Measuring the Surface–Surface Interactions Induced by Serum

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Measuring the Surface−Surface Interactions Induced by Serum Proteins in a Physiological Environment Zhaohui Wang,† Chuanxin He,‡ Xiangjun Gong,*,§ Jianqi Wang,† and To Ngai*,† †

Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, The People’s Republic of China College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, China 518060 § School of Materials Science and Engineering, South China University of Technology, Guangzhou, China 510640 ‡

ABSTRACT: In this work, we applied total internal reflection microscopy (TIRM) to directly measure the interactions between three different kinds of macroscopic surfaces: namely bare polystyrene (PS) particle and bare silica surface (bare-PS/ bare-silica), PS particle and silica surfaces both coated with bovine serum albumin (BSA) (BSA-PS/BSA-silica), and PS particle and silica surfaces both modified with polyethylene glycol (PEG) (PEG-PS/PEG-silica) polymers, in phosphate buffer solution (PBS) and fetal bovine serum (FBS). Our results showed that in PBS, all the bare-PS, BSA-PS, and PEG-PS particles were irreversibly deposited onto the bare silica surface or surfaces coated either with BSA or PEG. However, in FBS, the interaction potentials between the particle and surface exhibited both free-diffusing particle and stuck particle profiles. Dynamic light scattering (DLS) and elliposmeter measurements indicated that there was a layer of serum proteins adsorbed on the PS particle and silica surface. TIRM measurement revealed that such adsorbed serum proteins can mediate the surface−surface interactions by providing additional stabilization under certain conditions, but also promoting bridging effect between the two surfaces. The measured potential profile of the stuck particle in FBS thus was much wider than in PBS. These quantitative measurements provide insights that serum proteins adsorbed onto surfaces can regulate surface−surface interactions, thus leading to unique moving behavior and stability of colloidal particles in the serum environment.

1. INTRODUCTION Colloidal particles ranging from submicron1−3 to micron4−6 size have drawn great interests in various biomedical applications, such as drug delivery, diagnostics, biosensors and imaging. Once injected into the bloodstream environment, these colloidal particles will be exposed to a wide range of lipids, sugars, electrolytes and especially serum proteins.7 Within seconds, nonspecific protein deposition may occur, leading to the change of the surface properties, which has been shown to play an important role in determination of subsequent physiological events.8−10 In this complex environment, aggregation of colloids is a common phenomenon, which however has been often understated or overlooked.11 Actually, this phenomenon will significantly alter their expected analytical sensitivity, cytotoxicity, body circulation time or uptake amount.12−14 It has been extensively discussed that proteins existing in the physiological environments can significantly affect the aggregation behavior of colloidal particles, however, how protein adsorption leads to aggregation is not fully understood.11 From the aspect of colloids, it is wellknown that their stability is influenced by interactions including van der Waals (vdW) forces, electrostatic repulsion, steric repulsion et al. Our purpose of this work is to study what is happening to the surfaces in the physiological environment and how these serum proteins influence the colloidal interactions, hoping to give some fundamental understanding of serum © XXXX American Chemical Society

protein induced surface−surface interactions in the highly complex media. In the past two decades, in order to suppress or eliminate protein adsorption, the colloidal surfaces have been modified with the so-called “antifouling” polymers.15 Numerous functional surfaces have been developed for effective decrease of protein adsorption, including peptides,16 polysaccharide,17,18 phospholipids,19 poly(ethylene glycol) (PEG),20,21 polyglycerol (PG),22,23 zwitterionic polymers,24,25 and polyvinylpyrrolidones.26 Among them, PEG or “PEGylation” is the most common used one and has been considered to be the gold standard to resist protein adsorption and thus prolong the circulation life of colloidal particles. For example, Konstantin Sokolov et al. reported that PEG can stabilize the gold nanoparticle suspension against unwanted aggregation in biological media.27 Schmidt et al. tested the performance of polymer-drug conjugates in blood serum by dynamic light scattering (DLS), and showed that PEG densities influence the colloidal stabilities.28 However, how peglyation influences the colloidal interactions which in turn affect the stability of particles in serum environment is still a problem which needs to be further explored. Received: September 16, 2016 Published: October 30, 2016 A

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2. EXPERIMENTAL SECTION

Nowadays, surface force apparatus (SFA) or atomic force microscope (AFM) were commonly employed to study the protein and polymer involved surface−surface interactions. Surfaces including mica,29 silicon wafer,30 silica31 and also polystyrene32 have all been used. However, many of the macroscopic surface coatings were only tested in aqueous buffer with physiological salt concentrations or in single protein media. Actually, these simple systems do not accurately model the practical challenges presented by blood plasma and serum. The underlying principles of how serum proteins influence polymer-coated surfaces interactions in these biological fluids have, however, not been studied in detail. Compared with SFA and AFM, total internal reflection microscopy (TIRM) has been developed for passively measuring kBT-level interactions involving in colloidal systems. It measures the scattering of light by a spherical particle when it is illuminated by an evanescent wave. As the probe particles are purely excited by thermal energy, this noninvasive optical technique allows for a force resolution down to a few femtonewtons and is therefore suited to the study of extremely weak interactions relevant to the stability of colloidal dispersions. Everett et al. have used TIRM to measure the nonspecific interactions of bovine serum albumin (BSA) and PEG-coated silica surfaces in a phosphate buffer solution (PBS).33 Furthermore, Eichmann et al. applied this technique to detect the specific protein, Concanavalin A (ConA) decorated particles interacting with dextran-functionalized surfaces in the presence of glucose.34 All these studies demonstrate that TIRM has potentials to quantify the colloidal interactions in some biological systems. However, the extension of this technique to monitor the protein induced surface interactions in real physiological media has been so far neglected. For colloidal systems with known size, the interactions between a colloid and surface from TIRM can be transferred to colloid−colloid interactions based on the Derjaguin approximation.35 As a result, we can correlate the interactions to the scale of drug carrier and other biomedical devices in the body, by treating drug carrier and medical devices as colloidal particles. To understand the influence of serum proteins on colloidal and the subsequent interactions, we directly measure the interactions between three different kinds of surfaces: namely bare polystyrene (PS) particle and bare silica surface (bare-PS/ bare-silica), PS particle and silica surfaces both coated with BSA (BSA-PS/BSA-silica), and PS particle and silica surfaces both modified with PEG (PEG-PS/PEG-silica), in a fetal bovine serum (FBS) solution by TIRM. Here, the bare samples refer to surfaces without modification, while the BSA preadsorbed samples refer to surfaces which have already been covered by a kind of serum proteins. First, we studied the interactions between two bare surfaces (bare-PS/bare-silica) to see how serum proteins influence the surface−surface interactions without any modification. Second, we studied BSA-PS/BSAsilica interactions, in order to reveal what will happen if the surfaces have already been coated with the abundant protein, BSA, in serum. Third, we studied PEG-PS/PEG-silica interactions to examine how the PEG layer influence the surface−surface interactions in serum environment. Finally, the measurement of the asymmetric surfaces of BSA-PS/PEG-silica and PEG-PS/BSA-silica in FBS was done to check whether PEG modified surface can be stable when interacting with a precontaminated BSA surface.

2.1. Materials. Dichlorodimethylsilane (DCDMS) (99%) was purchased from ACROS ORGANICS and used without further purification. 3-Aminopropyltriethoxysilane (APTES) (98%) was purchased from IL USA. Polyethylene glycol (Mw ∼ 2000 g/mol) (PEG-2K) was purchased from Fluka. Glycein (99.1%) was purchased from Riedel-de Haen. Glutaric anhydride, sodium acetate, N,N′dicyclohexyl carbodiimide (DCC) (99%), N-Hydroxysuccinimide (NHS) (98%), bovine serum albumin (BSA) and phosphate buffered saline (PBS) tablets were all purchased from Sigma-Aldrich and used without further purification. Pluronic F108 (average Mn ∼ 14 600 g/ mol, PEO−PPO-PEO, PEO 82.5 wt %) was purchased from BASF SE. Fetal bovine serum (FBS), polystyrene sulfate latex spheres (PS) with a diameter of 5.6 μm (4% w/v) and 80 nm (8% w/v) were purchased from Life Technologies Corporation. Silica glass slides (BK-7) were purchased from Fischer Scientific Co.. Silicon wafers (single side polished), ⟨111⟩, N-type, diameter × thickness 5 in. × 0.5 mm, were purchased from Zhejiang Lijing Silicon Materials Co., China. 2.2. Surface Modification. 2.2.1. Synthesis of NHS-Activated PEG-COOH. PEG-2K (10.0 g) was dissolved in toluene (50 mL) and the moisture was removed by azeotropic distillation. Afterward, Glutaric anhydride (1.71 g) and sodium acetate (0.1230 g) were added to the solution. The reaction was allowed to proceed for 12 h under reflux. The reaction solution containing glutaric acid terminated PEG was cooled to 40 °C. DCC (4.21 g) and NHS (3.41 g) were then added and reacted for 4 h. The mixture was filtered to remove the dicyclohexylurea (DCU) and the filtrate was concentrated. NHS activated PEG-COOH was recovered by precipitation in cold hexane and dried in vacuum. 2.2.2. BSA-Adsorbed Glass Slides (BSA-Silica) and PS Particles (BSA-PS). Glass microscope slides were immersed in Piranha solution (H2SO4 (98% v/v): H2O2 (30% v/v) = 3:1) overnight and cleaned by repeatedly rinsing with deionized water (D.I. water). The treated slides were further cleaned by an ultraviolet (UV)-ozone plasma cleaner (Harrick Sci. Corp.). The cleaned slides were then modified to be hydrophobic by immersing in 1% (v/v) dichlorodimethylsilane in toluene for 1 h. After rinsed thoroughly with toluene, chloroform and acetone, they were dried with nitrogen gas. For adsorption of BSA, the slides were immersed in a solution of 1.0% (w/w) BSA in PBS buffer at room temperature overnight. After that, they were rinsed with PBS buffer and then washed with D.I. water thoroughly to remove excess BSA and dried with nitrogen gas. PS sulfate latexes (50 μL 0.04 g/mL) in two different sizes (5.6 μm for TIRM and 80 nm for dynamic light scattering in diameter) was diluted to 1 mL by PBS solution containing BSA of 10 mg/mL (in excess of a saturated case) and shook overnight. After that, they were purified by centrifugation for three times and dispersed in 1 mL PBS buffer to get the BSA-PS particle dispersion. 2.2.3. PEG-Modified Glass Slides (PEG-Silica) and Pluronic F108 Adsorbed PS Particles (PEG-PS). The slides were cleaned using the method described above and then modified with amino groups by immersion in 0.2% (v/v) APTES in toluene for 1 h. After rinsed thoroughly with toluene and acetone, they were dried with nitrogen gas. For the grafting of PEG, the slides were immersed in a solution of 1.0% (w/w) NHS activated PEG-COOH in PBS buffer at room temperature for overnight. Then the surface were rinsed with PBS buffer and residual NHS groups were quenched by addition of 1.0% (w/w) glycine.36 PS sulfate latexes (50 μL 0.04 g/mL) in two different sizes (5.6 μm for TIRM and 80 nm for dynamic light scattering in diameter) were diluted to 1 mL by PBS solution containing Pluronic F108 of 10 mg/mL which is in excess of saturated cases and shook for overnight. It was then purified by centrifugation for three times and dispersed in 1 mL PBS buffer. 2.3. Characterization. 2.3.1. Thickness of the Silicon Surfaces after Coating with the Polymers or Proteins by Ellipsometer. Silicon wafers were first modified to become hydrophobic and then adsorbed with BSA under the same condition as the preparation of the BSAcoated silica slides. Meanwhile, some of the silicon wafers were also modified to contain amino end-functional groups and finally reacted to result in PEG−coated surfaces. Since a thin layer of silica was always B

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net potential energy, Φ(h), for each freely diffusing particle contains three parts: gravity, van der Waals attraction and steric repulsion. As a result, Φ(h) can be described as,

formed on silicon surfaces, the adsorption behavior of BSA or PEG on silicon surface could be considered to be close to the case on silica glass slide.37 The thickness of the different modified layers on the silicon surface in the dried state were measured respectively in the ambient environment by an ellipsometer (SC600 Discrete Wavelength, Shanghai Sanco Instru., China) at the angle of 70° and wavelength of 632.8 nm. The phase difference and polarized amplitude component were used to calculate the thickness. The thickness of the SiO2 layer on the silicon wafer was measured as ∼2 nm. Cauchy model was used for measurement of the hydrophobic functionalized(DCDMS) surfaces, the adsorbed film of BSA, the amino group(APTES) layer, the reacted PEG layer and the FBS protein layer (refractive index for the DCDMS layer, BSA layer, APTES layer, PEG layer and the FBS protein layer were assumed as 1.430, 1.450, 1.433, 1.467, and 1.450 respectively). The data from six points of the samples were averaged. 2.3.2. Size Variation of BSA-PS and PEG-PS Particles before and after Immersion in FBS by DLS. The hydrodynamic radius () of bare PS particles (diameter ∼80 nm given by the supplier) and particles after coated with BSA or Pluronic F108 layers (BSA-PS or PEG-PS) were determined by portable DLS (Portable Particle Sizer, Jianke Instru., China). The size change of these three kinds of particles before and after immersion in FBS for 2 h was also measured to check the adsorption behavior of serum proteins on surfaces. Before measurement, all samples were centrifuged and washed by PBS buffer to remove excess protein/polymer, then redispersed in PBS buffer and filtered through a 0.5 μm filter membrane. Correlation functions were collected at 30° for 900 s, and analyzed by cumulant fit to yield the final size. 2.3.3. Total Internal Reflection Microscope (TIRM). The principle of TIRM has been described in many literatures.38 TIRM is a technique for the measurement of interaction energy between a colloid and a flat surface. In a TIRM measurement, an evanescent wave, which decays exponentially with the distance from the interface, is generated at the glass−water surface by a total internal reflection. When a micron-sized probe sphere moves closely to the surface, the sphere will scatter the evanescent wave. The scattered intensity (I) of the colloidal sphere has been shown to be proportional to that of the evanescent wave as38−40

I(h) = Ih → 0exp(− βh)

Φ(h) = ΦG(h) + ΦvdW (h) + ΦS(h)

The gravity, ΦG(h), of the particle in a fluidic medium is determined by its buoyant weight and given by

ΦG(h) = Gh =

ΦvdW (h) = − Aa(h + δ)−P

⎛ h⎞ ΦS(h) = B exp⎜⎜ − ⎟⎟ ⎝ δp ⎠

(6)

(7)

where B can be calculated by the free energy per unit area of a brush, δp is proportional to the thickness of the two layers. In TIRM measurements, three different kinds of particles (PS, BSAPS and PEG-PS) were first dispersed in either PBS or FBS and then injected into a sample cell containing bare bottom surface or surfaces coated with BSA proteins or PEG polymers. After setting up, at least 30 particles were randomly selected and interaction profiles between each selected particle and the corresponding bottom surface were measured, respectively. All TIRM experiments were performed at 25 °C.

(1)

3. RESULTS AND DISCUSSION 3.1. Adsorption Behavior of Serum Protein on Bare Silica, BSA-Silica, and PEG-Silica Surfaces. We modified the silica slide surfaces by coating with BSA or PEG and then examined the protein adsorption behaviors onto these surfaces in FBS. For silica surfaces coated with BSA molecules (BSAsilica), the surface was first modified to become hydrophobic, indicating by the increase of contact angle from about 3° to 98°. The hydrophobic silica slide was then immersed in BSA solution, a thin layer of BSA (∼2.6 nm) was determined by ellipsometry measurement in the dried state, the contact angle decreased to 67°. For the coating of silica surfaces with PEG polymer (PEG-silica), the slide was also first modified with APTES in order to introduce amino groups and then PEG was grafted onto the surface by reacting NHS activated PEGCOOH with amine groups, resulting in a polymer film thickness of ∼0.7 nm in the dried state. The adsorption behavior of serum proteins onto the bare silicon, BSA-silica, and PEG-silica is shown in Figure 1. It can be seen that the adsorbed layer thickness of the serum protein on bare silica, BSA-silica and PEG-silica are 2.2, 1.7, and 1.5 nm respectively, indicating that proteins can be adsorbed and deposited onto all three surfaces in the physiological meidum. However, PEGsilica surface has a better protein reduction (or antifouling) performance than the bare silica and BSA-silica surfaces.

(2)

where the refractive index for glass slide is 1.512 (n1) and the refractive index for FBS is 1.344 (n2) (measured by Abbe Refractometer). Note that He−Ne laser (λ = 632.8 nm) is used and the angle of incidence θ is 70°. Thus, according to eq 2, for FBS, the penetration depth is 109 nm. Measuring I(h) over time provides a nonintrusive method to determine the distance h between the probe sphere and the bottom surface. After equilibrium, the probable distribution of finding the particle at a certain distance to the wall, p(h) can be calculated from histogram I(h), which is on the other hand related to the interaction Φ(h) between the sphere and the surface by the Boltzmann distribution

⎡ Φ(h) ⎤ p(h) = A exp⎢ − ⎥ ⎣ kBT ⎦

(5)

where p is a noninteger power, δ is a surface roughness correction factor and A is an effective Hamaker constant.34 If the surfaces are under constrained equilibrium, the amount of adsorbed polymer is kept constant and remains kinetically trapped between the two approaching surfaces. The steric repulsion, ΦS(h), thus appears due to the overlapping of the adsorbed polymer layers. The arising interaction can be described by the classic Alexander-de Gennes model for polymeric brushes.41 According to previous calculations,34,40,42−48 the steric repulsion can be described as

λ 4π (n1 sin θ)2 − n22

4 3 πa (ρp − ρf )gh 3

where g is the gravity acceleration, a is the radius of the particle, ρp and ρf are the densities for particle and fluid, respectively. On the other hand, it has been reported that39 van der Waals attraction, ΦvdW(h), can be approximated with a fitted inverse power law as

where h is the separation distance between the colloidal sphere and the glass surface, β−1 is the characteristic penetration length, and Ih→0 is the immobilized particle intensity which can be obtained by depositing the colloidal sphere on the bottom surface with a salty solution (i.e., 100 mM NaCl). β−1 is described as β −1 =

(4)

(3)

where A is a constant normalizing the integrated distribution to unity. 2.4. Net Interaction Potentials. In physiological media, the electrostatic repulsion between the probe particle and surface can be ignored since the ionic strength of the solution is high. In this way, the C

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The adsorption behavior of serum proteins onto the bare PS, BSA-PS and PEG-PS particles were then studied by further dispersing these three different particles in FBS. After washing by PBS to remove excess protein/polymer, they were redispersed in PBS buffer. Figure 2 shows that the size of bare PS, BSA-PS, and PEG-PS was further increased by 9.3, 13.2, and 1.5 nm respectively, revealing that three PS particle surfaces were adsorbed with a layer of serum protein. As expected, although the adsorption of serum proteins was not completely suppressed, the PEG-PS particle showed the best antifouling performance among three. It is interesting to note that the amount of adsorbed serum protein is highest in BSAPS particle, revealing that surface coated with BSA molecules shows a much less ability in suppressing protein adsorption in serum even compared to bare PS surface. This is markedly different with the BSA-silica surface as mentioned in Figure 1. It has been argued that protein adsorption onto surface is highly dependent on the surface properties, leading to different surface coverage, conformation, and roughness.51 One likely explanation is that the adsorbed BSA film at the PS surface and at the silica surface has different properties. The adsorbed film at the silica surface has been definitely denaturized after the dry process while the adsorbed BSA film at the PS surface was always kept in the physiological environment. However, the origin of such different is yet clear in serum. 3.3. Measuring Interactions between Bare-PS/Bare Silica, BSA-PS/BSA-Silica, and PEG-PS/PEG-Silica Surfaces in FBS by TIRM. Figure 3 shows potential energy profiles for the bare PS and bare silica, BSA-PS and BSA-silica, as well as PEG-PS and PEG-silica in FBS as measured by TIRM. The potentials were computed from a Boltzmann inversion of histograms of the excursion normal for each particle and averaged over surface position. It can be seen that the potentials in Figure 3 are typically separated into two groups as (i) freely diffusing particles with very few binding events (green, blue and cyan points in Figure 3), and (ii) particles that are essentially irreversibly deposited for the 30 min measurement time (red points in Figure 3). By monitoring trajectories of 30 particles, the result shows that in terms of proportions of each type, the potential of bare-PS/base-silica has around 45% of the profiles corresponding to tethered or stuck particles (red data points in Figure 3a). The potential thus appears quite narrow as a result of minimal particle excursion normal to the silica surface. In contrast, the potential corresponds to freely diffusing particles experiences much greater excursion above the surface (i.e, ∼ 400 nm) (green points in Figure 3a). Surprisingly, the potentials for the BSA-PS/BSA-silica show that more than 80% of the BSA-particles were irreversibly deposited to the BSA-coated surface (Figure 3b), likely there is a strong binding force between the BSA-coated surfaces. However, for the case of PEG-PS/PEG-silica (Figure 3c), the majority of profiles correspond to freely diffusing particles with only around 20% stuck particles. The stuck ratio of the particles under three conditions implies that PEG modified surface is the most stable one while the BSA modified surface has very poor ability in resisting the deposition under the serum condition. It was reported that BSA physically adsorbed onto hydrophobic surface can hardly cover the full area of surface, thus the surface often shows a heterogeneous property, consisting of individual adsorbed proteins that are unevenly distributed.52 The presence of such patches of the proteins may promote bridging effect which induced destabilization.11 However, for the bare surface, a more homogeneous serum protein coverage could be formed

Figure 1. Thickness change of bare or modified silica surfaces after coated with BSA and PEG (red color) as well as the adsorbed serum protein layer on these three surfaces after immersion in FBS measured in the dried state (black color).

3.2. Adsorption Behavior of Serum Protein on the Bare PS, BSA-PS, and PEG-PS Particles. The adsorption behavior of serum proteins onto the bare PS and PS particles coated with BSA or PEG was monitored by DLS to check the particle size change after they were dispersed in FBS. Figure 2

Figure 2. Blue color indicates the measured hydrodynamic radius () of bare PS. The red color shows the increased thickness after PS particles coating with BSA and PEG in PBS. The black color shows the adsorbed serum protein layers on bare PS, BSA-PS, and PEG-PS particles in FBS.

shows that the measured hydrodynamic radius, < Rh>, for bare PS particles is 40.9 nm. After immersion in BSA, < Rh> increased to 54.0 nm, indicating that PS surfaces were decorated with multilayers of BSA molecules since the size of each BSA molecule is ∼7.0 nm.49 PS particles coated with PEG (PEG-PS) were obtained by mixing the particles with Pluronic F108 triblock copolymer in PBS buffer.50 DLS measurement shows that < Rh> increased from 40.9 to 44.0 nm (Figure 2), revealing that the triblock polymer was successfully attached to the PS surfaces, presumably with the hydrophobic PPO adsorbed at the PS surface while the hydrophilic PEO extends into the aqueous solution to provide a steric repulsion of the dispersed particles. D

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For the PEG-PS/PEG-silica surfaces, repulsive force is solely found with no obvious specific attraction. This result further confirms that PEG polymer modified surface shows the most stable behavior in the serum. Assuming that the bottom surface is obtained from the averaged intensity of stuck particles, Ih→0, in FBS, the absolute separation distance (hm) between the PEG-PS/PEG-silica was determined to be around 69.0 nm. However, for the bare-PS/bare-silica and BSA-PS/BSA-silica two systems, hm is less than 20.0 nm. Therefore, the absence of van der Waals attraction as illustrated in Figure 3c might be due to a long separation distance between the two PEG-coated surfaces. After removing the gravity, the remaining contributions from steric and van der Waals interactions in the potential profiles were well fitted by eq 6 and 7 as indicated by the black lines in Figure 3. The fitting results were also summarized in Table 1. Table 1. Fitted Parameters of Potential Profiles in Figure 3 Using Equations 6 and 7a bare-PS/bare-silica BSA-PS/BSA-silica PEG-PS/PEG-silica

δp/nm

A

p

9.6 12.8 13.1

0.357 0.410 N.A.

2.15 2.05 N.A.

a

Here, p is the power index of van der Waals interaction, A is an effective Hamaker constant, and δp is the steric parameter.

Note that the fitted van der Waals potential is in good agreement with previous measurement reported by Wu et al.,33 and here we have assumed that the roughness of the PS particle and silica slide surface is 20 nm in eq 6.32 The fitted steric parameter, δp, in eq 7 is related to the thickness of modified layer onto both PS and silica surfaces, which has been reported to be roughly proportional to L for two asymmetric surfaces.43 2π Therefore, for bare-PS/bare-silica, the adsorbed serum protein offers the steric repulsion between each other while for BSAPS/BSA-silica and PEG-PS/PEG-silica surfaces, steric repulsion is contributed by a combination of the modified BSA/PEG layer and the adsorbed serum protein layer. In other words, the adsorbed protein layer can significant mediate the interaction between two surfaces. In BSA-PS/BSA-silica case, BSA layer actually further promotes protein adsorption in the serum medium. Furthermore, we investigated the potential profiles of barePS/bare-silica, BSA-PS/BSA-silica, and PEG-PS/PEG-silica in PBS solution. Surprisingly, no freely diffusing particles were found in PBS, and all particles were irreversibly deposited onto the silica surfaces, even the surfaces coated with BSA or PEG. We argue that the different phenomenon in PBS and FBS might be related to the gravity of the particles. In FBS, the effective gravity of PS particle is 27 fN, which is around half of the value in PBS buffer (45 fN). Figure 4 shows the stuck particle potentials in PBS. They are very narrow, indicating the minimal particle excursions normal to the surface. However, a careful examination noted that the potentials of the stuck particles in FBS are a bit wider than in PBS, particularly at a potential energy of 3kBT. The widths of bare-PS/bare-silica, BSA-PS/BSA-silica, and PEG-PS/PEG-silica are 39.4, 37.2, and 48.2 nm, respectively, compared to 3.5, 8.5, and 8.5 nm in PBS. We conjecture that the reason might be the serum protein layers from FBS adsorbed onto the two surfaces, resulting in some residual soft repulsion. The equilibrium heights of the

Figure 3. Interaction profiles between two surfaces: (a) bare-PS/baresilica, (b) BSA-PS/BSA-silica, (c) PEG-PS/PEG-silica in FBS as measured by TIRM. The narrow potential profile with the red color points corresponds to the stuck particle profile, while the green, blue and cyan color points represent the profiles of freely diffusing particles. Note that gravity has been removed from the freely diffusing particle profiles and the marked number refers to the percentage of the particles that are freely diffusing above the surface. The corresponding inserted schematic graphs present the deposition behavior of the bare PS or coated PS particle onto the bottom surface. The equilibrium position of stuck particles in FBS was defined as the zero position for all these potential energy profiles so that the absolute height of freely diffusing particles above the surface can be shifted accordingly.

so as having a more stable behavior compared to the BSA pretreated system. We further theoretically fit the potential energy profiles of the freely diffusing particle in Figure 3 in order to understand the interaction forces that control the particle deposition to the surface. The fits to the green, blue and cyan profiles in Figure 3 correspond to unbound particles that experience sufficient Brownian excursion to escape the surface and the linear gravitational potential without any obvious binding. As a result, these profiles could be described with steric, van der Waals, and gravity potentials based on eq 4. Since the size and density of the used PS particle are known, the gravity potential was calculated based on eq 5 and subtracted from all potential profiles in Figure 3. It can be seen in Figure 3 (a) and (b) that after the subtraction, the bare-PS/bare-silica and BSA-PS/BSAsilica surfaces show an ∼1.0 kBT of attraction between the two surfaces, likely related to the van der Waals attraction. However, E

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the density of the brush, M is the molecular weight of the polymer chains, and d is the thickness of the brush in the dry state. Under our conditions, the graft density is calculated to be ∼0.4 chains/nm2. The graft density of Pluronic F108 adsorbed on the PS surface has been reported to be ∼0.27 chains/nm2.53 However, each Pluronic F108 chain has 129 PEG units for 1 grafting point, whereas for PEG-2K polymer loops, there are only ∼23 PEG units. The surface grafted with Pluronic F108 polymer chains has more antifouling functional groups, and that may be the reason that PEG-PS above the BSA-silica surfaces is more stable. Our preliminarily results conclude that the conformation of the adsorbed polymers can affect the stability of the material in the serum. However, it is still being researched with respect to systematically studying how the graft density and the graft length influence the surface−surface interactions.

Figure 4. Interaction profiles for stuck particles in PBS and FBS media, in which ○ refers to the stuck particle profile in PBS and □ refers to the stuck particle profile in FBS. Black refers to bare-PS/baresilica, red refers to BSA-PS/BSA-silica, and green refers to PEG-PS/ PEG-silica. The well width at 3kBT is marked with the dashed line.

4. CONCLUSIONS The interaction forces among three different kinds of macroscopic surfaces, namely, bare-PS/bare-silica, BSA-PS/ BSA-silica, and PEG-PS/PEG-silica in an FBS solution, were studied by TIRM to mimic the interaction of functional surfaces in a blood serum. DLS and ellipsometer measurements indicated that all three surfaces were adsorbed with a layer of serum protein when they were dispersed in the blood serum. Potential energy profiles measured by TIRM show that there was a weak attractive force between two surfaces in the barePS/bare-silica and BSA-PS/BSA-silica systems, whereas for the PEG-PS/PEG-silica surface, only a pure repulsive force was found. The result confirmed that the adsorbed serum proteins can mediate the surface−surface interactions. It not only provides additional stabilization under certain conditions but also promotes a bridging effect between the two surfaces. Moreover, our result showed for PEG-coated surfaces that the functionalized PS surface with Pluronic F108 was more effective at reducing protein adsorption than functionalizing the silica surface with PEG-2K. Also, the conformation of the adsorbed polymers could affect the stability of the material in the serum. The quantitative measurement by TIRM indicates that serum proteins adsorbed onto surfaces can regulate surface−surface interactions, thus leading to unique moving behavior and stability of colloidal particles under the serum conditions. The surface modification can vary the serum protein adsorption behavior, thus changing the moving behavior and stability of the surface-modified colloidal particles. This investigation into serum-protein-induced surface interactions thus will provide an empirical guideline to design functional surfaces for drug delivery, biomedical implants, and other clinical applications.

stuck particles, bare-PS, BSA-PS, and PEG-PS were determined to be 114.0, 109.0, and 62.0 nm, respectively, further confirming that all surfaces have been covered by a layer of proteins but PEG-PS particles adsorbed the least amount. The asymmetric cases, i.e., interactions of BSA-PS/PEG-silica and PEG-PS/BSA-silica in FBS, were also measured using TIRM. Figure 5 shows that for the BSA-PS/PEG-silica system,

Figure 5. Interaction profiles between asymmetric surfaces of PEGPS/BSA-silica and BSA-PS/PEG-silica in an FBS medium as measured by TIRM. The narrow potential profiles with the red color points and blue color points correspond to the stuck particle profiles of BSA-PS/ PEG-silica and PEG-PS/BSA-silica in FBS, respectively. The green color points represent the profiles of freely diffusing particles of PEGPS/BSA-silica. Note that gravity has been removed from the freely diffusing particle profiles and the marked number refers to the percentage of the particles that are freely diffusing above the surface.

all of the particles were unexpectedly deposited on a PEGcoated surface (the red points in Figure 5). However, for the PEG-PS/BSA-silica system, around 56% of the particles are freely diffusing. This ratio is lower than for the PEG-PS/PEGsilica system but is still much higher than for the BSA-PS/BSAsilica system. The net potential profile for the freely diffusing PEG-PS particles above the BSA-silica surface also exhibits only steric repulsion, which is similar to the PEG-PS/PEG-silica system. In addition, the estimated separation distance between two surfaces was determined to be ∼62.0 nm, which is comparable to the value of 69.0 nm in the PEG-PS/PEG-silica system. From these two experiments, we learned that functionalizing the PS surface with Pluronic F108 is more effective than functionalizing the silica surface in stabilizing the colloids. The graft density of PEG on the silica surfaces can be N ρd calculated by σ = AM , where NA is Avogadro’s number, ρ is

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The financial support of this work by the Hong Kong Special Administration Region (HKSAR) General Research Fund (CUHK14303715, 2130429), Collaborative Research Fund (CRF 2014/15-C4028-14GF), and the National Natural F

DOI: 10.1021/acs.langmuir.6b03420 Langmuir XXXX, XXX, XXX−XXX

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Langmuir

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Science Foundation of China (21404044) is gratefully acknowledged.

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