Ionic Strength-Responsive Binding between Nanoparticles and

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Ionic strength responsive binding between nanoparticles and proteins Xiaohan Wang, Shi Zhang, Yisheng Xu, Xiaotao Zhao, and Xuhong Guo Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00944 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 24, 2018

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Ionic strength responsive binding between nanoparticles and proteins

Xiaohan Wanga, Shi Zhanga, Yisheng Xua,b*, Xiaotao Zhaoa, Xuhong Guoa,b*

a

State Key Laboratory of Chemical Engineering, East China University of Science and Technology, 200237 Shanghai, P.R. China

b

Engineering Research Center of Materials Chemical Engineering of Xinjiang Bingtuan, Shihezi University, 832000 Xinjiang, P.R. China

*

To whom correspondence should be addressed. E-mail: [email protected]

(Yisheng Xu), [email protected] (Xuhong Guo)

ACS Paragon Plus Environment

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ABSTRACT: Electrostatic interaction is a strong, dominant non-specific interaction which was extensively studied in protein-NP interactions1-3, while the role of hydrophobic interaction arising from the abundant hydrophobic residues of globule proteins on the protein-NP binding between proteins and charged nanoparticles has rarely been studied. In this work, a series of positive charged magnetic nanoparticles (MNPs) were prepared

via atom transfer radical polymerization (ATRP) and surface hydrophobicity differentiation was achieved through post-polymerization quaternization by different halohydrocarbon. The ionic strength and hydrophobicity responsive binding of these MNPs toward β-lactoglobulin (BLG) was studied by both qualitative and quantitative methods including turbidimetric titration, dynamic light scattering (DLS) and isothermal titration calorimetry (ITC). Judged from the critical binding pH and binding constant for MNP-BLG complexation, the dependence of binding affinity on surface hydrophobicity exhibited an interesting shift with increasing ionic strength, which means that the MNPs with higher surface hydrophobicity exhibits weaker binding affinity at lower ionic strength but stronger affinity at higher ionic strength. This interesting observation could be attributed to the difference in ionic strength responsiveness for hydrophobic and electrostatic interactions. In this way, the well-tuned binding pattern could be achieved with optimized binding affinity by controlling the surface hydrophobicity of MNPs and ionic strength, thus endowing this system with great potential to fabricate separation and delivery system with high selectivity and efficiency.


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KEYWORDS: Magnetic nanoparticles; Protein binding; Ionic strength; Surface hydrophobicity

INTRODUCTION With the rapidly advancing nanotechnology, a broad range of functional nanoparticles (NPs) have been developed for various applications, especially biomedical applications to achieve enhanced therapeutic efficacy4-7. Therefore, these nanoparticles would interact with a vast diversity of biomolecules, such as proteins8-9, enzymes10, polysacchraides11 and nucleic acids12 for different biomedical purposes. Generally, the NP-biomolecules binding processes with high selectivity and tunable affinity need to rely on specific interactions such as antigen-antibody and avidin-biotin recognition, which is costly and greatly restricted by high specificity. Therefore,

a

fast,

flexible

and

cost-effective

approach

for

controllable

NP-biomolecules binding is vital for developing novel biomaterials with optimized selectivity. Nanoparticle-protein assemblies, driven by the non-covalent interaction present versatile and flexible scaffolds for various biomedical applications such as drug delivery13 and biosensing14. Based on previous reports15, electrostatic interactions, hydrophobic interactions, hydrogen bonding and cation bridging are the most common non-covalent interactions involved in the process of NP-protein binding. Among those interactions, electrostatic interaction between charged moieties of


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nanoparticles and “charge patches” of proteins16-17 plays a major role in dictating binding affinity between proteins and nanoparticles while hydrophobic interaction between nonpolar surface group of nanoparticles and hydrophobic domains of proteins could modulate the affinity of protein to NP surface through a cooperative effect18-19. It is noteworthy that both of these interactions are highly sensitive to external conditions such as pH and ionic strength, which could in turn serve as external parameters to modulate overall binding conditions. Moreover, surface hydrophobicity of nanoparticles, an internal determinant for hydrophobic interaction, has already been proven to tailor various biological behaviors and therapeutic actions, including selective binding with biomolecules20, cellular uptake21, anti-bacterial behaviors22 and immune response23. However, the controllable binding performances achieved by surface hydrophobicity are limited to mono-layer protected nanoparticles without further control by external conditions such as ionic strength, which has already been proven to exhibit limited degree of selectivity toward proteins24-26. Therefore, combining surface hydrophobicity and ionic strength as internal and external parameters respectively could potentially achieve optimized binding affinity and selectivity for various nanosized biomaterials. In addition, magnetic nanoparticles (MNPs) have exhibited great potential for pharmaceutical and biomedical purposes due to its non-toxicity, targeting ability and economy, so it is of promising prospect to develop functional MNPs with well-tuned binding performance.


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Herein, we utilized a series of positively charged magnetic nanoparticles of different surface hydrophobicity to study their bindings toward β-lactoglobulin (BLG), a model whey protein under different ionic strengths via both qualitative and quantitative methods including turbidimetric titration, dynamic light scattering (DLS) and isothermal titration calorimetry (ITC). Judged from the critical binding pH and binding constant for MNP-BLG complexation, the dependence of binding affinity on surface hydrophobicity exhibits an interesting shift with increasing ionic strength, which indicates that the MNPs with higher surface hydrophobicity exhibit weaker binding affinity to BLG at lower ionic strength but stronger affinity at higher ionic strength. This interesting observation could be attributed to the difference in ionic strength

responsiveness

for

hydrophobic

and

electrostatic

interactions.

Hydrophobicity, as an internal parameter could be conjugated with ionic strength to modify the overall binding process between nanoparticles and proteins for optimized affinity and selectivity. Compared to monolayer ligand modified gold nanoparticles, the polyelectrolyte attached to MNP surfaces could potentially exhibit greater hydrophobicity differentiation and the effect of surface hydrophobicity on binding affinity under different ionic strength was investigated on MNP systems. To the best of our knowledge, few literatures have systematically reported MNP-based biomaterials whose binding affinity could be tailored by simultaneous effect of surface functionality and external stimuli.

Therefore, this research can provide

potential insights on the structure-activity relationship of nanoparticles and the design


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of novel nanomaterials with tunable selectivity and affinity for targeted separation, purification and sensing applications.

EXPERIMENTAL SECTION Materials Ferrous sulfate heptahydrate, ferric chloride hexahydrate, oleic acid (OA), ammonium hydroxide (25 wt%), toluene, copper(I) chloride (99%), copper (II) chloride dihydrate and sodium phosphate were purchased from Sinopharm Chemical Regent Co., Ltd. 2-Bromo-2-methylpropionyl bromide, γ-aminopropyltriethoxysilane (APS),

triethylamine

(TEA)

were

bought

from

2-(Dimethylamino)ethyl

J&K

Chemical.

methacrylate(DMAEMA),

1,1,4,7,10,10-hexamethyltriethylenetetramine(HMTETA),

bromoethane,

1-bromobutane, 1-bromohexane, 1-bromooctane were purchased from Adamas-beta. DMAEMA, toluene, copper (I) chloride was purified according to our previous reports25. Copper (II) chloride dihydrate was dried under vacuum to move the hydrate water. β-Lactoglobulin (BLG, Mw = 18.3 kD, pI = 5.1) was purchased from Sigma-Aldrich and used without further purification. Water used in all experiments was purified by reverse osmosis in Milli-Q system (Millipore). Silane initiator 2-bromo-2-methyl-N-(3-(trimethoxysilyl)

propyl)

propanamide

synthesized as presented in our previous publication25.


(BMTP)

was

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Synthesis of OA modified MNPs The synthetic procedure was based on experimental protocols proposed in previous report27-28. 2.35 g ferrous sulfate heptahydrate and 4.10 g ferric chloride hexahydrate were dissolved in 100 mL deionized water and 25 mL ammonium hydroxide (25% wt) was subsequently added quickly into the solution under vigorous stirring (350 rpm) and nitrogen atmosphere. 1 mL oleic acid was dropwise added into the mixture after the reaction solution was heated to 80 °C. After 1h reaction, the OA-modified NPs was further purified by extraction in toluene for three times after mixing with 8.0 g NaCl . Finally, the products were dehydrated over anhydrous sodium sulfate and stored in toluene with solid content determined as 15 mg/mL.

Synthesis of initiator modified MNPs BMTP (2.38 g), triethylamine (4.0476 g) dissolved in 20 mL toluene and 30 mL OA modified MNPs solution (15 mg/mL) were mixed together in 100 mL round flask with mechanical stirring. After 48h reaction under nitrogen atmosphere, the BMTP modified MNPs were precipitated in 50 mL petroleum ether and redispersed in toluene. After repeating the precipitation-redispersion procedure for 3 times, the collected MNPs then were redispersed into 15 mL DMF and dialyzed in DMF for 2 days. Finally, the products were stored in DMF with solid content determined as 5 mg/mL..

Synthesis of PDMAEMA modified MNPs (MNP0)


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Firstly, 5 mL BMTP modified MNPs solution (10 mg/mL), 5.6 mL DMAEMA (33 mmol) and 10 mL DMF was deoxygenated for 30 min and stored under nitrogen before use. 28 µL HMTETA (0.1mmol) dissolved into 5 mL DMF was deoxygenated for another 30 min and added into the solution. The reaction mixture was further purged with nitrogen for 1 h, after which copper(I) chloride (8.8 mg, 0.08 mmol) and copper(I) chloride (2.2 mg, 0.016 mmol) were added under nitrogen. The reaction was carried out at 90 °C under mechanical stirring overnight and stopped by opening the flask to air. The obtained PDMA modified MNPs was purified by centrifugation and dialysis against DMF and water for 1 day and 3 days respectively. The solid product (~200 mg) was obtained after vacuum desiccation.

Synthesis of MNP1-MNP4 by quarternization 25 mg of PDMAEMA modified MNPs was dissolved in 5 mL isopropanol and mixed with 5 mL halohydrocarbon including bromoethane, 1-bromobutane, 1-bromohexane, 1-bromooctane for MNP1-4 respectively. The reaction was carried out in room temperature for 24, 24, 36 and 48 h for MNP1-4 respectively to ensure sufficient extent of reaction. The particles were collected by centrifugation and then dialyzed against water for 3 days for further purification. The solid product (~25 mg) was obtained after vacuum desiccation.

Turbidimetric titrations Turbidimetric titrations could help identify the critical pH of soluble


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complexation (pHc) and phase separation (pHφ) for MNP-BLG bindings for different MNP types and ionic strengths. The turbidity, reported as 100-%T, was measured by a Brinkmann PC 950 colorimeter, equipped with a 420 nm filter and a 2 cm path length fiber optics probe while pH was detected by a Thermo pH meter (Ross Ultra Combination pH, 8172ROSS Sure-Flow, Orion). The solutions of MNP1-MNP4 and BLG was made in 8 mL phosphate buffer (PB) separately with ionic strength adjusted to 5, 20, 50 and 100 mM respectively by adding NaCl. Before mixing, the pH of separate solution was adjusted to 3 as initial titration state. After colorimeter equilibration with deionized water, 0.1 M or 1M NaOH was added to the mixed solution of MNP and BLG with 16 mL to ensure pH increments of ~0.1 with stirring (stirring rate: 750 rpm) while pH and transmittance of the mixed solution was monitored and recorded at the same time.

Dynamic Light Scattering The average size of MNP0-MNP4 and the size changes for BLG-MNP binding during turbidimetric titration were characterized by PSS Nicomp 380 (scattering angle: 90°). Samples are taken from MNP-BLG solutions at desired conditions (pH, ionic strength) during turbidimetric titration and average diameters were obtained after three duplicates..

Isothermal Titration Calorimetry The isothermal titration calorimetry (ITC) could obtain thermodynamic profiles


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and binding parameters on the BLG-MNP bindings and it was conducted on MicroCal ITC200, GE Healthcare. The titrants of BLG and MNPs were prepared with phosphate buffer at pH of 7.5 and different ionic strengths of 5 mM, 20 mM, 50 mM and 100 mM separately and they should be degassed for 10 mins and filtered by 0.22 µm Millipore filters prior to use. After loading and equilibrating the instrument at 25 °C, 40 µL BLG solutions (0.2 mM) in injection syringe were injected into 200 µL of 3.5×10-5 mM MNPs solution in cell. The titration process was performed with 20 successive injections with injection interval of 120s and stirring rate of 750 rpm. The thermodynamic data was calibrated by subtracting dilution heats obtained by BLG-blank solution titration before fitting and analysis. ITC data analysis was typically performed by Microcal Origin software, in which a one-site binding model was employed to fit the thermodynamic profiles and calculate binding parameters including binding stoichiometry (n), binding constant (Kb), enthalpy change (∆H) and entropy change (∆S).

Other characterizations DelPhi V98.0 (Molecular Simulations Inc.) was used to calculate the electrostatic potential around BLG by the solution of the Nonlinear Poisson Boltzmann equation and display electrostatic potential around the protein. The crystal structure of BLG was obtained from the RCSB Protein Data Bank (http://www.rcsb.org). The spherical-smeared-charged model proposed by Tanford29 was used to calculate amino acid charges, utilizing the titration curve of each protein as previously described30-31.


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Infrared spectra were recorded on a Nicolet 5700 FT-IR spectrophotometer with samples dried and deposited on KBr pellets. X-ray powder diffraction (XRD) data were acquired on a Bruker D8 XRD diffractometer with Cu Kα radiation. The TEM samples were prepared by depositing magnetic nanoparticles on a carbon film coated copper grid and the images were acquired on a JEOL JEM2100F microscope operating at 200 kV. X-ray photoelectron spectroscopy (XPS) studies were conducted using a Thermo ESCALAB 250 spectrometer equipped with a monochromatic Al Kα X-ray source operating at a power of 150 W. The energy resolution for the wide scans was 1.0 eV. This was reduced to 0.1 eV for high-resolution scans. Core-line spectra were peak-fitted using XPSpeak 41 software, and all binding energies were referenced relative to the main hydrocarbon C 1s signal calibrated at 284.8 eV.

RESULTS AND DISCUSSION Synthesis and Characterization of Magnetic Nanoparticles The synthesis route utilized to prepare the cationic magnetic nanoparticles with different hydrophobicity included the following steps: the formation of magnetic nanoparticles

via

co-precipitation

method,

attachment

of

initiator

2-bromo-2-methyl-N-(3-(trimethoxysilyl)propyl) propenamide (BMTP), subsequent surface initiated ATRP of 2-(dimethylamino)ethyl methacrylate (DMAEMA), and quaternization of the grafted PDMA with halohydrocarbon with different length. Therefore, the as-synthesized MNPs could exhibit high positive charge (~+40 mV)


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and different hydrophobicity (referred to as MNP1-4). The chemical structure of the cationic polymer layers of the obtained MNPs is shown in Figure 1.

Figure 1. Chemical structures of positively charged magnetic nanoparticles of different surface hydrophobicity and its well-tuned binding with BLG.

Figure 2 shows the characterization results of the as-synthesized MNPs of different steps. FTIR spectra of the MNPs prepared in each step was shown in Figure 2 (a), in which all peaks at 586 cm-1 characteristic of magnetite could be clearly observed. After attachment of the initiator, the Si-O-Si vibration bond at 1000 cm-1 as well as amide II bond of the silane initiator at 1526 cm-1 emerged, replacing the original coordinated carboxyl group of OA at 1406 cm-1. Moreover, after surface initiated polymerization, the presence of PDMA layers could be confirmed by a


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characteristic C=O stretch vibration bond of PDMA at 1735 cm-1 as well as the enhanced symmetrical and asymmetrical CH2 and CH3 groups at 2850 and 2915 cm-1. Meanwhile, as shown in Figure 2 (b), the average size of PDMA modified MNPs (MNP0) reached 60 nm with an increase of 50 nm after polymerization as detected by DLS. After quaternization, the zeta potential of MNPs increased from -1.3 mV to around +40 mV (Figure S1) with average size further increasing to ~90 nm (Figure S2) due to the enhanced surface charge and electrostatic repulsion caused therefrom. It is noteworthy that the water solubility of the MNPs has also enhanced significantly after surface initiated ATRP and quaternization due to the attachment of hydrophilic PDMA chains as shown in Figure 2 (c). From TEM images in Figure 2 (d), the obtained quaternized MNPs exhibit relatively good size distribution with average core size of around 10 nm and the crystal lattice could be clearly observed in the enlarged version of a single MNP, demonstrating the preservation of crystal structure of MNPs which could be further confirmed by the presence of six characteristic peaks at 30.2° (220), 35.6° (311), 43.3° (400), 53.6° (422), 57.1° (511), 62.8° (440) of XRD results in Figure 2 (e).


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Figure 2. (a) FTIR spectra of OA (black), BMTP (red) and PDMA (blue) modified MNPs. (b) Hydrodynamic size of OA (black), BMTP (red) and PDMA (blue) modfied MNPs (c) Pictures of aqueous solution of OA (1), BMTP (2), PDMA (3) and final product MNP1 (4) (d) TEM images of MNP1 (e) XRD profile of OA (black), BMTP(red), PDMA (blue) modified MNPs and final product MNP1 (olive)

To isolate the effect of hydrophobicity, the degree of surface quaternization was detected by XPS since different cationic (N+) and neutral (N0) nitrogen atoms within the polymer shell can be distinguished and quantified by peak fitting32. Figure 3 shows representative N1s core-line spectra recorded for the PDMA modified MNPs (MNP 0) and a series of quaternized MNPs (MNP1-MNP4) by different halohydrocarbons. From obtained XPS spectra, similar surface quaternization degree


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is achieved (86-92%) within different MNPs, which explains the similar zeta potential at around +40 mV for MNP1-MNP4 (Figure S1). Table 1 lists the hydrodynamic diameter (Dh), zeta potential and quaternization degree (QD) of MNP-MNP4 detected by DLS and XPS. The similar size, surface charge and uniform quaternization degree enable surface hydrophobicity to be the only variable parameter for the following MNP-BLG binding.

Figure 3. N 1s spectra obtained for MNP1-MNP4 detected by XPS.


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Table 1. Hydrodynamic diameter (% number), zeta potential and quaternization degree (QD) of the MNPs

MNP

Dh (nm)

Zeta potential(mV)

QD(%)

MNP1

86.2±3.20

43.2±3.1

92%

MNP2

91.4±4.57

41.0±4.6

94%

MNP3

90.3±5.73

38.7±3.8

88%

MNP4

87.4±3.61

37.8±2.2

86%

Well-tuned binding between MNP and BLG The synthesized series of MNPs were subsequently utilized to study the effect of surface hydrophobicity and ionic strength on binding behaviors between MNPs and BLG. BLG was chosen because it is a model whey protein featuring concentrative negative patches as shown by electrostatic potential contours in Figure S3 and is appropriate choice to study binding behaviors of cationic particles. Both qualitative and quantitative methods are applied to investigate the electrostatic and hydrophobic effect on binding affinity by changing ionic strength and MNP types. Firstly, turbidimetric titration and DLS were conducted to determine the different stages and affinity for MNP-BLG binding qualitatively. The turbidimetric titration results of the binding between BLG and MNP1-4 at ionic strength from 5 mM to 100 mM were shown in Figure 4. Judged from the titration results of blank BLG and MNP solutions at the same experimental conditions (Figure S4-S5), we could know that the turbidity of BLG solutions as well as average size of MNPs doesn’t exhibit significant increase so the self-aggregation has been effectively suppressed and the titration curves


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between MNP and BLG are convincing. From these turbidimetric titration curves, the critical pH for the onset of binding (pHc, a rough reflection of binding affinity) and phase separation (pHφ, the charge neutralization pH where phase separation starts33) as well as three binding stages, i.e., absence of interaction, soluble complex formation and phase separation of insoluble complex can be clearly identified in all turbidimetric titration curves (Figure S6). The determined pHc and pHφ values for different types of MNPs and ionic strength are summarized in Figure 5.

Figure 4. Turbidimetric titration curves for BLG-MNP bindings at I=5 mM (a), 20 mM (b), 50 mM (c) and 100 mM (d). [BLG]=0.05mg/mL, [MNP] = 0.02mg/mL. Inset image is the enlarged local version for identification of pHc


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According to the phase boundary results obtained in Figure 5, non-monotonic dependence on ionic strength of binding affinity, identified by pHc and phase boundary for separation, identified by pHφ for all MNPs could be clearly observed, conforming to previous studies on binding behaviors between proteins and polyelectrolyte26. The non-monotonic binding behavior was explained by the electrostatic repulsions between MNPs in addition to short-range attraction and long-range repulsion (SALR) effect proposed by our group25. Since MNPs are grafted with polyelectrolyte chains with limited degree of freedom, spatial restriction mutually exerted by both NPs and proteins has to be taken into account in addition to the interplay between different surface functionalities15. Initial increase of ionic strength would lead to the closer distance and overlapping of potential layer around MNPs. However, the shrinkage of potential layers under further increase of ionic strength is insufficient to overlap and enhance the binding. Therefore, the binding affinity tends to exhibit a maximum at certain ionic strength.

Figure 5. pHc (a) and pHφ (b) of MNP-BLG binding under different ionic strengths as obtained from turbidimetric titrations. The lines are for visual guidance only.


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Moreover, with increasing ionic strength, the dependence of binding affinity on surface hydrophobicity turns out to exhibit an interesting shift. When the phase boundary of BLG and MNP 1-4 was compared at fixed ionic strength, the more hydrophobic nanoparticles seemed to exhibit weaker MNP-BLG binding affinity at lower ionic strengths (I = 5 mM and 20 mM) and stronger binding affinity at higher ionic strengths (I = 50 mM and 100 mM). From the DLS results in Figure 6, the curves of turbidity and size on pH exhibit similar tendency and the size of MNP-BLG complexation seemed larger with decreasing MNP surface hydrophobicity at fixed pH and low ionic strength, coinciding with the turbidimetric titration results. Previous reports have shown that hydrophobic interactions seem to enhance the overall binding affinity via short-range attraction between non-polar polymer chains and hydrophobic domains within proteins18, 34. Therefore, it is quite interesting to observe this different phenomenon in our experiments.


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Figure 6. (a) Hydrodynamic diameter and turbidity of BLG-MNP4 complexes as a function of pH at I=5 mM (b) Size of BLG-MNP complexes as a function of pH and surface hydrophobicity at I=5 mM.

To further investigate the effect of surface hydrophobicity and ionic strength on MNP-BLG binding, isothermal titration calorimetry was subsequently used to quantify thermodynamic parameters of binding process. Figure 7 represents typical isothermal curves for MNP-BLG binding at I = 5 mM and pH = 7.5. The pH is chosen to ensure strong binding affinity for convincing isothermal curve and allow sufficient binding discrimination between different MNPs for further application in bio-separation. Judged from the heat change in cell after each injection represented by original ITC curves, the MNP-BLG bindings are typical endothermic processes with entropy origin. Depending on MNP types and ionic strength, the thermodynamic profiles for MNP-BLG binding at I = 20 mM, 50mM and 100mM were obtained in Figure S7-S9 and one site binding, in which MNPs with cationic polymer shell are assumed to have multiple identical independent binding sites, is utilized to obtain thermodynamic parameters including enthalpy and entropy change as well as binding constants (Kb) for different MNP-BLG binding from fitting curves of isothermal titration results.


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Figure 7. Isothermal titration calorimetry data for the binding of MNP1 (a) to MNP4 (d) on BLG in PB buffer at ionic strength of 5 mM and pH of 7.5. 200 µL aliquot of 3.5 × 10−5 mM MNP solution was titrated with injections of BLG solution (40 µL of 0.2 mM)

Table 2. Thermodynamic parameters for MNP-BLG binding at I=5 mM

MNP

N

Kb ×106 (M-1)

△H (cal/mol)

MNP1

680±10.2

5.37±0.91

(8.34±0.11) ×103

61.4

MNP2

664±8.29

4.16±0.92

(9.50±0.19) ×103

63.8

MNP3

564±8.16

3.52±0.77

(1.23±0.025) ×104

70.1

MNP4

496±9.31

3.45±0.92

(1.34±0.035) ×104

59.9


△S (cal/mol/K)

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Judged from summarized thermodynamic parameters from Table 2 and Table S1, all of the MNP-BLG bindings are found to be entropy-driven processes in which the favorable entropy change (∆S>0) triumphs over unfavorable enthalpy gain (∆H>0), regardless of surface hydrophobicity and ionic strength. Moreover, the more hydrophobic nanoparticle, like MNP4, seems to exhibit more evident enthalpy and entropy change than the hydrophilic ones. The bindings between BLG and gold nanoparticles in previous reports have shown similar tendency20, 24 and these enthalpy or entropy driven processes can be explained by the overall complexation process considered as two simultaneous process shown in eqn (1) and eqn (2)35.

NP + BLG ⇌ NP − BLG

(1)

xH O + yH O ⇌ x + y − zH O + zH O (2) NP · xH O + BLG · yH O ⇌ NP − BLG · x + y − zH O + zH O (3) Where, H2OB, H2ON, H2ON-B refers to water molecule associated with protein, nanoparticle and protein-nanoparticle complex, respectively. The first process, involving non-covalent complexation between MNPs and BLG, is exothermic with negative ∆H, ∆S while the second process of water reorganization and release is endothermic with positive ∆H and ∆S. Therefore, whether the binding between NP and protein is endothermic or exothermic depends on which of the process mentioned above predominates during the overall complexation process. Therefore, it is clear that the second process plays a predominant role for all MNP-BLG bindings, and the more hydrophobic particles release more amount of water, leading to more enthalpy and entropy change. Previous studies have also


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shown that the sorption of alkylamine to indium tin oxide exhibited more positive ∆S and ∆H with increasing alkyl chain length, coinciding with our observation36.

Figure 8. Plot of binding constant (Kb) vs. ionic strength for different type of MNPs. The lines are for visual guidance only.

Furthermore, the plot of binding constants against ionic strength and particle types in Figure 8 displays the similar tendency with turbidimetric titration, i.e., non-monotonic ionic strength dependence of binding affinity and the shift of binding affinity-surface hydrophobicity dependence. This interesting tendency is due to the difference in ionic strength responsiveness for electrostatic and hydrophobic interactions. At lower ionic strengths (I = 5 mM, I = 20 mM), the electrostatic interaction plays a dominant role in dictating binding affinity and the interactions between quaternary ammonium cation of the MNPs and the anionic domains on BLG may be sterically diminished by the increasing carbon chain within tertiary amine of the polymer shell within MNPs20. However, after increasing ionic strength, the


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distance between MNP gets closer due to screening of electrostatic repulsions and the potential layers around MNPs overlap despite shrinkage. During this process, the hydrophobic effect begins to play a different role since the closer distance enables the non-polar molecules of MNPs to interact with hydrophobic domains within the protein and further accumulation of salt ions around NPs and proteins could weaken the electrostatic interaction and presumably cause a “salting out” effect. Moreover, the overlapping effect of potential layers is enhanced by hydrophobic effect since the additional carbon chains within MNPs could form physical crosslinks as reported before32. Ionic strength has already been shown to strengthen the short-range interaction such as cation-π interaction by shortening the distance between proteins and polyelectrolyte during complexation37-38. In our case, enhancement of hydrophobic interaction is realized by similar screening effect, which in turn strengthens the binding effect. In a word, the addition of salt leads to different response of electrostatic and hydrophobic interaction between MNP and BLG, which could explain the ionic strength responsive bindings observed by turbidimetric titration and isothermal titration calorimetry..

CONCLUSIONS In summary, a series of novel cationic magnetic nanoparticles with well-defined surfaces and different surface hydrophobicity were successfully prepared and the combined effect of surface hydrophobicity and ionic strength on binding patterns


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between BLG and MNPs were studied by turbidimetric titration, DLS and ITC. Both of the qualitative and quantitative analysis demonstrates a non-monotonic binding and a shifted affinity-hydrophobicity dependence for MNP-BLG complexation, which could be attributed to the difference in ionic strength responsiveness for hydrophobic and electrostatic interaction. Notably, even though quantification of the relative contribution of electrostatic vs hydrophobic interaction exactly still remains a challenge, the

well-tuned affinity could be achieved via proper balance of electrostatics, controlled by ionic strength and hydrophobicity, endowing this novel system with great potential in selective binding for separation and biosensing applications.

ACKNOWLEDGMENT We gratefully thank the financial support from the NSFC Grants (21676089 and 51773061), Shanghai talent development fund (2017038), the Fundamental Research Funds for the Central Universities (222201717013) , the 111 Project of the Ministry of Education of China (No. B08021).

Supporting Information Zeta potential Values and hydrodynamic sizes of the final products of MNPs (MNP0-MNP4), potential surfaces of BLG, isothermal calorimetry results and thermodynamic parameters for BLG-MNP binding are included in the supporting information.


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For Table of Contents Only


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Figure 1. Chemical structures of positively charged magnetic nanoparticles of different surface hydrophobicity and its well-tuned binding with BLG 190x142mm (300 x 300 DPI)


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Figure 2. (a) FTIR spectra of OA (black), BMTP (red) and PDMA (blue) modified MNPs. (b) Hydrodynamic size of OA (black), BMTP (red) and PDMA (blue) modfied MNPs (c) Pictures of aqueous solution of OA (1), BMTP (2), PDMA (3) and final product MNP1 (4) (d) TEM images of MNP1 (e) XRD profile of OA (black), BMTP(red), PDMA (blue) modified MNPs and final product MNP1 (olive) 190x142mm (300 x 300 DPI)


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Figure 3. N 1s spectra obtained for MNP1-MNP4 detected by XPS 190x142mm (300 x 300 DPI)


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Figure 4. Turbidimetric titrations for BLG-MNP bindings at I=5 mM (a), 20 mM (b), 50 mM (c) and 100 mM (d). [BLG]=0.05mg/mL, [MNP]=0.02mg/mL. Inset image is the enlarged local version for identification of pHc 190x142mm (300 x 300 DPI)


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Figure 5. pHc (a) and pHφ (b) of MNP-BLG binding under different ionic strengths as obtained from turbidimetric titrations. The lines are for visual guidance only. 190x142mm (300 x 300 DPI)


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Figure 6. (a) Hydrodynamic diameter and turbidity of BLG-MNP4 complexes as a function of pH at I=5 mM (b) Size of BLG-MNP complexes as a function of pH and surface hydrophobicity at I=5 mM. 190x142mm (300 x 300 DPI)


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Figure 7. Isothermal titration calorimetry results for BLG-MNP binding from MNP1 (a) to MNP4 (d) in phosphate buffer at I=5 mM at pH 7.5. 200 µL aliquot of 3.5 × 10−5 mM MNP solution was titrated with injections of BLG solution (40 µL of 0.2 mM) 190x142mm (300 x 300 DPI)


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Figure 8. Plot of binding constant (Kb) vs. ionic strength for different type of MNPs. The lines are for visual guidance only. 190x142mm (300 x 300 DPI)


Ionic Strength-Responsive Binding between Nanoparticles and

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