Bovine Serum Albumin Adsorption on TiO2 Colloids - ACS Publications

Feb 14, 2017 - Salzburg, Austria. ‡. Department of ... history of the material investigated. ... sample history of commercial TiO2 nanoparticle samp...
0 downloads 0 Views 815KB Size
Subscriber access provided by University of Newcastle, Australia

Article

Bovine serum albumin adsorption on TiO2 colloids: the effect of particle agglomeration and surface composition Augusto Márquez, Thomas Berger, Andrea Feinle, Nicola Huesing, Martin Himly, Albert Duschl, and Oliver Diwald Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03785 • Publication Date (Web): 14 Feb 2017 Downloaded from http://pubs.acs.org on February 21, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Bovine serum albumin adsorption on TiO2 colloids: the effect of particle agglomeration and surface composition Augusto Márquez,a Thomas Berger,a,* Andrea Feinle,a Nicola Hüsing,a Martin Himlyb, Albert Duschlb and Oliver Diwalda

a

Department of Chemistry and Physics of Materials, Paris Lodron University of Salzburg,

Hellbrunnerstrasse 34/III, A - 5020 Salzburg, Austria b

Department of Molecular Biology, Paris Lodron University of Salzburg, Hellbrunnerstraße 34/III,

A - 5020 Salzburg, Austria

* Corresponding author:

Phone: +43-662-8044-5931, Fax: +43-662-8044-622, [email protected]

1 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 25

Abstract Protein adsorption at nanostructured oxides strongly depends on synthesis conditions and sample history of the material investigated. We measured the adsorption of bovine serum albumin (BSA) to commercial Aeroxide TiO2 P25 nanoparticles in aqueous dispersions. Significant changes in adsorption capacity were induced by mild sample washing procedures and attributed to the structural modification of adsorbed water and surface hydroxyls. Motivated by the lack of information about the sample history of commercial TiO2 nanoparticle samples we used vapor phase grown TiO2 nanoparticles, a well-established model system for adsorption and photo-catalysis studies, and performed on this material for the first time a systematic and quantitative BSA adsorption study. After alternating vacuum and oxygen treatment of the nanoparticle powders at elevated temperatures for surface purification we determined size distributions covering both the size of the individualized nanoparticles and nanoparticle agglomerates using transmission electron microscopy (TEM), X-ray diffraction (XRD) and dynamic light scattering (DLS) in aqueous dispersion. Quantitative BSA adsorption measurements at different pH values and thus variable combinations of surface charged proteins and TiO2 nanoparticles revealed a consistent picture: BSA adsorbs only at the outer agglomerate surfaces without penetrating into the interior of the agglomerates. This process levels at coverages of single monolayers, which resist consecutive simple washing procedures. A detailed analysis of the protein specific IR amide bands reveals that the adsorption induced protein conformation change is associated with a decrease of the helical content. This study underlines that robust qualitative and quantitative statements about protein adsorption and corona formation require well documented and controllable surface properties of the nanomaterials involved.

2 ACS Paragon Plus Environment

Page 3 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1. Introduction Hybrid systems made up from combinations of proteins and engineered inorganic nanomaterials play an important role in biocatalysis,1,2 biosensors,3,4 protein separation,5–8 protein crystallization9 and nanomedicine.10–12 Their functionality relies on a complex interaction pattern between these two entirely different classes of matter. For this reason enormous efforts have been devoted to the study of the chemical, physical and biological factors governing the interaction of biomolecules with inorganic nanomaterials.13–19 In addition, related research is motivated by the critical evaluation of health and safety issues which arise from intentional or unintentional exposure of biological systems to engineered nanomaterials.20,21 In biological media the properties of colloidal nanoparticles are subject to protein adsorption producing a protein corona around the particles, which determines the biological identity of the inorganic core component.22,23 Related characterization approaches differ in their level of simplification. Analytical experiments which resemble realistic biological situations typically render the various materials characterization steps extremely complex and difficult. This is mainly due to challenges in quantitative analysis and reproducibility.16,20 Strongly simplified model experiments which address the interaction between well characterized nanoparticle colloids with specified types of proteins, on the other hand, provide means for a quantitative assessment of biological processes at the solid/liquid interface.17 The central questions in this context are, whether the particular type of interaction studied represents a determining step in the overall biological situation and whether the associated level of complexity can be increased in a rational way towards realistic environments. Clearly, the identification of the fundamental factors that govern the activity of nanomaterials in biological environments is complicated by the vast array of system variables. These are associated with the properties of the nanoparticles, those of the biological entity, the surrounding continuous phase and, last but not least, the complex interaction of all these factors. Key to 3 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 25

protein adsorption on nanoparticles are their chemical surface composition, size distribution functions, surface charge, structure and stability of the agglomerates. As even subtle differences in materials synthesis or processing may give rise to significant variations, this complexity has raised the concern that any finding for a given materials’ system is of limited generality.13,17,18 Protein specific contributions to adsorption arise from their composition, conformation and structural plasticity, size, and again from surface charge. Last but not least, the surrounding medium, its ionic strength, pH value and temperature represent a third set of external parameters.13–16,18 Several guidelines for benchmarking the behavior of nanoparticles have been proposed both for simplified model systems but also for materials in realistic biological environments.17–19,21 The latter systems are complex and require to systematically address the various combinations of proteins and nanoparticles. Subtle changes of the surface composition, the particle dispersion and related dynamics entail major differences in the physicochemical24 and biological properties25,26 of at first sight equivalent particle systems (i.e. particles of comparable size and shape, bulk composition and structure).18 Furthermore, as pointed out above, these particle characteristics may behave dynamically in response to changes in the surrounding environment, which deserves special attention. The necessary set of physico-chemical parameters addressed should comply with the claims and conclusions of the respective study.21 The combination of an initial, an in situ and a final characterization of the materials is desirable not only for following dynamic changes of the system properties but also for studying long term aging effects.18,19 In any case, there is general agreement in the field of bio-nano interactions that more systematic and quantitative studies on reference systems are needed.17 In the here presented model study we investigate the effect of the adsorption of bovine serum albumin (BSA) on the agglomeration and dispersion state of TiO2 nanoparticles, which also includes the reverse effect particles can have on protein conformation. We have paid particular 4 ACS Paragon Plus Environment

Page 5 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

attention to the control of surface composition, size distribution and aggregation state of the original TiO2 nanoparticle powder, which has previously been established as robust model system for adsorption and photocatalysis27–34 and for defect engineering of functional nanomaterials.35 Upon transfer of vapor phase-grown nanoparticles into aqueous dispersion we characterize the size and surface charge of the resulting particle agglomerates by dynamic light scattering. Protein adsorption is studied both qualitatively and quantitatively in a combined spectroscopic, thermogravimetric and light scattering approach. 2. Experimental 2.1 Nanoparticle synthesis For exploratory experiments we used commercial Aeroxide TiO2 P25 (specific surface area: 50 m2, primary particle size: 21 nm, crystal structure: ~80% anatase and ~20% rutile)36 prior to and after treatment in alkaline solution. In the latter case the as received powder was dispersed in aqueous 1 M NaOH solution and stirred for 12 h at room temperature. Afterwards the dispersion was centrifuged (EBA 20 centrifuge, Hettich, 6000 rpm) and the powder was washed repeatedly with H2O until the supernatant reached a neutral pH. Once the powder was immersed and converted into aqueous particle dispersions, we avoided any drying of the sample which is typically associated with formation of solid-solid interfaces upon conversion of agglomerates into aggregates.37 Anatase TiO2 nanocrystals were prepared by metal organic chemical vapor synthesis (MOCVS) based on the decomposition of titanium (IV) isopropoxide at T = 1073 K in a hot wall reactor system.27,28 For purification, the obtained powder samples were subjected to thermal treatment under high vacuum conditions (p < 10-5 mbar). First, the powder sample was heated to T = 873 K using a rate of r ≤ 5 K min-1. Subsequent oxidation with O2 at this temperature was applied to remove organic remnants from the precursor material and to guarantee the stoichiometric 5 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 25

composition of the oxide. The absence of any organic remnant following such a post-synthesis treatment was evidenced by IR spectroscopy.28 XRD analysis yields an anatase/rutile ratio of 96:4 and an average particle size of 11 nm.35 As demonstrated by transmission electron microscopy (TEM), the majority of crystallites display a highly irregular, spherical shape after such activation.27,28 The size of the nanocrystals ranges from 8 to 18 nm with a mean diameter of 13 nm. The specific surface area deduced from the TEM mean diameter value corresponds to 121 m2 g-1 and is in good agreement with results from nitrogen sorption measurements (130 ± 13 m2 g-1).27 2.2 Preparation and characterization of particle dispersions The size distribution and zeta potential of agglomerates was determined for aqueous TiO2 particle dispersions with a concentration of 0.1 mg mL-1. To ensure sufficient particle dispersion an ultrasonic finger (UP200St, Hielscher Ultrasonics GmbH) was used while the dispersion was cooled in an ice bath to prevent heating via mechanical sample agitation. Protein adsorption was performed at room temperature by mixing 15 mL of TiO2 particle dispersions with 5 mL of protein solutions of different concentration (final particle concentration of [TiO2] = 0.1 mg mL-1). The dispersion was then mechanically stirred for 12 h. A Zetasizer Nano ZSP ZEN5600 (Malvern Instruments) was used to determine the size distribution of dispersed TiO2 particles, proteins and protein/TiO2 heteroaggregates by dynamic light scattering (DLS) as well as the zeta potentials by laser Doppler electrophoresis. The pH dependence of the zeta potential was recorded using an autotitrator (MPT-2, Malvern Instruments) and diluted HCl and NaOH titrants. The titration curves are constructed from two independent measurements. Starting from circumneutral conditions (aqueous dispersions of protein, TiO2 particles or protein/TiO2 heteroaggregates) one titration was performed into the acidic and another one into the basic pH region.

6 ACS Paragon Plus Environment

Page 7 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

2.3 Quantitative analysis of protein adsorption - Thermogravimetric measurements TiO2 particle dispersions were treated ultrasonically for 30 minutes as described above. Protein adsorption was performed at room temperature by mixing the TiO2 particle dispersions with protein solutions of different concentration (final particle concentration [TiO 2] = 1 mg mL-1) followed by mechanical stirring at room temperature for 12 h. The protein/TiO2 heteroaggregates were separated from the aqueous solution by centrifugation (EBA 20 centrifuge, Hettich, 6000 rpm) and washed twice with H2O. The solid fraction was vacuum dried for 3h at room temperature using a membrane pump. For thermogravimetric analysis (TGA) the sample was heated in synthetic air from room temperature to 1000 °C (r = 10 °C min-1) and the mass difference was recorded (STA 449 F3 Jupiter, Netzsch). 2.4 Study of protein conformation – ATR-FTIR-spectroscopy For IR measurements an attenuated total reflection (ATR) unit (PIKE Technologies, Veemax II) was attached to a Bruker Vertex 70 FTIR spectrometer equipped with a MCT detector. The measurements were performed at an incident angle of 55 ° using a hemispherical ZnSe prism. Spectra were obtained by averaging 100 scans at a resolution of 4 cm-1 and are represented as –log(R/R0), where R and R0 are the reflectance values corresponding to the single beam spectra recorded for sample and reference, respectively. A TiO2 film was immobilized on the ATR prism by doctor blade deposition using Scotch tape as spacer. First, 40 µL of a 0.4 M TiO2 nanoparticle dispersion was applied per cm2 of prism surface and spread over it. The resulting film was then dried in a nitrogen stream. Afterwards, the IR cell was assembled by pressing a glass cell against the pre-coated prism using a Teflon ring as the junction. Finally, the cell was filled with water or aqueous protein solution to measure the background or sample spectrum, respectively.

7 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 25

3. Results and Discussion 3.1 Protein adsorption on commercial Aeroxide TiO2 P25 aggregates As outlined in the introduction protein adsorption is influenced by sample history covering synthesis, processing and sample storage. For a first crude estimate for the effect of related effects we used commercially available Aeroxide TiO2 P25 powderi and analyzed the pH dependent development of the zeta potential (Figure 1a). Starting from purely aqueous dispersions we performed one titration into the acidic pH range by stepwise addition of HCl solution. In a second experiment the dispersion was titrated into the basic pH region by adding NaOH solution aliquots. The isoelectric point pHIEP of TiO2 P25 was found to be at pH 4.5 (Figure 1a), which is significantly lower than the pHIEP of ~ 6.3 as reported in literature.38 At circumneutral conditions the zeta potential is approximately - 20 mV. Alkaline treatment of the powder with a 1 M NaOH aqueous solution followed by extensive washing with water shifts the pHIEP to 5.5 – 6.0, i.e. towards the reported literature value. For aqueous suspensions of TiO2 particles after NaOH treatment and washing we observed a decreased zeta potential value of ~ – 10 mV. Whereas the primary particle size of TiO2 P25 is 21 nm,36 hydrodynamic diameter of particle dispersions peak at 90 - 100 nm (Figure 1b). These diameters are attributed to particle aggregates that cannot be broken up by ultrasonic treatment.39 Both hydrodynamic diameter and colloidal stability remain essentially unaffected by NaOH treatment (Figure 1b).

i

stored for ~ 1 year in the laboratory shelf after delivery

8 ACS Paragon Plus Environment

Page 9 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 1: (a,b) Colloidal properties of as received and NaOH-treated commercial Aeroxide TiO2 P25 ([TiO2] = 0.1 mg mL-1): Dependence of the zeta potential on solution pH (a) and hydrodynamic diameters of particle aggregates determined at different times following ultrasonic treatment (b). The inset shows digital images of the two samples at different times following ultrasonic treatment. (c) Thermogravimetric profiles of pure and BSA covered Aeroxide TiO2 P25 (as received and NaOH-treated). The protein was adsorbed from aqueous suspension: [TiO2] = 1 mg mL-1, [BSA] = 200 µg mL-1, adsorption time: 18 h. After adsorption the particles were washed repeatedly with water and dried in vacuum prior to thermogravimetric analysis in synthetic air. 9 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 25

BSA (molar weight: 66433 g mol-1) has an isoelectric point of pHIEP = 4.840 and at circumneutral conditions carries an almost uniform negative charge41 (- 30 mV at pH 7). Thermogravimetric analyses of TiO2 P25 powders were performed to investigate whether the NaOH treatmentinduced changes of the oxide surface charge, i.e. the shift from – 20 mV to – 10 mV under circumneutral conditions, (Figure 1a) impacts BSA adsorption. As a result, we found that the TG profile of protein-free TiO2 P25 particles does not change: upon heating to 250 °C the sample mass decreases for the as received and the NaOH treated samples by ~ 1.5 % as a result of water desorption, but remains essentially constant thereafter (Figure 1c). Following BSA adsorption from aqueous suspensions ([TiO2] = 1 mg mL-1, [BSA] = 200 µg mL-1, adsorption time: 18 h) an additional mass loss is observed in the temperature range between 250 and 600 °C and attributed to the decomposition of adsorbed protein. As a main result, the amount of surface-bound BSA depends significantly on sample pretreatment. One would expect that negatively charged BSA molecules (zeta potential: - 30 mV) experience a weaker electrostatic repulsion from the less negatively charged surface of NaOH-treated particles (zeta potential: - 10 mV). However, the amount of adsorbed BSA accounts only for 66 % of the amount that was adsorbed on the untreated particles. This leads us to the conclusion that electrostatic repulsion is not the dominant factor to explain the modified adsorption behavior. TiO2 P25 powders are prepared from TiCl4 in a hydrogen flame36 and for this reason contain significant amounts of residual chloride ( 40 µg mL-1 the slope of the two curves is virtually identical indicating that BSA adsorption to TiO2 agglomerates levels at higher concentrations. This is perfectly in line with the constant hydrodynamic diameter of BSA/TiO2 composites observed for concentrations [BSA] > 20 µg mL-1 (Table S1, Figure 3b). Considering TiO2 agglomerates with a size of 80 nm and a solid fraction of 0.5 (i.e. 50 % porosity)37 we estimated the number of BSA molecules per agglomerate for low BSA concentrations (samples B and C, Table S1). Complementing the Bradford assay, we also used thermogravimetric analysis (both at low and high BSA concentrations; samples B, C and E) and determined the mass fraction of adsorbed protein. For this purpose we evaluated in the TGA profiles the mass loss in the temperature range between 250 and 600 °C. Both analytical approaches yield consistent results (Table S1): about 760 BSA molecules were found to be adsorbed per TiO2 agglomerate from a 20 µg mL-1 solution (sample C). A doubling of the BSA concentration in solution (sample E) leads only to a moderate increase to 920 BSA molecules / TiO2 agglomerate (Figure 3c).

15 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 25

Next we roughly estimated the number of BSA molecules forming a protein monolayer at the external surface of a TiO2 agglomerate. For this purpose, agglomerates were modeled by spheres with a diameter of 80 nm (i.e. the hydrodynamic diameter determined by DLS, Figure 2). The required adsorption area per BSA molecule was approximated by the projection area of a spherical molecule with a diameter of 6 nm (i.e. the hydrodynamic diameter of BSA in solution, Figure 2) to a planar surface. This analysis yields a theoretical monolayer coverage of 710 BSA molecules per TiO2 agglomerate. From comparison with the experimentally determined limit of ~ 900 BSA molecules / TiO2 agglomerate we conclude that BSA adsorption occurs predominantly at the external surface of TiO2 agglomerates and protein penetration into the intra-agglomerate pores seems to be inhibited (Figure 3c and scheme in Figure 2). 3.3 Structural changes of BSA adsorbed on porous films of vapor phase-grown TiO2 nanoparticles The adsorption of BSA on porous films of vapor phase-grown TiO2 nanoparticles was investigated by ATR-IR spectroscopy, which is well-suited for the study of dynamic processes at the solid-liquid interface in biological fluids.46 A porous film of TiO2 nanoparticles was immobilized on the ATR prism from aqueous dispersion and dried in a nitrogen stream as described in the Experimental Section. Since the surface charge of both the protein and the oxide depends on solution pH (Figure 4) we performed pH-dependent measurements. While the TiO2 surface is essentially neutral in purely aqueous conditions, BSA molecules carry a negative surface charge with typical zeta potential values of – 25 mV. In acidic solution both particles and proteins are positively charged with zeta potential values of + 40 mV at pH 3 (Figure 4). In addition to surface charge, the pH value determines the conformational state of BSA. Mainly composed of -helix segments the secondary structure of BSA features also smaller contributions from turn and extended chain structures but does not contain -sheet elements.47 At low pH reversible conformational conversion of the normal (N) form into the fast (F) (below pH 16 ACS Paragon Plus Environment

Page 17 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

4) and moreover into the expanded (E) form (below pH 3) is associated with a continuous decrease of the protein’s helical content and an opening of the molecule.48 The ATR-IR spectra of BSA adsorbed onto the TiO2 film from circumneutral (pH 6.2) and acidic (pH 3) aqueous solution (Figure 5a) contain protein-specific bands at 1645 cm-1 (amide I) and 1545 cm-1 (amide II) together with less intense bands at 3280 cm-1 (amide A), 3030 cm-1 (amide B) and between 3000 and 2800 cm-1 ((C-H)).46,49–51The band intensities were found to be more than five times higher for the measurements in circumneutral environment. The intensity evolution of the protein-specific bands during the initial stages of BSA adsorption is highlighted in Figure 5b. IR spectra of free BSA in solution are shown in Figure S3.

17 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 25

Figure 5: (a) ATR-IR spectra of BSA adsorbed at pH 6.2 and pH 3.0 on porous films of TiO2 nanoparticles ([BSA] = 200 µg mL-1, [TiO2] = 1.3 mg mL-1, adsorption time: 8 h), background spectra: TiO2 nanoparticle film in contact with the protein-free solution at pH 6.2 (black) and at pH 3.0 (red), respectively. Inset: second derivative of the spectral region featuring the amide I band of free and adsorbed BSA. b) Intensity evolution of the protein-specific amide II band in the initial stage of BSA adsorption.

18 ACS Paragon Plus Environment

Page 19 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Electrostatic interactions between protein and hydrophilic oxide surfaces control the amount of BSA adsorption.45,52 In systems of oppositely charged oxide particles and proteins the saturation coverage is a result of the counteracting interplay between electrostatic particles, and protein attraction and the repulsion between equally charged adsorbed proteins.15,53 The significantly higher amount of BSA being adsorbed on the TiO2 film at circumneutral conditions (Figure 5) is in sound agreement with the electrostatic interaction arguments: at pH 6.2 TiO2 particles are almost neutral, while the zeta potential of BSA molecules is moderately negative (- 25 mV). At pH 3, on the other hand, both particles and proteins carry a high positive charge (+ 40 mV). A strong repulsion between particles and proteins, on the one hand, and between the adsorbed proteins, on the other, explains the significantly lower limit of protein adsorption at pH 3. In addition, as will be outlined below, the space requirements of the adsorption site at the oxide particle surface are higher for the extended form of BSA. A detailed analysis of ATR-IR spectra was performed to explore adsorption-induced changes in protein conformation. The amide I band shows a high sensitivity to the structure of the protein backbone, since different secondary structures contribute to the amide I band in a narrow wavenumber range.49,50,54 The second derivative of BSA spectra related to the protein in solution and in the adsorbed state are shown in the inset of Figure 5 for circumneutral and acidic conditions. Two main features are attributed to -helix and random structures (1650 cm-1) and short-segment chains connecting -helical segments or -sheets (1630 cm-1).47,55,56 For proteins in solution the relative contribution of the feature at 1650 cm-1 decreases in the acidic environment, whereas the contribution at 1630 cm-1 increases. This effect originates from the decrease of the protein’s helical content upon the conformational transition from the N-form to the F- or E-form of BSA.48 Exactly this trend in the development of relative intensities between the contributions at 1650 and 1630 cm-1 is also observed for the adsorption of BSA on TiO2 at constant pH values being more pronounced at acidic conditions. These results point to a change 19 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 25

of the protein structure such as the adsorption-induced decrease of the -helix content in the BSA molecule.

24,41,57,58

The extended (E) form of the protein at pH 3 seems to facilitate such an

adsorption-induced structural modification. 4. Conclusions The heterogeneously distributed properties of commercial metal oxide nanoparticle powders render the identification of factors that determine protein adsorption and - consequently - the biological activity difficult, if not impossible. For a first rational assessment of protein adsorption on TiO2 nanoparticle powders we employed vapor phase-grown nanoparticles as particulate model systems. Powder processing at high vacuum conditions and in oxygen assures contaminant-free surfaces and yields an ensemble of isolated spherical nanoparticles with a size distribution of individual particles below 20 nm. The transfer of the vapor phase-grown particles into aqueous colloidal dispersion leads to TiO2 agglomerates with a diameter of approximately 80 nm. BSA adsorption onto these agglomerates saturates at a monolayer and at the same time gives rise to the electrostatic stabilization of BSA/TiO2 composites. Importantly, protein adsorption does not disintegrate the pre-existing TiO2 agglomerates and protein penetration into the intra-agglomerate pores seems to be inhibited. ATR-IR spectroscopy revealed characteristic changes in the amide stretching vibration region that are indicative of the decrease of the relative content of -helix components. In an acidic environment (pH 3) where the proteins in solution adopt an extended form facilitating the adsorption-induced structural protein modification at the TiO2 surface, this effect is more pronounced. Our study underlines the potential and necessity of using well-defined nanoparticle powders to establish a qualitative and quantitative assessment of the interaction between inorganic nanomaterials and biomolecules. Starting from such well-defined model conditions an increase of the systems’ complexity may be achieved by stepwise changes of the sample treatment protocols, on the one hand, and composition of the aqueous environment, on the other. Only 20 ACS Paragon Plus Environment

Page 21 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

such a bottom-up approach in scientific enquiry can provide a sufficiently firm and reliable base for physical, chemical and biological statements about the microscopic interaction between proteins and nanomaterials as well as for safety-by-design approaches. Acknowledgements We thank Dr. Michael Elsässer for performing the TGA measurements and Krisztina Kocsis for assistance with DLS experiments. This work was financially supported by the University of Salzburg within the Allergy-Cancer-BioNano (ACBN) Research initiative. Supporting Information Additional experimental details, particle size distribution functions for anatase TiO 2 nanoparticles and agglomerates, results from Bradford analysis and IR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

21 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 25

References (1) Zhou Z., Hartmann M. Progress in enzyme immobilization in ordered mesoporous materials and related applications: Chemical Society Reviews 2013, 42, 3894–3912. (2) Fried D.I., Brieler F.J., Fröba M. Designing Inorganic Porous Materials for Enzyme Adsorption and Applications in Biocatalysis: ChemCatChem 2013, 5, 862–884. (3) Bhakta, S. A., Evans, E., Benavidez, T. E., Garcia, C. D. Protein adsorption onto nanomaterials for the development of biosensors and analytical devices: A review: Analytica Chimica Acta 2015, 872, 7–25. (4) Topoglidis, E., Cass, A., O'Regan, B., Durrant, J. R. Immobilisation and bioelectrochemistry of proteins on nanoporous TiO2 and ZnO films: Journal of Electroanalytical Chemistry 2001, 517, 20–27. (5) Hartmann, M. Ordered mesoporous materials for bioadsorption and biocatalysis: Chemistry of Materials 2005, 17, 4577–4593. (6) Katiyar A., Pinto N.G. Visualization of size-selective protein separations on spherical mesoporous silicates: Small 2006, 2, 644–648. (7) Lu, Z., Ye, M., Li, N., Zhong, W., Yin, Y. Self-assembled TiO2 nanocrystal clusters for selective enrichment of intact phosphorylated proteins: Angewandte Chemie - International Edition 2010, 49, 1862–1866. (8) Moerz, S. T., Huber, P. pH-Dependent Selective Protein Adsorption into Mesoporous Silica: Journal of Physical Chemistry C 2015, 119, 27072–27079. (9) Chayen N.E., Saridakis E., Sear R.P. Experiment and theory for heterogeneous nucleation of protein crystals in a porous medium: Proceedings of the National Academy of Sciences of the United States of America 2006, 103, 597–601. (10) Maiolo, D., Del Pino, P., Metrangolo, P., Parak, W. J., Baldelli Bombelli, F. Nanomedicine delivery: Does protein corona route to the target or off road?: Nanomedicine 2015, 10, 3231–3247. (11) Min, Y., Caster, J. M., Eblan, M. J., Wang, A. Z. Clinical Translation of Nanomedicine: Chemical Reviews 2015, 115, 11147–11190. (12) Wang, Y., Santos, A., Evdokiou, A., Losic, D. An overview of nanotoxicity and nanomedicine research: Principles, progress and implications for cancer therapy: Journal of Materials Chemistry B 2015, 3, 7153–7172. (13) Nel, A. E., Mädler, L., Velegol, D., Xia, T., Hoek, E., Somasundaran, P., Klaessig, F., Castranova, V., Thompson, M. Understanding biophysicochemical interactions at the nanobio interface: Nature Materials 2009, 8, 543–557. (14) Mahmoudi, M., Lynch, I., Ejtehadi, M. R., Monopoli, M. P., Bombelli, F. B., Laurent, S. Protein-nanoparticle interactions: Opportunities and challenges: Chemical Reviews 2011, 111, 5610–5637. (15) Rabe M., Verdes D., Seeger S. Understanding protein adsorption phenomena at solid surfaces: Advances in Colloid and Interface Science 2011, 162, 87–106. (16) Walkey, C. D., Chan, W. Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment: Chemical Society Reviews 2012, 41, 2780–2799. (17) Pino, P. D., Pelaz, B., Zhang, Q., Maffre, P., Nienhaus, G. U., Parak, W. J. Protein corona formation around nanoparticles - From the past to the future: Materials Horizons 2014, 1, 301–313. (18) Kumar, A., Das, S., Munusamy, P., Self, W., Baer, D. R., Sayle, D. C., Seal, S. Behavior of nanoceria in biologically-relevant environments: Environmental Science: Nano 2014, 1, 516–532. (19) Mudunkotuwa, I. A., Grassian, V. H. Biological and environmental media control oxide nanoparticle surface composition: The roles of biological components (proteins and amino 22 ACS Paragon Plus Environment

Page 23 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

acids), inorganic oxyanions and humic acid: Environmental Science: Nano 2015, 2, 429– 439. (20) Valsami-Jones, E., Lynch, I. How safe are nanomaterials?: Science 2015, 350, 388–389. (21) Nel, A. E., Parak, W. J., Chan, W., Xia, T., Hersam, M. C., Brinker, C. J., Zink, J. I., Pinkerton, K. E., Baer, D. R., Weiss, P. S. Where Are We Heading in Nanotechnology Environmental Health and Safety and Materials Characterization?: ACS Nano 2015, 9, 5627–5630. (22) Lynch, I., Cedervall, T., Lundqvist, M., Cabaleiro-Lago, C., Linse, S., Dawson, K. A. The nanoparticle-protein complex as a biological entity; a complex fluids and surface science challenge for the 21st century: Advances in Colloid and Interface Science 2007, 134-135, 167–174. (23) Vidic, J., Haque, F., Guigner, J. M., Vidy, A., Chevalier, C., Stankic, S. Effects of water and cell culture media on the physicochemical properties of ZnMgO nanoparticles and their toxicity toward mammalian cells. Langmuir 2014, 30, 11366–11374. (24) Catalano, F., Alberto, G., Ivanchenko, P., Dovbeshko, G., Martra, G. Effect of Silica Surface Properties on the Formation of Multilayer or Submonolayer Protein Hard Corona: Albumin Adsorption on Pyrolytic and Colloidal SiO2 Nanoparticles: Journal of Physical Chemistry C 2015, 119, 26493–26505. (25) Zhang H., Dunphy D.R., Jiang X., Meng H., Sun B., Tarn D., Xue M., Wang X., Lin S., Ji Z., Li R., Garcia F.L., Yang J., Kirk M.L., Xia T., Zink J.I., Nel A., Brinker C.J. Processing pathway dependence of amorphous silica nanoparticle toxicity: Colloidal vs pyrolytic: Journal of the American Chemical Society 2012, 134, 15790–15804. (26) Warheit, D. B., Webb, T. R., Reed, K. L., Frerichs, S., Sayes, C. M. Pulmonary toxicity study in rats with three forms of ultrafine-TiO2 particles: Differential responses related to surface properties: Toxicology 2007, 230, 90–104. (27) Berger, T., Sterrer, M., Diwald, O., Knözinger, E., Panayotov, D., Thompson, T. L., Yates Jr., J. T. Light-induced charge separation in anatase TiO2 particles: Journal of Physical Chemistry B 2005, 109, 6061–6068. (28) Berger, T., Sterrer, M., Diwald, O., Knözinger, E. Charge trapping and photoadsorption of O2 on dehydroxylated TiO2 nanocrystals - An electron paramagnetic resonance study: ChemPhysChem 2005, 6, 2104–2112. (29) Berger, T., Diwald, O., Knözinger, E., Sterrer, M., Yates Jr., J. T. UV induced local heating effects in TiO2 nanocrystals. Physical Chemistry Chemical Physics 2006, 8, 1822–1826. (30) Berger, T., Diwald, O., Knözinger, E., Napoli, F., Chiesa, M., Giamello, E. Hydrogen activation at TiO2 anatase nanocrystals. Chemical Physics 2007, 339, 138–145. (31) Berger T., Diwald O. Defects in metal oxide nanoparticle powders, 2015. (32) Baumann, S. O., Elser, M. J., Auer, M., Bernardi, J., Hüsing, N., Diwald, O. Solid-solid interface formation in TiO2 nanoparticle networks. Langmuir 2011, 27, 1946–1953. (33) Siedl, N., Elser, M. J., Halwax, E., Bernardi, J., Diwald, O. When fewer photons do more: A comparative O2 photoadsorption study on vapor-deposited TiO2 and ZrO2 nanocrystal Ensembles. Journal of Physical Chemistry C 2009, 113, 9175–9181. (34) Siedl, N., Baumann, S. O., Elser, M. J., Diwald, O. Particle networks from powder mixtures: Generation of TiO2-SnO2 heterojunctions via surface charge-induced heteroaggregation. Journal of Physical Chemistry C 2012, 116, 22967–22973. (35) Elser, M. J., Diwald, O. Facilitated lattice oxygen depletion in consolidated TiO 2 nanocrystal ensembles: A quantitative spectroscopic O2 adsorption study. Journal of Physical Chemistry C 2012, 116, 2896–2903. (36) Evonik Industries 2015, 07/2015. (37) Elser, M. J., Berger, T., Brandhuber, D., Bernardi, J., Diwald, O., Knözinger, E. Particles coming together: Electron centers in adjoined TiO2 nanocrystals: Journal of Physical Chemistry B 2006, 110, 7605–7608. 23 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 25

(38) Kosmulski, M. pH-dependent surface charging and points of zero charge. IV. Update and new approach. Journal of Colloid and Interface Science 2009, 337, 439–448. (39) Jiang, J., Oberdörster, G., Biswas, P. Characterization of size, surface charge, and agglomeration state of nanoparticle dispersions for toxicological studies. Journal of Nanoparticle Research 2009, 11, 77–89. (40) Carter, D. C., Ho, J. X. Structure of serum albumin. Advances in Protein Chemistry 1994, 45, 153–203. (41) Turci F., Ghibaudi E., Colonna M., Boscolo B., Fenoglio I., Fubini B. An integrated approach to the study of the interaction between proteins and nanoparticles: Langmuir 2010, 26, 8336–8346. (42) Martra, G. Lewis acid and base sites at the surface of microcrystalline TiO 2 anatase: relationships between surface morphology and chemical behaviour: Applied Catalysis A: General 2000, 200, 275–285. (43) Minella, M., Faga, M. G., Maurino, V., Minero, C., Pelizzetti, E., Coluccia, S., Martra, G. Effect of fluorination on the surface properties of titania P25 powder: An FTIR study. Langmuir 2010, 26, 2521–2527. (44) Berger, T., Diwald, O. “Traps and Interfaces in Photocatalysis: Model Studies on TiO2 Particle Systems” in “Photocatalysis: Fundamentals and Perspectives” Editors J. Schneider, D. Bahnemann, J. Ye, G. Li Puma, D. Dionysiou (RSC Energy and Environment Series) 2016, 14, 185-217. (45) Rezwan, K., Studart, A. R., Vörös, J., Gauckler, L. J. Change of ζ potential of biocompatible colloidal oxide particles upon adsorption of bovine serum albumin and lysozyme: Journal of Physical Chemistry B 2005, 109, 14469–14474. (46) Mudunkotuwa, I. A., Minshid, A. A., Grassian, V. H. ATR-FTIR spectroscopy as a tool to probe surface adsorption on nanoparticles at the liquid-solid interface in environmentally and biologically relevant media: Analyst 2014, 139, 870–881. (47) Murayama, K., Tomida, M. Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy. Biochemistry 2004, 43, 11526–11532. (48) Lundblad R. L. Biotechnology of Plasma Proteins; CRC Press: London, UK, 2012. (49) Barth, A., Zscherp, C. What vibrations tell us about proteins. Quarterly Reviews of Biophysics 2002, 35, 369–430. (50) Barth, A. Infrared spectroscopy of proteins. Biochimica et Biophysica Acta - Bioenergetics 2007, 1767, 1073–1101. (51) Glassford, S. E., Byrne, B., Kazarian, S. G. Recent applications of ATR FTIR spectroscopy and imaging to proteins. Biochimica et Biophysica Acta - Proteins and Proteomics 2013, 1834, 2849–2858. (52) McUmber, A. C., Randolph, T. W., Schwartz, D. K. Electrostatic Interactions Influence Protein Adsorption (but Not Desorption) at the Silica-Aqueous Interface: Journal of Physical Chemistry Letters 2015, 6, 2583–2587. (53) Kumar, S., Aswal, V. K., Callow, P. pH-dependent interaction and resultant structures of silica nanoparticles and lysozyme protein: Langmuir 2014, 30, 1588–1598. (54) Arrondo, J., Muga, A., Castresana, J., Goñi, F. M. Quantitative studies of the structure of proteins in solution by Fourier-transform infrared spectroscopy. Progress in Biophysics and Molecular Biology 1993, 59, 23–56. (55) Bouhekka, A., Bürgi, T. In situ ATR-IR spectroscopy study of adsorbed protein: Visible light denaturation of bovine serum albumin on TiO2. Applied Surface Science 2012, 261, 369– 374. (56) Lu, R., Li, W.-W., Katzir, A., Raichlin, Y., Yu, H.-Q., Mizaikoff, B. Probing the secondary structure of bovine serum albumin during heat-induced denaturation using mid-infrared fiberoptic sensors. Analyst 2015, 140, 765–770. 24 ACS Paragon Plus Environment

Page 25 of 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(57) Simón-Vázquez, R., Lozano-Fernández, T., Peleteiro-Olmedo, M., González-Fernández, T. Conformational changes in human plasma proteins induced by metal oxide nanoparticles: Colloids and Surfaces B: Biointerfaces 2014, 113, 198–206. (58) Liu W., Rose J., Plantevin S., Auffan M., Bottero J.-Y., Vidaud C. Protein corona formation for nanomaterials and proteins of a similar size: Hard or soft corona?: Nanoscale 2013, 5, 1658–1665.

TOC graphic:

25 ACS Paragon Plus Environment