(c) clearly shows that the BSA molecules are attached with ZnO NPs (Fig. 2(d)). Inset of Fig. 2(d) shows the HRTEM of pure BSA. The dimension of BSA is almost same to that of BSA attached to the ZnO NPs. The schematic of the BSA-ZnO NPs corona formation is shown in Fig. S3 (see supplementary material51). The BSA-ZnO NPs binding kinetics equilibrium has been analyzed by fluorescence quenching measurements. The addition of ZnO NPs of different concentrations (CZnO) with BSA results a change in the maximum fluorescence emission spectrum intensity (Imax), suggesting the occurrence of fluorescence quenching process (Fig. 3). The quenching occurs via the adsorption and interaction of the Tryptophan residues23 accessible to the metallic surface of the ZnO NPs. A small red shift (2 nm) of TRY emission with CZnO ¼ 0.1 mg/ml also signifies the unfolding as well as denaturation of BSA in the presence of ZnO NPs.35,36 The strength and association cooperativity of the adsorption of BSA onto the ZnO NPs are analyzed by determining the Hill coefficient using the well known Hill equation given by Q ¼ ðI0 IÞ=I0 ;

(2)

Q=Qmax ¼ ½NPn =ðkn D þ ½NPn Þ:

(3)

Here, I and I0 are the fluorescence intensities in the absence and in the presence of NPs, respectively; Qmax is the saturation value of Q, relative intensity; kD is the proteinnanoparticle equilibrium constant (dissociation constant); and n is the Hill coefficient.34,37,38 Details regarding the Hill equation and different parameters are discussed in the supplementary material.51 Inset of Fig. 3 shows Hill plots of BSA-ZnO binding measurements. The parameters obtained from the analyzed data are summarized in Table I (see supplementary material51). We have found cooperative binding (n > 1) for TRYZnO complex. Binding of TRY with ZnO NPs induces the BSA molecules to organize at the surface boundaries of ZnO

FIG. 3. Fluorescence spectra of BSA and BSA-ZnO complex (a) pure BSA with CBSA ¼ 0.01 mg/ml, (b)–(d) BSA-ZnO NPs complex with CBSA ¼ 0.01 mg/ ml and CZnO ¼ 0.01, 0.03, 0.06, and 0. 1 mg/ml. Inset shows the fluorescence quenching plot of BSA with CBSA ¼ 0.01 mg/ml and CZnO ¼ 0.01, 0.03, 0.06, and 0. 1 mg/ml. Red lines represent the curves fitted by Eq. (2).

NPs. Therefore, we have found an enhancement of the cooperativity of the TRY-BSA binding transition. Therefore, TRY moieties are the most favorable binding sites of BSA with ZnO NPs. The fluorescence quenching measurements are also done with pure TRY (see Fig. S4 of supplementary material51) and result supports the argument (inset of Fig. S4 and Table II) (see supplementary material51). CD spectroscopy is used to analyze the conformational change of secondary structure of BSA in interaction with ZnO. ZnO NPs binding associated unfolding of BSA is analyzed by fitting the CD spectra with K2D2 software39 (see Fig. S5 in supplementary material51). The positive peak at 190 nm and two negative peaks at 208 and 222 nm state that BSA is a-helix rich protein (see Fig. S5 in supplementary material51). The K2D2 fitting gives that pure BSA is composed of 67.45% a-helix40–42 and 3.24% b-sheet, matched with the reported earlier values. CD spectrum of BSA-ZnO NPs complex shows the decrement of the ellipticity of the above mentioned peak. K2D2 fitting of CD spectrum of BSA-ZnO NPs complex states that the interaction of ZnO decreases the a-helix component of BSA to 61.33% along with increases b-sheet component of BSA to 4.06%. Therefore, the unfolding/conformational change of BSA occurs in the presence of ZnO NPs. However, the information of the existence of intra and intermolecular aggregation of BSA cannot be calculated from CD spectroscopy. The FTIR analysis of amide I band of protein can give the information on the secondary structure of protein along with intra and intermolecular aggregates.43–50 Therefore, ZnO NPs binding associated unfolding of BSA is also analyzed by fitting the amide I band (1600–1700 cm1) of FTIR spectrum and summarized in Table II (see Fig. S6 and Table II in supplementary material51). The fitting result of normalized amide-I peak of pure BSA is reported in our earlier literature.13 The summary of fitting results implies that the BSA is composed of high amount of a-helix (62.82%) and of small amount of b-sheet.13,45 The small change of values of a and b components of BSA in FTIR spectrum than CD spectrum is due to solidification of the samples on silicon wafer for studying FTIR spectroscopy. FTIR spectra of pure BSA reveal the presence of inter-molecular aggregates (A1) ¼ 17.34% and intra-molecular aggregates (A2) ¼ 2.66%.13 The decrement of a-component with a large increment of intra-molecular aggregation (44.30%) is observed (see Table II in supplementary material51). The b/a ratio is 0.282, greater than pure BSA (0.198), implies the conversion of some a helix into b sheet also agrees the result obtained from the study of CD spectroscopy, qualitatively. Therefore, the reorganization phenomena of BSA found by studying the time variation absorption spectroscopy lead to the conformational change/ unfolding of BSA. The interaction as well as the formation of BSA-ZnO NPs bioconjugate has been investigated. The blue shift of absorption peak of ZnO, in ZnO NPs-BSA system, confirms the interaction of ZnO NPs with BSA. The results from the time dependent absorption spectroscopy confirm the interaction of BSA with the surface of ZnO NPs through the formation of stable hard corona. The surface binding along with reorganization of BSA on the surface of ZnO NPs forms stable “hard corona.” The time constants for surface binding


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and reorganization are found to be 1.10 min and 70.68 min, respectively. The BSA molecules upon interaction with ZnO NPs are branched into aggregated fibrillar structure by unfolding as observed from CD, FTIR spectroscopy and HRTEM image. The change of intensity of the fluorescence emission peak of the ZnO NPs-BSA conjugated system confirms the occurrence of fluorescence quenching in the bioconjugation process via interaction of the TRY residues accessible to the metallic surface of the ZnO NPs. We have found that the TRY moieties are the most favorable binding sites of BSA with ZnO NPs, and the cooperative binding of TRY with ZnO NPs induces the BSA molecules to organize at the surface boundaries of ZnO NPs. A small amount of conversion of a-helix to b-sheet and resultant conformational change/unfolding of BSA is also found in the presence of ZnO NPS. The authors would like to acknowledge DST-FIST sponsored Department of Physics & Technophysics for providing various instrumental facilities. Dr. S. Saha and Dr. T. Kamilya thank UGC for providing funding in MRP F-No. 41-1401/2012(SR). Thanks also go to Dr. Prabir Pal, Department of Spectroscopy, IACS and the authority of the IACS for providing the central instrumental facility of CD. 1

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