Temperature-Triggered Protein Adsorption on ... - ACS Publications

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Temperature-Triggered Protein Adsorption on Polymer-Coated Nanoparticles in Serum Olga Koshkina,§,#,† Thomas Lang,§,# Raphael Thiermann, § Dominic Docter,// Roland Stauber,// Christian Secker,v Helmut Schlaad,∆ Steffen Weidner,# Benjamin Mohr,§ Michael Maskos,*, §,‡, Annabelle Bertin#,◊,‡ §

Fraunhofer ICT-IMM, Carl-Zeiss-Str. 18-20, 55129 Mainz, Germany, #BAM Federal Institute

for Materials Research and Testing, Unter den Eichen 87, 12205 Berlin, Germany, //Molecular and Cellular Oncology/Department of Nanobiomedicine, University Medical Center of Johannes Gutenberg-University Mainz, Langenbeckstraße 1, 55101 Mainz, Germany, vMax Planck Institute of Colloids and Interfaces, Department of Colloid Chemistry, Research Campus Golm, 14424 Potsdam, Germany, ∆University of Potsdam, Institute of Chemistry, Karl-LiebknechtStraße 24-25, 14476 Potsdam, Germany, ◊Free University Berlin, Institute of Chemistry and Biochemistry – Organic Chemistry, Takustraße 3, 14195 Berlin, Germany

ABSTRACT: The protein corona, which forms on the nanoparticle’s surface in most biological media, determines the nanoparticle’s physicochemical characteristics. The formation of the protein corona has a significant impact on the biodistribution and clearance of nanoparticles in vivo. Therefore, the ability to influence the formation of the protein corona is essential for most biomedical applications to include drug delivery and imaging. In this study, we investigate the protein adsorption on nanoparticles with a hydrodynamic radius of 30 nm and a coating of

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thermoresponsive poly(2-isopropyl-2-oxazoline) in serum. Using multi-angle dynamic light scattering (DLS) we demonstrate that heating of the nanoparticles above their phase separation temperature induces formation of agglomerates, with a hydrodynamic radius of 1 µm. In serum, noticeably, stronger agglomeration occurs already at lower temperatures compared to serum-free conditions. Cryogenic transmission electron microscopy (cryo-TEM) revealed a high pack density of agglomerates when serum was not present. In contrast, in the presence of serum agglomerated nanoparticles were loosely packed, indicating that proteins are intercalated between them. Moreover, an increase in protein-content is observed upon heating, confirming that protein adsorption is induced by alteration of surface during phase separation. After cooling and switching back the surface most of the agglomerates were dissolved and the main fraction returned to the original size of approximately 30 nm as shown by asymmetrical flow-field flow fractionation (AF-FFF) and DLS. Furthermore, the amounts of adsorbed proteins are similar before and after heating the nanoparticles above their phase separation temperature. Overall, our results demonstrate that the thermoresponsivity of the polymer coating enables turning on and off the corona-formation on nanoparticles in situ. As the local heating of body areas can be easily done in vivo, the thermoresponsive coating could potentially be used to induce the agglomeration of nanoparticles and proteins and accumulation of nanoparticles in a targeted body region.

INTRODUCTION Nanoparticles have a wide range of potential applications in biomedical sciences to include targeted drug delivery systems and contrast agents for imaging methods such as magnetic resonance imaging or fluorescence spectroscopy.1,

2, 3, 4

Aiming at therapeutic drug delivery

systems, nanoparticles are most commonly intravenously injected into the blood stream at which


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point they immediately begin to interact with a very complex biological milieu.5, 6 Biomolecules such as lipids, sugars and especially serum proteins adsorb onto the surface of the nanoparticle to form the so-called “protein corona”.7 The protein corona can change how the body interacts with a nanoparticle because the nanoparticle’s size and surface characteristics such as charge or targeting molecules can be altered. Therefore, crucial implications with respect to cellular uptake as well as for biodistribution and clearance of nanoparticles can be expected in vivo.7, 8, 9 Studies comparing nanoparticles varying in size,10,

11,

12,

13

shape,14 charge,15,

16

and

hydrophobicity17 have demonstrated that the interplay of physicochemical properties between nanoparticles and serum proteins determine the composition of the protein corona and the kinetics of corona formation. Moreover, the composition of corona depends on the composition of medium18 is dynamic and can alter its properties over time17, 19 or when the composition of the medium changes.20 The protein corona is usually subdivided in two parts: proteins which are strongly and weakly bound to the particle’s surface are known as the “hard” and “soft” coronas, respectively.17, 21, 22 However, in our opinion the transition between this two parts of corona is only vaguely defined; thus, it is difficult to differentiate between these two parts of corona. The protein corona is what the body physically “sees” and hence its composition will affect biological responses a nanoparticle may have.22 In vitro models have shown that the protein corona impacts particle cytotoxicity,14, 23 cellular uptake,23, 24 cell death at early exposure of time25 and can modulate prothrombogenic properties.26 Apparently, controlling the formation of the protein corona is necessary for successful application of a nanomaterial in vivo and should help to better influence the biodistribution or organ accumulation of nanoparticles.


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Thermoresponsive polymers are promising candidates for investigating controlled protein corona formation.27,

28, 29, 30

Those polymers, which have a lower critical solution temperature

(LCST), are mostly hydrophobic above the LCST while below the LCST they are mostly hydrophilic. In recent years poly(2-oxazoline)s (POx), a class of bioinspired synthetic thermoresponsive pseudo-peptide polymers, have gained attention as biomaterials31,

32

due to

their biocompatibility and low toxicity.33 Although, POx can be degraded via a radical mechanism,34 in a manner similar to PEG, they show low organ accumulation32, 35, 36 and high stability against hydrolysis to monomers.37 Therefore, POx appear more promising for in human use than for example poly(N-isopropylacrylamide), which application is often limited by possible organ accumulation and cytotoxicity of monomeric N-isopropylacrylamide.38, 39, 40 The ability of POx to adsorb and desorb single proteins on flat surfaces above and below LCST has already been demonstrated.41, 42 However, it should also be noted that curvature of the nanoparticles has impact on the corona formation.19,

25

Therefore, for applications of these

polymers as a coating of nanoparticles for in vivo use we must now understand how protein adsorption occurs on the curvature of a nanoparticle in the presence of the full range of biomolecules found in serum. In this study we use poly(2-isopropyl-2-oxazoline) (PiPOx) as a thermoresponsive surface modification of nanoparticles. The LCST of PiPOx is around 40 °C, depending on the polymer chain length, composition, or salinity of medium, and is close to the body temperature.43, 44, 45 Using serum as a medium we demonstrate that the protein adsorption is strongly suppressed at temperatures below LCST. Above LCST protein adsorption takes place and nanoparticle-protein agglomerates are formed. While cooling back to room temperature the proteins desorb from the


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surface, so that the adsorption appears to be a reversible process. Thus, the protein adsorption could be switched on and off in situ using temperature as an external stimulus.

EXPERIMENTAL SECTION Materials. Water was purified with ultrapure water system from Sartorius. All chemicals were used

as

received:

Triethoxyemethylsilane

(T),

diethoxydimethylsilane

(D),

p-

chlorophenylmethyl-triethoxysilane (ClBz-T), 3-(triethoxysilyl)propylsuccinic anhydride and PyBOP® were purchased from ABCR, Germany, N,N-diisopropylethylamine (DIEA), rhodamine B, maleimide, dithiothreitol (DTT), bromophenol blue from Sigma-Aldrich, Germany, tris(hydroxymethyl)aminomethane hydrochloride (TRIS-HCl) from Carl-Roth, Germany. All solvents were obtained from Sigma-Aldrich, Germany, Applichem, Germany or Carl-Roth, Germany in at least p. a. quality. The Gibco® RPMI 1640 was purchased from Life Technologies and FCS gold from GE Healthcare. Dialysis was carried out with Spectrapor 6 regenerated cellulose membrane, MWCO 15 kDa from Spectrumlabs, USA. N-(2-aminoethyl)maleinimide was synthesized as described by Antczak et al.46 and rhodamine B-labeled silane monomer by procedure previously described by our group.47 The synthesis of thiol-terminated poly(2-isopropyl-2-oxazoline) is described in Supporting Information (Scheme S1 and experimental procedure). Characterization Methods. The investigation of particle size in aqueous solution was performed by multi angle dynamic light scattering (DLS) using an ALV/CGS-3 Compact Goniometer System, with HeNe Laser (λ0 = 633 nm). All samples were filtered (Millex-LCR filter, 0.45 µm pore size) before measurement. The measurements were carried out at scattering angles θ = 26-150° in 4 or 8 angular steps. The data analysis was carried out with HDRC


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software, which was kindly provided by Manfred Schmidt, University Mainz, Germany. For determination of hydrodynamic radii in water or serum-free RPMI, the autocorrelation functions were evaluated by applying a biexponential fit. The size in serum-containing medium at T = 293 K was calculated using the multicomponent analysis, the autocorrelation function of nanoparticle-protein mixture was spited in a triexponential fit function for serum (known component) and biexponential fit function for nanoparticles (unknown component).48 Triexponential fitting was applied to evaluate the size in serum-containing medium at different temperatures.49 To obtain information on polydispersity µ2(90°) values were derived from a cumulant fit at 90°. The final particles size was obtained by extrapolation of apparent Rh values at different scattering angles qà0, where q is the absolute value of the scattering vector.50 Asymmetrical flow field-flow fractionation (AF-FFF) was conducted with AF-FFF system 2.0 with a PMMA plate from Consenxus. This system consists of constaMETRIC® flow pump (Thermos Separations), WellChrom Micro-Star K-100 injection pump (Knauer) a channel with 190 µm spacer, a Liqui-Flow® (Bronkhorst Hi-Tech). The separation was carried out using a polyethersulfone membrane at the cross flow gradient of 2.5-0 mL/min. For detection a 486absorption detector (Waters) at λ = 560 nm or fluorescence detector (Merck-Hitachi F1110) with excitation at 540 nm and detection at 580 nm were used. Particles were incubated in medium at room temperature or at 37 °C for 15 min prior to measurements. Transmission electron microscopy (TEM) was measured with EM 420K (Philips), equipped with LaB6-cathode and slow scan 1K CCD camera at acceleration voltage of 120 kV. For the sample preparation the particles were deposited from solution with c = 2.5 mg/mL on carbon coated copper grid.


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Cryogenic TEM imaging was performed by means of a Zeiss Libra® 120 under liquid N2 cryo conditions on holey carbon-coated copper grids after freezing the solution at controlled temperature (T > LCST) in liquid ethane at -180°C. The microscope was used at 120 kV acceleration voltage and the images were taken with a CCD camera. The concentration of samples was 0.25 mg/mL. The incubation time above the cloud point was 15 min. ζ-Potential was obtained with Zetasizer Nano from Malvern using a dip cell. The measurements were carried out at 5 mM sodium chloride solution at pH ≈ 7 at c = 0.25 mg/mL. Thermogravimetric analysis (TGA) was measured with TG/DTA 220 from Seiko using a platinum pan as a reference. The measurements were carried out under synthetic air atmosphere between room temperature and 600 °C with a heating rate of 10 K/min and between 600 °C and 800 °C under nitrogen atmosphere with a heating rate of 20 K/min. The grafting density was calculated from the difference in weight loss between particles before and after functionalization. MALDI-ToF mass spectrometry was carried out with Autoflex III Smartbeam from Bruker using

trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile

(DCTB)

as

matrix. Synthesis of nanoparticles. The synthesis of carboxy-modified rhodamine B labeled poly(organosiloxane) core-shell nanoparticles was performed as described previously in aqueous emulsion with dodecylbenzenesulfonic acid as the surfactant using a slightly modified procedure (Scheme S2 in Supporting Information).47, 51 The composition of monomers for the core was 7 g trimethoxymethylsilane

(T),

5g

diethoxydimethylsilane

(D),

3g

(p-

chloromethyl)phenyltrimethoxysilane and 0.5 g rhodamine B-(p-trimethoxysilyl)benzylester. For subsequent polycondensation of carboxy-modified shell 2 g T, 3 g D and 5 g 3(triethoxysilyl)propylsuccinic

anhydride

were

used.

After

end

capping

with


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trimethylethoxysilane nanoparticles were purified by dialysis against methanol and transferred in THF by dialysis for modification with polymer. 120 mg of carboxy-modified nanoparticles in 3.86 mL THF from dialysis were diluted with 4 mL DMF and reacted with 0.30 g (1.17 mmol) N-(2-aminoethyl)-maleiimide using DIEA as catalyst and 0.41 g (1.56 mmol) PyBOP®52 as a coupling agent under N2-atmosphere for 2 days. After the reaction, particles were purified by dialysis against DMF yielding a solution of maleimido-functionalized particles. The concentration of particles in the resulting solution was determined gravimetrically (5.7 mg/mL). Finally, 2.6 mL of particle solution were degassed with nitrogen followed by addition of 150 mg PiPOx and 20 µL DIEA in DMF. After stirring under N2 atmosphere over night water soluble particles were obtained, which were purified by dialysis against water. Incubation of nanoparticles with serum and isolation of nanoparticles with “hard” corona. All experiments were conducted at least twice to ensure reproducibility. The ratio of total particle-surface area to serum was kept the same for the two different particles to ensure comparability between the results as described previously.10 Particle suspensions were incubated with an equal amount of serum for 1 h at 20 °C and 37 °C (total volume 500 μL). The samples were centrifuged to pellet the particle-protein complexes (10 min at 12 000 rpm/4 °C). The pellet was resuspended in PBS buffer, transferred to a new vial, and centrifuged again to pellet the particle-protein complexes (10 min at 12 000 rpm/4 °C); this procedure was repeated three times. After the third washing step, the supernatant did not contain any detectable amount of proteins (SDS PAGE as a control). Proteins were eluted from the particles by adding SDS-sample buffer (62.5 mM Tris-HCl pH 6.8; 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue) to the pellet and incubating at 95 °C for 5 min.


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Protein separation by 1-D gel electrophoresis.25,

53

For 1D gel electrophoresis, 20 μL of

recovered nanoparticles in SDS-sample buffer were separated on a 12% SDS-polyacrylamide gel. Gels were run at the constant voltage of 200 V for 35 min and stained with Coomassie Brilliant Blue R-250 (Bio-Rad). Protein quantification was performed using the BioRad Protein Assay (Bio-Rad) as reported.

RESULTS AND DISCUSSION PiPOxylated nanoparticles in water. In this study we used fluorescent poly(organosiloxane) (POS) core-shell nanoparticles for the subsequent modification with polymer.54 These initial carboxy-functionalized nanoparticles have a hydrodynamic radius of 17 nm with second cumulant value µ2(90°) = 0.12 (DLS in methanol with 5 mM KBr). These nanoparticles were first modified with maleimido-groups and then coupled to thiol-functionalized poly(2-isopropyl2-oxazoline) (PiPOx) (Supporting Information, Scheme S2). The PiPOx with approximately 67 monomer units has a cloud point of 41 °C, as determined by turbidimetry.55 We decided to use this polymer to ensure that no additional denaturation of serum proteins by temperature takes place. Figure 1 shows the schematic structure (a) and the transmission electron microscopy (TEM) image (b) of the PiPOxylated nanoparticles.


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Figure 1. a. Schematic illustration of PiPOx coated, fluorescent poly(organosiloxane) nanoparticles. b. TEM image of nanoparticles deposited from water. Scale bar 100 nm. Table 1. Characterization of PiPOxylated nanoparticles for protein adsorption studies R (TEM)

(9.1 ± 1.7) nm

Rh (DLS) (µ2)

31.1 nm (0.13)

Rh (AF-FFF)

27 nm

ζ-potential

(-4 ± 1) mV

Grafting density of PiPOx (TGA)

0.1 µmol / m2

Cloud point (turbidimetry, transmittance 80%)

34 °C

Agglomeration temperature (DLS)

30 °C

To obtain information on characteristics of the nanoparticles, we characterized them with a variety of methods without serum proteins. The results are summarized in Table 1. According to TEM, the nanoparticles are ~9 nm in size (radius), which is smaller than the hydrodynamic radius obtained by DLS. This effect might be caused by shrinking of poly(organosiloxane)-


10

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network and lack of contrast of collapsed of the PiPOx-shell after drying for TEM.56 However, µ2(90°) value obtained by cumulant analysis of the autocorrelation function is 0.13, which could indicate a broad size distribution of the nanoparticles caused by agglomeration. The fitting of autocorrelation function at θ = 90° with triexponential fit reveals that also smaller size fraction with 2% of the total amplitude present in the solution (Supporting Information, Figure S5). This small size fraction, which might be free polymer, contributes to the increase of µ2(90°) value. The fractionation of particles by AF-FFF provides only one size fraction with a radius of 27 nm, which is similar to radius from DLS (Supporting Information, Figure S7, left). The tailing of the elution profile might have been caused by either slight agglomeration of the nanoparticles or by nanoparticle-membrane interactions. As no second fraction could be detected, we assumed that the concentration of any kind of agglomerates could be disregarded for protein adsorption studies. Since PiPOx is an uncharged polymer, the zeta potential is close to zero. The cloud point of PiPOxylated nanoparticles was examined by turbidimetry and DLS (Supporting Information, Figure S6). Both methods demonstrate lower cloud point of PiPOXylated nanoparticles, compared to 41 °C for free polymer in aqueous solution. This behavior can be explained by the decrease of flexibility of polymer chains after grafting of polymer onto the surface. Additionally, the phase separation temperature of PiPOx also depends on the polymer concentration and decreases when the concentration increase.44 The local concentration of polymer chains on the nanoparticle surface could be higher than in the solution, which would also lead to changes in the phase separation behavior. However, further investigation of phase transition on the molecular level, including study of possible cooperative behavior of polymer chains, is needed to explain this change of cloud point upon grafting of polymer onto nanoparticles. The slight discrepancy between DLS and turbidimery is due to the difference in measurement setup and measured


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variables. In addition, the fractionation of particles by AF-FFF reveals that the particles have the same size before and after heating to 37 °C (Supporting Information, Figure S7). PiPOxylated nanoparticles in biological media below phase separation temperature. Since the colloidal stability of nanoparticles in biological media can be lowered especially with high salinity, we initially characterized the nanoparticles in the serum-free and serum-containing RPMI medium at room temperature.

Table 2. Characterization of nanoparticles in protein-free and protein-containing media by DLS. Sample / medium

Rh / nm

µ2(90°)

(fit function)

cumulant fit

NPs in RPMI 1640

30.9 (biexponential)

0.14

NPs in RPMI 1640 + 5% FCS

39 (multicomponent48)

0.39

5% FCS in RPMI without NPs 13.3 (triexponential)

0.33

DLS results (Table 2) demonstrate that the nanoparticles remain stable under physiological conditions in RPMI 1640 medium with or without proteins. In serum-free medium the size and the µ2(90°) value are comparable to those in water. In the serum-containing medium the size increases by 9 nm. The increase of hydrodynamic size indicates that protein adsorption or agglomeration of nanoparticles has taken place in the presence of serum. Obviously, the mixture of serum-containing medium includes proteins of different sizes and has a broad size distribution, as indicated by a high µ2(90°) value. Although, we extracted the autocorrelation function of serum from the autocorrelation function of nanoparticles using the multicomponent analysis,48 we confirmed the DLS data by an additional method. To do so, we


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fractionated nanoparticles-protein mixture by AF-FFF. In contrast to DLS, the AF-FFF elugrams yield similar elution profiles after incubation in serum-free and serum-containing media (Figure 2). Therefore, we conclude that few aggregates are present in the protein-containing solution and the cause of the averaged size increase observed with DLS is due to the R6 dependency of the scattering intensity.50 Consequently, the size of the main fraction of the nanoparticles did not change after incubation with serum and no protein corona was formed.

27 nm NPs in RPMI NPs in RPMI with serum

1.0

Fluorescence

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0.8 0.6 0.4 0.2 0.0 200

600

1000

1400

t/s

Figure 2. AF-FFF elution profiles of nanoparticles in serum free (blue line) and serumcontaining medium (red line) yield similar elution profiles. The normalized intensity of fluorescence detector is plotted.

Heating the nanoparticles above their cloud point. After having investigated the system below the LCST we studied the thermoresponsive behavior of PiPOxylated nanoparticles in serum-free and serum-containing medium using multi-angle DLS, AF-FFF, and SDS-PAGE. Figure 3 shows the dependency of the hydrodynamic radius on temperature obtained by DLS in serum-free and serum-containing medium (see also Table S1 in Supporting Information). Below the phase separation temperature, the size of nanoparticles remains almost constant in both


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media. As soon as the phase separation temperature is reached, the polymer coating of the particles alters its properties from mostly hydrophilic to mostly hydrophobic. Due to this switching of polymer coating to hydrophobic, the nanoparticles tend to decrease their surface area in the polar solvent leading to increase of the hydrodynamic size due to the aggregation. 1200 1000

NPs in RPMI with serum NPs in RPMI

800

Rh / nm

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

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600 400 200 0 22 24 26 28 30 32 34 36 38

T / °C

Figure 3. Temperature-dependent DLS of the PiPOxylated nanoparticles in serum-free (blue Model Polynomial Equation Adj. R-Square

y =0,74156 A2 + (A1-A2)/(1 + exp((x-x

circles) and serum-containing media (red squares). The evaluation of autocorrelation function 0)/dx)) Value Rh

Intercept

Standard Error

17376,99854

was carried out with triexponential fit in serum-containing fit in Adj.medium R-Squareand biexponential 0,99645 Rh B1 -1314,66657 Rh

B2

Rh

A2

serum-free medium. Lines are guides for eyes; y-axis starts Rh at -50 nm for A1 better

407,76379 Value Standard Err 24,65233 7,12762 visibility of10,0253 the 10,179 1058,36727

data points. Due to adsorption of proteins the size of nanoparticles in protein-containing medium Rh x0 31,5565 Rh

dx

5789,49394

0,21817

increases stronger directly after the phase separation at 31 °C.

The phase transition temperature of the nanoparticles in both media is ~31 °C (slightly higher than in water, Table 1). The slight increase in phase transition temperature can be attributed to the presence of salts and other compounds to include sugars at isotonic concentration in the medium. Remarkably, in the presence of proteins the behavior of the nanoparticles is different after reaching the phase transition temperature, as the increase in radius is much greater. For


14

23,552

0,023

0,021

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example, the radius of nanoparticles in serum-free medium at 31.5 °C is 51.9 nm. In serum containing medium a radius of 510 nm or a factor of 10 times higher was detected. As the incubation times in both media were similar the faster increase of size in serum-containing medium can be related to interactions of nanoparticles with serum proteins. Possible effects, which cause this behavior, are the adsorption of serum proteins on the nanoparticle’s surface or additional destabilization of nanoparticles by protein at temperatures close to cloud point temperature due to some depletion effects. To study this behavior further we visualized the agglomerates in both media by cryogenic TEM (cryo-TEM) after incubation above the cloud point temperature (Figure 4). Serum proteins themselves are not visible in cryo-TEM due to the lack of contrast. However, it is notable that the agglomerates in serum-free and serum-containing media have different packing densities. The agglomerates in serum-free medium are densely packed with small distances between single particles. In contrast, agglomerates in serum-containing medium are packed loosely with larger distances between single particles. The relatively low packing density indicates that proteins are included in agglomerates. Apparently, it is protein adsorption rather than the depletion effects, which causes the pronounced agglomeration in the presence of serum proteins. Similar to aggregation of two polymers57 above their phase separation temperature, hydrophobic interactions between polymer coating and hydrophobic protein domains could induce increased protein adsorption and formation of agglomerates of several nanoparticles and serum proteins. In contrast, the agglomeration in serum-free medium is caused by energetically unfavorable interactions between the polymer chains and a bad solvent, which results in an entanglement of polymer shells5 and agglomerates with higher packing density.


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Figure 4. Cryo-TEM images of agglomerates isolated above phase separation temperature: (a) serum-free medium overview, (b) serum-free medium detail, (c) serum-containing medium overview and (d) serum-containing medium detail. Scale bar = 100 nm.

To further study this behavior, we isolated nanoparticle-protein-agglomerates, which are formed above the cloud point, and nanoparticles with adsorbed proteins below cloud point and


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compared their protein signatures by SDS-PAGE and protein contents by photometric assay. For isolation we used the procedure, which was previously described for isolation of silica nanoparticles with their “hard” corona.53 As an additional control we used amorphous, chargestabilized silica nanoparticles AmSil20, which are non-thermoresponsive and agglomerate in presence of proteins at room temperature.10, 25, 49, 53 To ensure comparability between results, the ratio of total particle surface area to serum was kept constant. The resulting SDS-PAGE (Figure 5) demonstrates that the protein signature of agglomerates (POS@PiPOx) after incubation at 37 °C is generally similar to the one obtained after the incubation at room temperature. In both cases bovine serum albumin appears to be the main fraction at 66 kDa.10, 11, 25 Compared to the control nanoparticle AmSil20, fewer proteins were eluted from PiPOxylated POS particles (POS@PiPOx) after either incubation at room temperature or at 37 °C and a different protein signature was obtained due to different surface properties. As expected from cryo-TEM mages, the signal intensity of proteins eluted from PiPOxylated nanoparticles at 37 °C is higher than after incubation at room temperature, indicating the increased protein adsorption. Photometric determination of protein content using protein assay shows that the amount of adsorbed proteins nearly increases by a factor of two (Table 3). Even if these results are associated with distinct uncertainty below phase separation due to very low protein adsorption on hydrophilic surface, the increase in protein content confirms our belief that phase separation and switching the surface from hydrophilic to hydrophobic triggers the adsorption of proteins.


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Figure 5. Separation of adsorbed proteins by SDS PAGE at 20 °C and at 37 °C.

Table 3. Quantification of adsorbed proteins at 20 ° and at 37 °C after isolation of nanoparticles with hard corona using colorimetric assay.a 20 °C

37 °C

sample

mProteine/1 NP / mg

mProteine/1 NP / mg

POS@PiPOx

5.6 × 10-14 ± 1.1 ×10-14

9.9 × 10-14 ± 1.0 ×10-15

AmSil20

1.1 × 10-13 ± 5.0 ×10-15

1.1 × 10-13 ± 4.1 ×10-15

a

Values are mean from three independent experiments recalculated in mass of proteins per one nanoparticle (NP). The error is calculated according to Gaussian error propagation.

Switching back the polymer coating. After cooling of the nanoparticles to room temperature the radius of the nanoparticles decreases to 97 nm in serum-free and to 161 nm in serumcontaining medium. The DLS measurements in the presence of proteins showed a broad size distribution of the sample, as indicated by the strong angular dependency of the apparent


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diffusion coefficient (Supporting Information, Figure S9). Thus, the exact determination of size by DLS is not possible. To determine the size of nanoparticles after cooling them to room temperature, we fractionated them by AF-FFF. Figure 6 compares the elution profiles of nanoparticles after incubation in serum-free and serum-containing medium at room temperature and at 37 °C. Fractionation of both samples, before and after heating to 37 °C, yielded only one fraction with an approximate radius of 30 nm. However, the elution profile of nanoparticles, which were incubated at 37 °C, show more pronounced tailing than of those which were incubated at room temperature. This tailing might in general have been caused by either nanoparticle-membrane interactions or agglomeration of the particles. The aggregates, which were already detected by DLS, are not visible in the AF-FFF: as already mentioned in the previous section, DLS is highly sensitive to molecular weight and size due to the R6 dependency of scattering intensity. Therefore it displays higher sensitivity for larger particles than the AF-FFF detectors, which are linearly scaled with the concentration of fluorescent dye. Nevertheless, the main fraction of nanoparticles has similar size before and after reaching the phase separation temperature. This observation demonstrates that the temperature-induced adsorption of proteins is reversible and can be switched on and off by changing the temperature.

b

a

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NPs in RPMI before heating after heating

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NPs in RPMI with serum before heating after heating

-3

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Figure 6. Fractionation of the PiPOxylated nanoparticles after incubation in serum-free (a) and serum-containing (b) medium before (blue line or dark-red line) and after (green line or light-red line) heating above phase separation temperature detected by fluorescence detector. The hydrodynamic sizes of the main fractions are similar after incubation at 37 °C and at room temperature. Tailing indicates the agglomeration of the nanoparticles. The reversibility of the protein adsorption can be further confirmed by the SDS-PAGE and protein assay. The SDS-PAGE of nanoparticles, which were incubated with serum at 37 °C and cooled to room temperature (Figure 7) demonstrates that the protein signature does not change compared to nanoparticles, which were incubated at room temperature. Again fewer proteins were eluted from PiPOxylated particles compared to the control nanoparticle AmSil20. The photometric quantification of proteins reveals that the amount of proteins in the corona of PiPOxylated nanoparticles after incubation at 37 °C and cooling (Table 4) does not differ significantly from the amounts obtained at room temperature (5.6 × 10-14 mg/NP, compare Table 3). Even though these results are again accompanied by some uncertainty, they demonstrate that the proteins desorb from the surface after cooling of nanoparticles to room temperature. This desorption of proteins correspondingly leads to the decrease in size, which is detected by AFFFF. Hence, we conclude that the protein adsorption on the PiPOxylated nanoparticles can be switched on and off by changing the temperature as an external stimulus.


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Figure 7. Separation of the proteins after incubation of nanoparticles at 37 °C followed by cooling to room temperature (POS@PiPOx37à20°C) compared to nanoparticles incubated with serum at 20 °C (POS@PIPOx 20°C).

Table 4. Amounts of proteins per nanoparticle in the hard corona determined by photometric assay after incubation of nanoparticles at 37 °C followed by cooling to room temperature.a sample

mProteine/1 NP / mg

POS@PiPOx

4.0 × 10-14 ± 1.1 × 10-14

AmSil20

1.1 × 10-13 ± 4.8 ×10-15

a

Values are mean from three independent experiments recalculated in mass of proteins per one nanoparticle. The error is calculated according to Gaussian error propagation.

CONCLUSION AND OUTLOOK


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The thermoresponsive properties of PiPOx allows for the manipulation of the protein corona on nanoparticles by variation of the temperature. In particular, DLS and cryo-TEM demonstrate differences in agglomeration behavior and structure of agglomerates in serum-free and serumcontaining medium upon heating nanoparticles above their phase separation temperature. Moreover, an increase in the amount of protein was detected above the LCST, confirming that phase separation of PiPOx-coating triggers the adsorption of serum proteins. Additionally, we showed that this temperature induced protein adsorption is a reversible process. Although DLS in serum showed the presence of agglomerates larger than 100 nm after cooling to the room temperature, AF-FFF demonstrated that the largest fraction of nanoparticles has the same size of approximately 30 nm before and after incubation with proteins at 37 °C. Furthermore, the amount of protein was similar before and after heating. Thus, the proteins adsorb on the surface of the nanoparticles at temperature above the cloud point and desorb after cooling below the cloud point. The polymer-coated particles used in this study have a cloud point below the body temperature, what could be a disadvantage for applications in vivo. However, the phase separation temperature of polymer depends on the polymer chain length and can be further adjusted by changing the composition.44, 58, 59 The thermoresponsive coating could be applied as a new targeting strategy of nanoparticles, where local heating could increase the agglomeration of nanoparticles with proteins or cells followed by accumulation of nanoparticle in the targeted organ.

ASSOCIATED CONTENT Supporting information


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Synthesis schemes of thiol-terminated PiPOx and PiPOxylated nanoparticles, additional experimental procedures, DLS, turbidimetry, AF-FFF data of nanoparticles in water and detailed DLS data in serum-free and serum-containing medium. This material is available free of charge via the Internet at http://pubs.acs.org. AUTOR INFORMATION Corresponding Author [email protected] Present Address †

Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud

University Medical Center, Geert Grooteplein 26/28, 6525 GA Nijmegen, The Netherlands. Author Contributions ‡

These authors contributed equally.

Funding Sources This work was supported by DFG SPP1313 Biological Responses to Nanoscale Particles, Peter und Traudl Engelhorn Foundation and Fonds der Chemischen Industrie. ACKNOWLEDGMENT The authors kindly acknowledge Andreas Thünemann (BAM) for the possibility to carry out the experiments at BAM Federal Institute for Materials Research and Testing, Manfred Schmidt (University of Mainz) for providing the HDRC software for the analysis of DLS data, Sascha


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Prentzel (University of Potsdam) for turbidimetry measurements, Christina Diehl for synthesis of PiPOx, Ulrike Braun and Dietmar Neubert (BAM) for TGA measurements, Christoph Bantz for general support and Michael Gradzielski (Technical University of Berlin) for fruitful discussions. REFERENCES (1)

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