Synthesis of Metal@Protein@Polymer Nanoparticles with Distinct

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Synthesis of Metal@Protein@Polymer Nanoparticles with Distinct Interfacial and Phase-Transfer Behavior Christian Goldhahn, Jonas Schubert, Helmut Schlaad, James K. Ferri, Andreas Fery, and Munish Chanana Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b02314 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 5, 2018

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Chemistry of Materials

Synthesis of Metal@Protein@Polymer Nanoparticles with Distinct Interfacial and Phase-Transfer Behavior Christian Goldhahn1,2,‡ , Jonas Schubert 3,4‡, Helmut Schlaad5, James K. Ferri6, Andreas Fery3,4 and Munish Chanana*1,2,7 1

Institute of Building Materials, ETH Zürich, 8093, Zürich, Switzerland.

2

Department of Physical Chemistry II, University of Bayreuth, 95447 Bayreuth, Germany

3

Leibniz Institut für Polymerforschung Dresden, Institute for Physical Chemistry and Polymer

Physics, Dresden, 01069 Dresden, Germany. 4

Physical Chemistry of Polymer Materials, Technische Universität Dresden, 01062 Dresden,

Germany 5

Institute of Chemistry, University of Potsdam, 14476 Potsdam, Germany

6

Department of Chemical and Life Science Engineering, Richmond, VA, USA.

7

Swiss Wood Solutions AG, 8093 Zurich, Switzerland.

*Corresponding Author: [email protected] ‡

Authors contributed equally.

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0a. TOC

0b. Abstract In this study, we present a novel and highly facile method for the synthesis of multiresponsive plasmonic nanoparticles with an interesting interfacial behavior. We used thiol-initiated photopolymerization technique to graft poly(N-isopropylacrylamide) onto the surface of protein-coated gold nanoparticles. The combination of the protein bovine serum albumin with the thermoresponsive polymer leads to smart hybrid nanoparticles, which show a stimuli-responsive behavior of their aggregation and a precisely controllable phase transfer behavior. Three interconnected stimuli, namely temperature, ionic strength, and pH, were identified as property tuning switches. The aggregation was completely reversible and was quantified by determining Smoluchowski’s instability ratios with time resolved dynamic light scattering. The tunable hydrophobicity via the three stimuli was used to study interfacial activity and phase transfer behavior of the nanoparticles at an octanol/water interface. Depending on the type of coating (i.e. protein or protein/polymer) as well as the three external stimuli, the nanoparticles either remained in the aqueous phase (aggregated or nonaggregated), accumulated at the oil/water interface, wet the glass wall between the glass vial and the octanol phase, or even crossed the oil/water interface. Such smart and interfacially active nanoparticles with external triggers that are capable of crossing oil/water interfaces under physiological conditions open up new avenues for a variety of applications ranging from the development of drug-delivery nanosystems across biological barriers to the preparation of new catalytic materials. 1. Introduction Nanoparticles (NPs) that can selectively adsorb or even cross oil-water interfaces triggered by a specific combination of various stimuli are highly interesting for various 2 ACS Paragon Plus Environment

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life science and industrial applications. They could be used in drug delivery across biological barriers,1 biosensing,2 interfacial or bulk catalysis,3 emulsion stabilization,45

and fabrication of 2D/3D nanocrystal assemblies6-9 with special electrical,10

magnetic,11 and optical properties.12 Such systems can change their physicochemical properties, such as charge, charge density or hydrophilicity/hydrophobicity-balance simultaneously or successively upon environmental changes. The stimuli for these changes can be temperature,13 pH,14 ionic strength15, light16 or gases (e.g. CO217). Hence, these systems are designated to selectively operate or conduct chemical reactions18 in aqueous phase,19-20 oil phase21 or at an oil-water interface.18,

22

A

precise control over the interfacial properties of such nanosystems is highly important for enabling sophisticated performances in fields of (biphasic) catalysis and drug delivery across biological barriers23, particularly at various physiological conditions (different pH values and salt concentrations). Hereby the chemical composition and the architecture24-25 of the organic coating is prevailing the interfacial activity and controlled phase transfer of such colloidal systems. A general strategy for the fabrication of such interfacially active systems that can cross oil-water interfaces is the grafting of non-ionic surfactants or non-ionic hydrophilic polymers26-28 on the particle surface. This can be done either in graftingto29, grafting-through30 or in grafting-from31 fashion. Such coatings can undergo a hydrophilic/hydrophobic transition upon an external stimulus such as temperature or ionic strength. Hence, when an oil-water interface is provided, they can readily adsorb at the interface or even cross it.26-28 By incorporating weak acidic or basic functional groups such as carboxylic or amino groups, a pH-responsive switch can be integrated in such colloidal systems.32 By fine tuning the environmental parameters such as pH, temperature and ionic strength, the hydrophilicity/hydrophobicity balance as well as the balance between the charged and uncharged state of NPs can be precisely controlled. The result is a switchable system in regard to reversible aggregation,33 interfacial adsorption34-35 or phase transfer behaviour27, 36 with various interconnected switches, usually temperature and pH or temperature and ionic strength.37 In this context, we previously reported on a protein/polymer-coated dual-responsive NP system, with an inter-dependency of the two stimuli pH (owed to protein coating) and

temperature

(owed

to

the

thermo-sensitive 3

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polymer

coating).

These


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protein/polymer-coated NPs aggregated and disaggregated reversibly in certain combinations of pH and temperature.37 They were prepared by grafting a thermosensitive

polymer,

i.e.,

poly[oligo(ethylene

glycol)

methacrylate-co-2-(2-

methoxyethoxy)ethyl methacrylate] (poly(OEGMA-co-MEO2MA) on bovine serum albumin (BSA)-coated gold NPs via surface-initiated atomic transfer radical polymerization (SI-ATRP). Although, ATRP is one of the most popular and versatile controlled polymerization techniques for grafting polymers onto surfaces and other polymers, it also features certain drawbacks. The use of toxic transition metal catalysts, e.g. Cu(II) complexes, represents one of the major drawbacks of this technique, particularly in biomedical and food grade applications.38 Such metal catalysts are difficult to completely remove from the final product, most often due to their strong adsorption or interference with the other synthesis educts and products. In our previous report,37 the Cu catalyst interfered strongly with the BSA-coating of the Au@BSA NPs as well as with that of the final Au@BSA@Poly(OEGMAMEO2MA-) NPs. Hence, a tedious purification procedure was required, in order to remove the Cu catalyst completely from the final product. Furthermore, for performing ATRP on the protein-coated NPs, an initiator site has to be introduced to the coating. This represents an additional reaction step, making the whole reaction process more arduous. In this work, we present a more compelling approach, i.e. protein-initiated photopolymerization for the synthesis of a protein/polymer-coated NP system, which is far simpler than ATRP and easily scalable. Here, neither a metal catalyst, which interferes with the protein coating, nor any pre-modification of the protein coating (e.g. initiator-coupling reaction) is necessary for the polymerization. We synthesized a novel protein/polymer-coated NP system (Au@BSA@Poly(Nisopropylacrylamide)(PNIPAM)), by grafting PNIPAM from BSA-coated gold NPs, simply by irradiating Au@BSA NPs (UVA-light, 320 nm < λ < 420 nm) in the presence of the NIPAM monomer. The resulting BSA/PNIPAM-coated NPs are responsive towards the stimuli pH, ionic strength and temperature (multi-responsive). Furthermore, they are highly interfacially active. The hydrophilic/hydrophobic phase transition of the NPs can thereby be fine-tuned under different combinations of the three stimuli. The degree of their reversible agglomeration and the degree of their adsorption at the oil-water interface (if provided) can be controlled. Moreover, by 4 ACS Paragon Plus Environment

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further increasing the hydrophobicity of the NPs, they can even cross the oil-water interface into the oil phase. By the best of our knowledge, a responsive colloidal system with multiple inter-connected switches and a precise control over NP aggregation, interfacial adsorption and their controlled phase transfer across the interfaces has not been reported yet. Thus, the BSA/PNIPAM-coated Au NPs represent a sophisticated class of interfacially active and multi-responsive plasmonic nanosystems, which can be synthesized

in

large

scales

simply

via

protein-initiated

free

radical

photopolymerization. 2. Results and Discussion The multi-responsive and interfacially active protein/polymer-coated gold NPs, Au@BSA@PNIPAM NPs, were synthesized through photopolymerization of NIPAM initiated from the thiol groups of the protein-coating (~ 24 BSA molecules)19 on the Au@BSA NP surface (Figure 1 A). For the core we selected Au NPs because of their plasmonic properties which allows easy monitoring of their colloidal stability (aggregation/disaggregation processes) and facile quantification of the NP concentration during the phase transfer processes.39 We selected BSA for the protein coating, since the resulting Au@BSA NPs exhibit a high colloidal stability (at pH above and below pI) and pH-responsive behavior.40, 37 Au@BSA NPs exhibit positive (+25 mV) and negative (-50 mV) surface charges below and above the isoelectric point of Au@BSA (pI = 4.6-4.8)37, 41, respectively. At pH = pI the overall charge of the protein-coated NPs is zero and the hydrophobic interactions increase, leading to a reversible agglomeration of the NPs.37 However, unlike the protein BSA, the BSAcoated Au NPs lack the intrinsic thermoresponsiveness. Therefore, the Au@BSA NPs do not agglomerate and disagglomerate reversibly upon temperature changes up to 80°C.37 The plausible reason for this behavior is the increased stability against temperature and pH of proteins upon their immobilization on solid surfaces.42-43 To impart thermoresponsiveness to these NPs, we grafted the thermoresponsive polymer PNIPAM44 onto the pH-responsive Au@BSA NPs directly from the protein layer of the BSA-coated NPs without using any metal catalyst and any premodification of the protein coating. The thiol groups45-46 of BSA (BSA contains 35 cysteine groups with 17 disulfides and at least 1 thiol group47) can form thiyl-radicals 5 ACS Paragon Plus Environment


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when irradiated with UV-light. The radicals can then be used to initiate the free radical polymerization of vinyl monomers. 46, 48-52 In comparison to the previously mentioned Au@BSA@MEO2MA-OEGMA/BSA NP system, we chose PNIPAM as the thermo-sensitive polymer instead of poly(OEGMAMEO2MA), since PNIPAM exhibits a sharper phase-transition at 32 °C, its lower critical solution temperature (LCST).53 For the synthesis of the Au@BSA@PNIPAM NPs, citrate-stabilized gold NPs (RTEM ~ (6.75 ± 0.84) nm, LSPRmax = 519 nm) were coated with the protein BSA as previously reported.7, 37 The Au@BSA NPs showed a LSPR band with a maximum at 525 nm. The red shift of 6 nm compared to the citrate-stabilized Au NPs is caused by the refractive index changes upon successful protein coating of the NPs without any aggregation (Figure 1 B). The purified BSA-coated NPs were then mixed with an aqueous solution of the monomer NIPAM. After removing the oxygen by three freezepump-thaw cycles or 30 min degassing with argon, the solution was irradiated with UVA-light (320 nm < λ < 420 nm) for 24 hours at room temperature. In this process, the UV light creates thiyl radicals from the thiol groups of the protein, which initiate a free radical photopolymerization of NIPAM directly from the protein surface. The final purified Au@BSA@PNIPAM NP dispersions exhibited similar color as the Au@BSA NP and a low change in the LSPR band (red shift ~ 2-3 nm, no broadening, LSPRmax = 527 nm, Figure 1B). This indicates a successful PNIPAM coating without aggregation and without a significant change of the refractive index around the NPs. Figure 1C shows the TEM images of Au@BSA@PNIPAM NPs in dried state. The thickness of the organic coating was measured to be (3.7 ± 0.6) nm (Figure 1), which is significantly higher than that of Au@BSA NPs (1-2 nm7, 37). Also the hydrodynamic radius of the NPs increased after the PNIPAM grafting (Figure 3B). Depending on the synthesis batch, hydrodynamic shell thicknesses of PNIPAM of 1- 14 nm were measured by dynamic light scattering (DLS; for detailed results as well as calculations of the molar mass of PNIPAM see Experimental section and Table S1 in SI). For the time-resolved DLS measurements, the Au@BSA@PNIPAM NPs with a PNIPAM

shell

thickness

of

4nm

(Au@BSA@PNIPAM4)

(Au@BSA@PNIPAM11) were used (Figure 3).


and

11nm

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Figure 1: (A): Schematic illustration of the UV-light induced, thiol-initiated free radical polymerization of NIPAM onto the NP surface. (B): UV/Vis-spectrum of the synthesized protein-coated NPs (black line) in comparison to the UV/Vis spectrum of the synthesized polymer-protein-gold hybrid NPs (magenta line). The peak maximum of Au@BSA@PNIPAM NP is at 527 nm and therefore 2 nm red-shifted in comparison to the maximum of Au@BSA NP, which is at 525 nm. This red-shift corresponds to a change in the refractive index around the particle, indicating polymer-formation. (C): TEM-image of the synthesized polymer-protein-gold hybrid nanoparticles, stained with I2. The organic layer is well visible as a grey corona around the black metal cores and is approximately 4 nm in the dry state (scale bar depicts 50 nm). The inset shows the magnification of one individual particle.

The PNIPAM shell leads to a pronounced change in the colloidal stability depending on pH, temperature, ionic strength and the polymer shell thickness. The behavior of the protein/polymer-coated NPs suggests the presence of an interplay between electrostatic and steric stabilization forces. As previously reported,

37, 40, 54

the

protein-layer provides electrosteric (electrostatic and steric) stabilization to the NPs. The PNIPAM shell in this case, makes an additional contribution to the steric stabilization, depending on the shell thickness. In contrast to the Au@BSA NPs, that reversibly agglomerate at pH 4.7 = pI

37

, the

polymer-protein hybrid NPs were stable over the whole pH range at temperatures below the LCSTPNIPAM (here room temperature, Figure 2 B, upper panel). When the 7 ACS Paragon Plus Environment


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pH is above or below the pI of BSA, the protein layer is charged and therefore provides electrostatic repulsion between the particles.37,

40

At pH ≈ pI, although the

inner protein layer is not charged, the NPs are still stable and individually dispersed. This effect is owed to the additional steric stabilization provided by the solvated PNIPAM shell. When heated to T > LCSTPNIPAM (e.g. 50 °C, Figure 2B, lower panel), the NP dispersions in the pH region below the pI of BSA showed an intense color change to purple, indicating NP agglomeration. The LSPR band exhibits a pronounced red shift of 32-68 nm, accompanied by a strong broadening of the spectrum. Here, the hydrophobic interactions between the polymer chains predominate which leads to a collapse of the polymer shell and hence steric stabilization vanishes, leading to NP agglomeration. The effect is completely reversible over multiple cycles. When cooled down to T < LCST (Figure 2 C) the dispersions regain their original color, as well as the spectral shape and position (no spectral broadening and shift, Figure 2 B, SI Figure S2 and Video V1). Since the thermoresponsive behavior of Au@BSA@PNIPAM NPs is pH-dependent, we investigated their thermoresponse at pH 2, 4.7 and 7.4. Therein pH 4.7 represents the pI and pH 2 and 7.4 (physiological conditions) are exactly 2.7 units below and above the pI. We performed temperature-dependent DLS measurements to determine the lowest critical aggregation temperature (temperature at which the NPs

start

aggregating,

i.e.

sudden

size

increase

of

the

NPs)

of

the

Au@BSA@PNIPAM NPs (Figure 2 D and E). For pH 2 and 4.7 this value can be extracted from the point, where the hydrodynamic radius increases. This increase is due to the aggregation of the NPs, caused by the collapse of the PNIPAM coating. Interestingly, for pH 7.4 (very low ionic strength, < 1mM), the NPs do not aggregate, but the collapse of PNIPAM shell can be recognized by a decrease of the hydrodynamic radius from 20 to 17 nm, which is consistent with literature.37, 55 As all solutions were prepared by adjusting MilliQ-Water with NaOH or HCl, a gradient of ionic strength is induced by the pH adjustments. At pH 2 the ionic strength of the NP dispersion is per se 12 mM, which is sufficiently high for affecting the aggregation behavior of the NPs. In order to compare the aggregation behavior at different pH values, the ionic strength must be kept constant. By adjusting the same ionic strength (12mM) for pH 7.4, we also observed NP aggregation at pH 7.4 and T > LCST 8 ACS Paragon Plus Environment

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(Figure 2 D and E). At pH > pI, the protein layer of the NPs is sufficiently charged (negative),37 providing electrostatic repulsion to the NPs. Consequently, NP agglomeration at T > LCSTPNIPAM is prevented (Figure 2 D (triangles)) and E). At pH ≈ pI (Figure 2 D (squares)) and pH < pI (Figure 2 D (diamonds)) the electrostatic repulsion

provided

by

the

protein

layer

is

not

sufficient37,

40

and

the

Au@BSA@PNIPAM NPs agglomerate when the temperature is raised above LCSTPNIPAM and the PNIPAM shell becomes hydrophobic. Hence, the responsive behavior of the NPs, including their colloidal stability, does not solely depend on pH but is interconnected with the effects induced by the ionic strength.

Figure 2: (A): Optical photographs of particle suspensions at different pH-values and at two different temperatures (RT and 50 °C). The particle agglomeration at pH ≤ pI and elevated temperature is well visible as the suspension changes its color from red to purple due to interparticle plasmonic coupling. (B): UV/Vis spectra of the synthesized hybrid particles at pH = 4.7 and different temperatures. The color change at the particle agglomeration is characterized by a red-shifted and broadened LSPR-peak at 50 °C (red line) compared to the narrow LSPR-peak



at room temperature (cyan line). The blue line shows the spectrum after cooling down the suspension back to RT and indicates that the agglomeration process is completely reversible by cooling down the suspension. (C): The reversibility is presented over four heating and cooling cycles. The graph shows the LSPR-position in dependence of the temperature at pH 4.7. (D): DLS-data of Au@BSA@PNIPAM4nm NPs at different pH-values in dependence of the temperature. At pH ≤ pI the hydrodynamic diameters increase due to particle agglomeration. At pH 7.4 no such agglomeration occurs. (E) Detailed look on the DLS-data of the synthesized Au@BSA@PNIPAM NPs at pH 7.4 in dependence of the temperature. As the temperature increases, the hydrodynamic radius (RH) decreases as a result of the polymer collapse. This behavior is characteristic for particles coated with temperature-responsive polymers and does not depend form the polymer shell thickness.

In order to separate and isolate the effects of pH from that of ionic strength, the ionic strength of the dispersions has to be adjusted as such that it is sufficiently high to ensure the effects (i.e. aggregation) induced by the ionic strength consistently. We chose an ionic strength of 150 mM, which is sufficiently high for inducing aggregation, but also corresponds to the physiological conditions. Hence, any differences in the responsive behavior of the Au@BSA@PNIPAM NPs at this ionic strength would be then ascribed to the effects of pH. Furthermore, as the aggregation of Au@BSA@PNIPAM NPs is induced or prevented by the outer polymer (PNIPAM) shell of the NPs, it is plausible that also the thickness of the PNIPAM shell (the additional sterically stabilizing layer) could play an important role on the aggregation behavior of the NPs. For the quantification of the influences of the given parameters, i.e. ionic strength, pH and the PNIPAM shell thickness, on the aggregation behavior of the NPs, Smoluchowski’s theory of aggregation can be applied. Therein, the stability ratio W is defined, which is the value for the probability of aggregation of two single NPs to dimers (Figure 3A). Often the inverse stability ratio W-1 is used to express the colloidal potential between two particles in terms of dimer generation rate:

Herein, k11 is the measured aggregation rate constant, whereas k(11)fast is the aggregation rate in the diffusion limited regime. 56-58 In this work, we measured k(11)fast at 50°C – i.e. a rapidly aggregating system. Salt dependent measurements show that the diffusion limited regime is in the range of 50-500 mM ionic strength (Figure S3). Hence, for the measurements we chose an ionic strength of 150 mM, which also corresponds to physiological conditions. To measure the aggregation rate constants, we investigated the aggregation kinetics of Au@BSA@PNIPAM NPs with PNIPAM-coatings of different thicknesses. 10 ACS Paragon Plus Environment

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Measurements were executed in dependence of temperature and pH at constant ionic strength (150mM) via time resolved DLS measurements of the hydrodynamic size of the NPs. By plotting the hydrodynamic radius RH against time, we see a linear increase in the beginning of the aggregation process (Figure 3C,D). For the formation of dimers (i.e. first aggregation species) a size increase of up to 1.4 times of the NPs radius is assumed.56-57 With the slope m of the first increase of the hydrodynamic radius (Figure 3C,D) k11 is calculated by dividing m by an optical factor O,59 the NPs concentration (CNPs) and the initial hydrodynamic radius RH,1.

The employed Au@BSA@PNIPAM NP thicknesses of the PNIPAM shell, were 4 nm (Au@BSA@PNIPAM4nm) and 11 nm (Au@BSA@PNIPAM11nm) (Figure 3B). The inverse stability ratio W-1 was calculated for the NP-systems for pH 2, 4.7 and 7.4 at the constant ionic strength of 150 mM. Time resolved DLS measurements were performed at 35 °C, 40 °C and 50 °C, as for temperatures below the LCST (i.e.,

Synthesis of Metal@Protein@Polymer Nanoparticles with Distinct

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