Understanding the Robust Physisorption between Bovine Serum

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Understanding the Robust Physisorption between Bovine Serum Albumin and Amphiphilic Polymer Coated Nanoparticles Yushuang Liu, Ruibo Zhong, Ping Zhang, Yuxing Ma, Xiaoling Yun, Pei Gong, Jianmin Wei, Xinmin Zhao, and Feng Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b08386 • Publication Date (Web): 31 Dec 2015 Downloaded from http://pubs.acs.org on January 5, 2016

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ACS Applied Materials & Interfaces

Understanding the Robust Physisorption between Bovine Serum Albumin and Amphiphilic Polymer Coated Nanoparticles Yushuang Liu#, Ruibo Zhong#, Ping Zhang, Yuxing Ma, Xiaoling Yun, Pei Gong, Jianmin Wei, Xinmin Zhao and Feng Zhang* *Agricultural Nanocenter, School of Life Science, Inner Mongolia Agricultural University, 306 Zhaowuda Road, Hohhot 010018, China. # These authors contribute equally. Abstract: The robust physisorption between nanoparticles (NPs) and proteins has attracted increasing attention due to the significance for both conjugation techniques and protein’s corona formation at the bio-nano interface. In the present study, we firstly explored the possible binding sites of the bovine serum albumin (BSA) on amphiphilic polymer coated gold nanoparticles (AP-AuNPs). By using mass spectrometry, a 105-amino-acid peptide (12.2 kDa) is discovered as the possible “epitope” responsible for the robust physisorption between BSA and AP-AuNPs. Secondly, with the help of nanometal surface energy transfer (NSET) theory, we further found that the epitope peptide could insert at least 2.9 nm into the organic molecular layers of AP-AuNPs when the robust conjugates formed, which indicates how such a long epitope peptide can be accommodated by AP-AuNPs and resist protease’s digestion. These findings might shed

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light on a new strategy for studying interactions between proteins and NPs, and further guide the rational design of NPs for safe and effective biomedical applications. Keywords: Gold nanoparticle, BSA, Amphiphilic polymer, Binding site, Nanometal surface energy transfer

INTRODUCTION Nanoparticles (NPs) have been considered as multifunctional imaging, diagnostic and therapeutic agents, and their exterior surfaces dictate the behavior of these systems with the outside world 1. However, the effective application of NPs is hampered by limited understanding and control over their interactions with complex biological systems 2. When NPs are internalized by cells or upon contact with biological fluids, on which a number of proteins have been identified to form a “corona” structure

2-5

, which crucially affects the nano-bio interface

6-7

and

eventually changes the NPs’ fate 8. The long-lived protein corona, so-called hard corona, is not only the determinant to the NP’s biological identity as it is what the cell “see” and interact with 5, 9-10

, but also holds promise as building blocks for nanofabrication due to functional versatilities

of proteins. The thermodynamic and kinetic parameters of the protein corona formation on NPs have been extensively studied

11-17

and well-reviewed

18-19

for different protein-NP systems. To

better understand and further precisely control the nano-bio interface, the higher resolved binding “epitope” 20 and the formation mechanism of NPs’ protein corona (including protein-NP conjugates) are currently recognized as crucial issues that need addressing urgently. Up to now, different methods have been developed to study the structural features and the docking information between proteins and NPs. X-ray crystallography can obtain the full atomistic structures of isolated proteins and their complexes, however, this methodology usually


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requires large quantity of samples for analysis, and the preparation of diffraction-quality crystals is often the bottleneck

21

. A few attempts achieved the protein-NP interface by

hydrogen/deuterium (H/D) exchange nuclear magnetic resonance (NMR) 22, however, one of the major drawbacks of this method is the size of proteins that can be measured: proteins of up to 50 kDa are quite standard nowadays and systems of up to 900 kDa can be accessible with appropriate labelling and pulse sequences 23, which is technically demanding and require delicate treatment of proteins 24. Alternatively, mass spectrometry (MS) has been capable of determining the protein-to-protein binding sites coupled with chemical crosslinking method

25-27

, which has

also been applied to explore the binding epitope of protein to NPs 28-29. Because the protein corona is formed just by physical interactions, so it is also easy for the proteins to desorb from NPs under mild conditions. In the present study, we in vitro built up a model of NP-protein conjugate/corona system by selecting two typical elements: the well-studied bovine serum albumin (BSA) protein and the amphiphilic polymer coated AuNPs (AP-AuNPs), which we previously found they can form so robust conjugates/corona that the resulting BSAn-AP-AuNPs can even resist gel electrophoresis (GE) for several times

30

. With the aim to understand this phenomenon on a

nanometer/molecular level, a combination of proteinase digestion, polyacrylamide GE (PAGE), nanometal surface energy transfer (NSET)

31-32

and MS was employed to explore the binding

sites of BSA on AP-AuNPs, and a possible binding model was also discussed.

EXPERIMENTAL SECTION 1. Chemicals and Reagents


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All the chemical regents were purchased from Sigma-Aldrich. Trypsin (lyophilized powder, 23.3 kDa from porcine/bovine, > 250 National Formulary Unit (N.F.U)/mg) was purchased from AMRESCO Corporation. Proteinase K (28.9 kDa, lyophilized powder) was purchased from Roche Corporation. Pepsin (lyophilized powder，800-2500 µ/mg) and BSA (lyophilized powder, > 98%) were attained from Sigma-Aldrich Corporation. The dialysis membranes (Mw cut-off: 8-14 kD) were bought from Spectrum company. Ultrafilters (membrane Mw cut off: 100 kDa) were bought from Sartorius Stedim company. Deionized water (18.2 MΩ•cm) used for all experiments was obtained from a Milli-Q system (Millipore, Bedford, MA). All the proteins were dissolved in Milli-Q water to be stock solution with a concentration of 600 µM. EDC stock solution (768 mM) was prepared freshly before use by dissolving 10 mg EDC in 67.9 µL Milli-Q water. 30% sodium dodecyl sulfonate (SDS) stock solution was prepared by dissolving 30 mg SDS in 100 µL Milli-Q water.

2. Synthesis and Characterization of 5 nm-diameter Hydrophobic AuNPs

Synthesis method was followed previously published protocols

30, 33-34

. Briefly, 2.17 g

tetraoctylammonium bromide was dissolved in 80 mL toluene. 300 mg tetrachloroauric acid was dissolved in 25 mL Milli-Q water. The two solutions were mixed and gently shaken for about 5 min until the yellow color disappeared from the aqueous phase and the toluene phase became red colored. 334 mg sodium borohydride dissolved in 25 mL Milli-Q water and added drop-wisely to the Au solution in the round flask within 1 minute immediately. The solution was stirred at room temperature for about 1 hour. Discarded the aqueous phase. The left solution was stirred overnight. Next day after adding 10 mL of 1-dodecanethiol, the mixture was heated at 65 °C for3 hours in a water bath. Then the solution was filled with methanol in a volume ratio of 1:1 and centrifuged at


4

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2000 rpm for 5 min. The dodecanethiol and tetraoctylammonium containing supernatant was discarded and the precipitated NPs were collected and re-dissolved in chloroform. The diameter of hydrophobic gold NPs was estimated to ~ 5 nm by dynamic light scattering method (DLS, Malvern Instruments, ZetaSizer S90). The concentration of AuNPs was determined to 10 µM by spectrometer with an absorption coefficient of 1.67 × 107 M-1cm-1.

3. Synthesis of Amphiphilic Polymer (AP)

This was also followed a previous published protocols

30, 33-34

. Briefly, 2.7 g (15 mmol)

dodecylamine powder was dissolved in 100 mL THF and poured the solution into 3.084 g (20 mmol) poly(isobutylene-alt-maleic anhydride) in a round flask. This mixture was heated at 60 °C for 1 hour with stirring. Then the solution was completely dried by evaporation and the remaining powder of the amphiphilic polymer was re-dissolved in 40 mL chloroform to a final monomer (including an anhydride ring) concentration Cmonomer of 0.5 M.

4. Amphiphilic Polymer Coated AuNPs (AP-AuNPs)

To transfer the hydrophobic AuNPs into water-soluble, a universal polymer coating procedure was followed the previous published protocols 30, 33-34. In short, the AP solution mixed with the above hydrophobic AuNPs in a molar ratio of 200 polymer monomer units per nm2 of NP’s effective surface area (Aeff = 4•π•(deff/2)2). The solvent was slowly evaporated under reduced pressure until the sample was completely dried. The remaining solid film in the flask containing NPs was re-dissolved in SB12 buffer (sodium borate 50 mM, pH 12). After polymer coating the diameter of AuNPs was measured to ~12 nm by DLS (Malvern Instruments, Zeta Sizer S90). The concentration of AuNPs was adjusted to 6 µM.


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5. Gel electrophoresis (GE)

2% agarose gels was prepared by dissolving 1.4 g agarose gel into 70 mL 1 × TBE buffer (89 mM Tris-borate and 2 mM EDTA, pH 8.3; Sigma-Aldrich) and heating in a microwave oven. All the samples were mixed with about 25% gel loading buffer (40% glycerol in 1 × TBE). The agarose GE was conducted under 4 V/cm on 2% agarose gel in 1 × TBE buffer for 60 min. AP-AuNPs were purified by 2% agarose gel electrophoresis as reported by previously reported 30. 5% and 15% PAG were commercial available and purchased from Solarbio Company (Beijing, China). And the PAGE was firstly conducted 70 min under 55 V on 5% PAG (for concentrating purpose), and then run again for 100 min under 120 V on 15% PAG (for separation purpose) in 1× SDS-PAGE buffer (Tris-glycine, pH 8.3).

6. Preparation of the BSAn-AP-AuNP Corona/Conjugates In order to obtain the optimal BSA/AP-AuNP mixing ratio, 10 PCR tubes were numbered 1~10. Firstly, added 10 µL AP-AuNPs solution into each tube. Secondly, different volumes of proteins were added by an increasing ratio into each tube. The reaction volume was fixed 30 µL. The ratio of molecule number and volume (µL) of AP-AuNPs, protein and water was listed in Table S1. All of the incubation experiments were conducted under room temperature overnight, and the BSA was dissolved in Milli-Q water, and the AP-AuNPs were in SB9 buffer. Secondly, the AP-AuNPs and their conjugates with BSA were extracted from the cut bands (containing the desired samples) sealed in dialysis membranes by elution and finally concentrated again by ultrafiltration (Mw Cut off 100 kDa, Satorius Corporation) according to a previous published protocol 30, 33.

7. Optical Characterization


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Room-temperature optical absorption spectra were obtained with an ultraviolet visible (UV-vis) spectrophotometer (U-2900, Hitachi). Circular dichroism (CD) spectra were taken on an Applied Photophysics Chirascan spectropolarimeter (Applied Photophysics, Surrey, UK) under nitrogen flow. Samples with the BSA concentration adjusted to the same 0.15 mg/mL were measured by a 1-mm quartz cell. All CD spectra were taken in a wavelength range of 185 - 260 nm, and each spectrum was the accumulation of three scans. The steady fluorescence spectra of all samples were recorded in a fluorescence spectrometer (Fluorolog®-MAX 4, Horiba) equipped with a 1.0-cm quartz cell. For measuring fluorescence, both excitation and emission slits were set up to 5 nm. The fluorescence lifetime was collected on an Edinburgh Instrument (FS5) equipped with a cooled R928P photo-counting photomultiplier detector. The samples were excited at 280 nm using a picosecond pulsed light-emitting diode (EPLED). The repetition rate is 2 MHz. The fluorescence decays were analyzed using OriginPro 8 SR0 software (Originlab Corporation, v8.0724). The following equation was used to analyze the experimental time-resolved fluorescence decays, I(t):

= b + ∑ α exp −

(1)

Here, n is the number of discrete decay components, b is a baseline correction, and α and τ are the pre-exponential factors and excited-state fluorescence lifetimes associated with the ith component, respectively 35. For multi-exponential fluorescence decays, the average lifetime, 〈τ〉, was calculated from 〈τ〉 = ∑

(2)

= / ∑

(3)


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Where is the corresponding relative amplitude of the lifetime component . 8. Proteinase Selection and Digestion Assay

Normally 3 kinds of common proteinases: pepsin, proteinase K and trypsin were popular to digest proteins, however, their preferential digestion sites may cause deviations to estimating the binding sites. To rule out such deviations, the best protease fitting for this purpose should leave the shortest peptide on AP-AuNPs after digestion, which can be checked by PAGE and further selected to prepare the samples for MS analysis. The samples of AP-AuNPs mixed with excess BSA for a saturated adsorption state were directly incubated with different proteinases in the PBS (phosphate buffered saline, 100 mM, pH 7.5) to a total volume of 30 µL at 37 °C overnight. To conduct a digestion assay, a series different enzyme/AP-AuNP ratios as listed in Table S2 were used to get the optimized proteinase/AP-AuNP molar ratios, and the final optimized ones to get the binding peptide samples were used as 100/1, 500/1, and 1000/1 for proteinase K, trypsin, and pepsin, respectively. In essence, due to its primary site of synthesis and activity is in the stomach (pH 1.5 to 2), pepsin exhibits a maximal activity at pH 2.0 between 37 - 42 °C and is inactive at pH 6.5 and above, which was obsoleted after the first GE test (Figure S1A). Compared with pepsin, both proteinase K and trypsin have the optimal operating pH of ~ 8 and the optimal operating temperature of ~ 37 °C, under these conditions their optimized digestion molar ratios of protease/BSAn-AP-AuNPs were evaluated by GE. The peptide-AP-AuNP samples were purified and collected by a Sephacryl S-500 column equipped with a HPLC system (Agilent 1260) with a flow phase of SB9 buffer and the flow rate of 1 mL/min, and the spectra was monitored at 520 nm.

9. Peptide Releasing From The AP-AuNPs


8

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After HPLC purification, the peptide-AP-AuNP samples were firstly concentrated to about 1 µM by 100 kDa ultrafiltration, and then were mixed with 10% sodium dodecyl sulfate (SDS) by a volume ratio of 1:1 for 1 hour to remove all the possible adsorbates. The solutions were then directly run on PAGE or agarose GE without any other treatments. The PAGE bands were cut and subjected for MS analysis.

10. In Gel Digestion

According to the previous protocol

36

, the gel spot was washed twice by pure water and then

destained with coomassie brilliant blue (CBB) destaining solution (25 mM NH4HCO3, 50% acetonitrile (ACN)) at room temperature for 30 min. Later the destained solution was removed and the dehydration solution #1 (50% ACN) was added for 30 min, and then replaced by dehydration solution #2 (100% ACN) for 30 min, and then replaced by 10 µL working solution (0.02 µg/µL trypsin in cover solution (25 mM NH4HCO3, 10% ACN) for 30 min, then covered with the cover solution for overnight (about 16 hours) digestion at 37 oC. After the digestion, the supernatant was transferred into another tube, and the extracting solution (5% trifluoroacetic acid (TFA), 67% ACN) was added to the left gel at 37 oC for 30 min, then centrifuged for 5 min at 5000 ×g. Finally, the peptide extracts of the gel spot were combined and then completely lyophilized.

11. MS Analysis

The lyophilized powders were re-suspended with 5 µL 0.1% TFA followed by mixing in 1:1 volume ratio with a matrix consisting of a saturated solution of α-cyano-4-hydroxycinnamic acid (CHCA, >99%) in 50% ACN, 0.1% TFA. 1 µL mixture was spotted on a stainless steel sample


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target plate. Peptide MS and MS/MS were performed on an ABI 5800 MALDI-TOF/TOF Plus mass spectrometer (Applied Biosystems, Foster City, USA). Data were acquired in a positive MS reflector by using a CalMix5 standard to calibrate the instrument (ABI5800 Calibration Mixture), and PMF scanning range was 800 - 3500 Da, and then 10 largest peaks in strength were selected for the MS/MS conducting. Both the MS and MS/MS data were integrated and processed by using the GPS Explorer V3.6 software (Applied Biosystems, USA) with default parameters; Based on combined MS and MS/MS spectra, proteins were successfully identified based on 95% or higher confidence interval of their scores in the MASCOT V2.3 search engine (Matrix Science Ltd., London, U.K.) by using the following search parameters: NCBInr-Metazoa database; trypsin as the digestion enzyme; one missed cleavage site; fixed modifications of Carbamidomethyl (C); partial modifications of Acetyl (Protein N-term), Deamidated (NQ), Dioxidation (W), Oxidation (M); 100 ppm for precursor ion tolerance and 0.5 Dalton for fragment ion tolerance.

12. Calculation of The Distance Between BSA and AP-AuNPs

The distance (d) between the fluorophores (Trp) of BSA and the surface of AP-AuNPs can be calculated by equation 1.

=

# & !" % #$

*.--./0 1#

d* = +

23# 24 54

where

(

= 1−

$

/7

6

(4)

(5)

is the quantum efficiency of energy transfer/quenching, and the d* value is a

convenient value to calculate for a dye-metal system, yielding the distance at which a dye will


10

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display equal probabilities for energy transfer and spontaneous emission. For lifetime quenching, which compared the measured decay rate for a BSAn-AP-AuNP system (τ8 ) versus the observed decay rate for the identical BSA in the absence of AP-AuNP (τ* ). For the Persson model, the d0 value can be calculated by equation 2 37. Where c is the speed of light (3× 10* cm ∙ s ? ), ΦA is the quantum yield of the donor (BSA = 0.118). ωA and ωD are the angular frequencies for donor (BSA, 6.8 × 10. s ? ) and bulk gold (8.4 × 10. s ?), respectively, and k D is the Fermi wavevector for bulk gold (1.2 × 10I cm?) 31-32. 13. Influence of Ibuprofen and SDS on BSA-AP-AuNP Conjugates

Ibuprofen (>98%) was dissolved in 50% ethanol (HPLC grade) and SDS was dissolved in water to make the stock solutions with the same concentration of 50 mM. 10 µL of the stock solutions were half-to-half diluted to mix with 10 µL BSA stock solution with a fixed concentration of 600 µM to get the molar ratios of SDS or ibuprofen to BSA as shown in Table S3. After overnight incubation, 10 µL AP-AuNPs with a fixed concentration of 1.2 µM was added into the above mixtures to get a total volume of 30 µL and incubated at room temperature overnight. The final samples were run on 2% agarose gel for 60 min.

RESULTS AND DISCUSSION In practice, the robust BSA-AP-AuNP conjugate/corona (Figure 1A) can be obtained from the overnight incubation at a BSA/AP-AuNP molar ratio of 500/1 by following the protocol previously developed in our lab

30

. Different from the other corona system, the current BSA

number in a corona can be controlled by tuning the protein/NP incubation ratio, and the discrete BSAn-AP-AuNP (where “n” denotes the number of BSA in this complex) conjugates can be


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readily obtained by agarose GE (Figure 1A). After the proteinase digestions (Figure 1B and supporting information Figure S1), the epitope peptides were capable of well releasing from AP-AuNPs via treating the complex samples with SDS solution (Figure 1C). Because the AP-AuNPs are too large to enter the polyacrylamide gel (PAG), so the binding peptides can directly run PAGE without further purifications. Figure 1D clearly showed a single peptide with the apparent molecular weight (Mw) of ~ 13 kDa on the PAG, which indicated that proteinase K was more effective for the digestion than trypsin (supporting information Figure S3) under its optimized digestion condition (supporting information Figure S1B).

Figure 1. Gel electrophoresis images for optimizing the preparation and enzymatic digestion of BSA-AP-AuNP conjugate/corona and the Mw evaluation of the epitope peptides. A) The agarose gel image of the series test for preparing BSA-AP-AuNP conjugates/corona. From left to right, the molar ratios of BSA/AP-AuNP are 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500, respectively. B) The agarose gel image for the dependence of proteinase K digestion efficiency on the molar ratio of enzyme/BSAn-AP-AuNP. From left to right, the 1st lane was the AP-AuNP


12

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as a control, and from the 2nd to last lanes were the BSAn-AP-AuNP conjugates digested by proteinase K with a regularly increasing enzyme/BSAn-AP-AuNP molar ratios of 0, 0.01, 0.02, 0.05, 0.10, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100, respectively. C) The agarose gel image for checking whether the epitope peptide released from AP-AuNPs. From left to right, they were AP-AuNPs as control, the BSAn-AP-AuNP conjugates after proteinase K digestion, AP-AuNPs mixed with 10% SDS solution, the BSAn-AP-AuNP conjugates after proteinase K digestion and treated by 10% SDS solution, respectively. D) The PAGE image for examining the epitope peptides released from AP-AuNPs. From left to right, the 1st lane was the protein standard markers as indicated by the arrows with the known Mw, and the 2nd lane was the BSA before digestion as a control, and the 3rd and the 4th lanes were proteinase K and the epitope peptides released from AP-AuNPs after proteinase K digestion, respectively. To further identify the binding epitope at amino acid scale, the PAG bands containing the peptides released from AP-AuNPs were again digested by trypsin in gel and further analyzed by MS and MS/MS by following the published protocol

36

. Figure 2A showed the typical MS

analysis results of the binding peptides released from AP-AuNPs after proteinase K digestion. Three peptides with m/z values of 1408.6176, 2440.1565 and 2477.1677 were found with a relatively high abundance in the MS analysis diagram (Figure 2A). Their sequences were further analyzed by MS/MS (Figure 2B, 2C, and 2D) and confirmed to 3 peptides which perfectly matched 3 fragments in the amino acid sequence of BSA (Figure 3) as: DAFLGSFLYEYSR (323 - 335, P-13 for short), LGEYGFQNALIVR (397 – 409, P-13’ for short) and KVPQVSTPTLVEVSR (413 – 427, P-15 for short), respectively. In combination with the PAGE result of the released peptides from AP-AuNPs, the single band after proteinase K digestion indicated that the binding site should be a single consecutive peptide (Figure 2D). Given the


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binding peptide including all of these 3 fragments and the interval fragments, the whole single peptide with a total 105 amino acid residues (323 – 427, P-e for short) showed a Mw of 12.2 kDa, which almost perfectly matches with the estimated Mw (~ 13 kDa) of the single band from PAGE (Figure 1D). Hence, the P-e is the possible binding epitope that should be response for the robust BSAn-AP-AuNP conjugates/corona formation via physisorption.

Figure 2. The MS and MS/MS analysis of the epitope peptide from proteinase K digestion. A) The MS analysis of fragments after trypsin digestion of the epitope peptide released from AP-AuNPs. B, C, and D) were the further MS/MS analysis of the fragments from A) with m/z of 1408.6176, 2440.1565 and 2477.1677, respectively. It

is

interesting that

the P-15

fragment

is

exactly the same fragment

of

KVPQVSTPTLVEVSR (436 - 451, P-15h for short) as the binding epitope of human serum


14

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albumin (HSA) on polyacrylic acid (PAA)-coated Fe3O4 NPs 28. We attributed this to that BSA and HSA share a very high homology (76%) and similar conformations (supporting information Figure S2) in bulk solution, in addition to the similar dense carboxylic groups modified NP’s surfaces of these two NP-systems. Moreover, the physisorption might be the prerequisite for the covalent

conjugation

especially

when

using

a

zero-crosslinker

(e.g.

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC), so that the same physisorption also involved before the crosslinking and even promoted the EDC chemistry 30. Nevertheless, how to understand such a 105-amino-acid peptide (P-e) as the binding epitope of BSAn-AP-AuNP conjugates/corona? In other words, how could the NP’s surface accommodate such a long peptide and protect it from digesting by proteinases?

Figure 3. The epitope peptide in BSA’s amino acid sequence derived from proteinase K digestion. The red coloured sequences in A) were the peptide’s fragments confirmed by MS/MS, and the yellow coloured regions indicated in both A) and B) (3D structure from NCBI) were the whole epitope peptide deduced by the comprehensive consideration of both MS and PAGE


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results.

The surface inhibition to the enzymatic activity could be neglected, this is due to the following reasons: 1) The previous published results showed only a 15-peptide (P-15h) of HSA left on PAA coated Fe3O4 NPs after the trypsin digestion 28, however our trypsin digestion results and the corresponding MS analysis gave out even longer (than the proteinase K’s digestion results) binding peptide (supporting information Figure S3 – S6); 2) The adsorbed BSA didn’t significantly change its conformation by CD measurements (Figure 4A) 30. So the anti-digestion phenomenon of the long epitope peptide could be resulted from that this epitope peptide inserted so deep into the bush of AP layers (the thickness was determined to ~ 3.1 nm by comparing the hydrodynamic diameter of AuNPs before and after AP coating 30) on AuNPs that the proteinases cannot digest it due to the steric hindrance effect. This could be better supported by a control experiment to use proteinases to digest the conjugates of citrate protected AuNPs bound with BSA, however, it was a pity that the citrate protected AuNPs seriously precipitated after they mixed with BSA proteins (even as low as 10% of that mixing ratio for AP-AuNP binding experiments), which was probably due to the strong electrostatic interaction between citrate protected AuNPs and BSA disturbing the colloidal stability of the formed complexes. Other deliberate designs/modifications of the AP molecule could be also interesting projects worthy of investigating in the future, since the length of hydrophobic chain (dedecylamine) will also alter the thickness of AP layers on the AuNPs and even the colloidal stability of AP-AuNPs, which has been studied previously 38. Because of the hyper-complexity and the ultra-small size of the BSAn-AP-AuNP, this hypothesis is hard to be directly verified from the current high-resolution microscopies like scanning probe microscopy (SPM) 39-41 and electron microscopy (EM). Instead,


16

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it could be useful to better understand the robust physisorption if the protein “inserting” effect can be proved.

Figure 4. Optical characterization of protein-NP conjugates/corona. A) The comparison of BSA’s CD spectra upon mixing AP-AuNPs with different BSA/AP-AuNP molar ratios as indicated as AP-AuNP (control), BSA (control), 3, 4, 5. B) The fluorescence decay curves for BSAn-AP-AuNP, pure BSA and their corresponding multi-exponential fits. C) The steady fluorescence spectra of pure BSA and BSAn-AP-AuNPs. D) The normalized UV-vis absorption spectrum (AP-AuNP’s Abs) and the photoluminescence spectrum of BSA (BSA’s PL). The distance between BSA and AP-AuNP can be estimated by using an optical ruler, a dipole-surface type energy transfer from a molecular dipole to a nanometal surface that more


17


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Page 18 of 33

than doubles the traditional Fӧrster range (22 nm) and follows a d-4 distance dependence

31-32

,

which has been successfully applied to analyze the distance between fluorophores and mental NPs

42-45

. From both the time-resolved and steady fluorescence measurements (Figure 4B and

4C), the quenched fluorescence and reduced lifetime indicated it happened an energy transfer from BSA to AP-AuNPs, which is understandable from the spectra overlap

30, 34, 46-47

between

BSA’s PL and the AP-AuNP’s absorption (Figure 4D). So by using Persson model

37

, the

distance between BSA and AP-AuNP was estimated to 2.8 nm, which is less than 3.5 nm (the thickness of organic layers including both the AP (3.1 nm) and the dodecanethiol monolayer (0.4 nm) on AuNPs). By comprehensive consideration of both the intact BSA’s conformation and the nearest distance of 2.2 nm from one of Trp residues to the binding site of BSA (measured by Pymol software, Figure 5), it can be concluded that the BSA molecule inserted ~ 2.9 nm (= 3.5 nm - 2.8 nm + 2.2 nm) into the organic layers of AP-AuNPs.


18

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Figure 5. The distance from BSA’s tryptophan residues to the nearest binding site of AP-AuNPs. By using Pymol software, the indicated distances between two Trp residues and the binding peptide’s edge are 2.199 nm and 4.263 nm, respectively. Due to the P-e contains the subdomain IIIA, so-called drug-binding site 2 which acts as a putative binding site for drugs with acidic or electronegative features like ibuprofen and naproxen 28, 48, so the colocalization of the binding sites of ibuprofen and the AP-AuNPs on BSA will expect a competitive binding test between the AP-AuNPs and ibuprofen. The corresponding experimental results showed that the ibuprofen gradually inhibited the formation of BSAn-AP-AuNPs with an increasing ibuprofen/BSA mixing molar ratio (Figure 6A and 6B).


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This could be due to the following two points: On one hand, the ibuprofen binding changed the protein’s conformational structure, however the albumin only occurred very little conformational change upon binding of ibuprofen

49

; On the other hand, the ibuprofen’s binding changed the

charge state of P-e: the carboxylic groups of both ibuprofen and AP will produce repulsive force to each other. Since SDS can also bind to BSA and with more (nonspecific) binding sites, a further comparison test between SDS and ibuprofen was conducted and the results showed that SDS can inhibit the formation of BSAn-AP-AuNPs more significantly than ibuprofen molecules and started from a relatively lower SDS/AP-AuNP mixing molar ratio (Figure 6A and 6B), which indicated: 1) The hydrophobic interaction plays the main role in the conjugate/corona formation

30

(P-e

contains

47

hydrophobic

amino

acid

residues

(47.6%),

16

amine/imidazole-containing residues (15.2%) and 16 carboxylic group-containing residues (15.2%)), which can be effectively destroyed by the introduction of negative charges; 2) The binding sites of P-e to AP-AuNPs could be more than one as specific as the drug-binding site 2, which further supports the presence of such a long binding peptide in the current BSAn-AP-AuNP conjugate/corona system.

Figure 6. Influence of Ibuprofen and SDS on the formation of BSAn-AP-AuNP


20

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conjugates/corona by physisorption. A) The agarose gel images for the BSA corona formation on AP-AuNPs competed with ibuprofen (upper) or SDS (down), respectively. The LAP-AuNPs and LBSAn-AP-AuNPs indicated the mobility lengths of AP-AuNPs and BSAn-AP-AuNPs (the most retarded band), respectively. B) The plots of the relative mobility ratios of LBSAn-AP-AuNP /LAP-AuNPs against the concentration ratio of SDS or ibuprofen to BSA. In combination with all of the following findings in the strong physisorption mediated protein-NP system: 1) BSAn-AP-AuNPs are so robust that can resist several times of GE

30

; 2)

The epitope peptide is nearly 20% of the BSA amino acid sequence with an anti-proteinase digestion effect; 3) The BSA inserted about 2.9 nm into the organic layers of AP-AuNPs; we propose a possible binding model named “mortise-tenon joint”, which has been used for several thousand years by woodworkers around the world to assemble pieces of wood. If ignored the protein-NP’s dynamic binding procedure which might include the groove’s formation on the AP-AuNPs and the P-e inserting into it upon physically binding with BSA, Figure 7 depicted a possible final state of BSAn-AP-AuNP conjugate/corona by using an idealized mortise-tenon joint model, in which the 3.5 nm thickness

30

of the organic layers of AP-AuNPs ensure and

endow the deep mortise grooves formed upon contact with BSA protein, into which about 1/5 BSA structure as the tenon tongue inserted, and the whole structure was not only stabilized by the mechanical force but also enhanced by the hydrophobic interaction 30 like the adhesive used by carpenters. The binding site for such a mortise-tenon joint model should be transient in a way that the mortise was shaped just after the physisorption was established. In other words, the protein-shaped grooves were most likely absent on pure AP-AuNPs and they also would disappear after the proteins were completely washed off. Due to the steric hindrance produced with this mortise-tenon joint model, the proteinases are too big (~ 2.3 times Mw of P-e) to enter


21


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Page 22 of 33

into the mortise holes for a completely enzymatic digestion. Moreover, both the hydrophobic interaction built up on a large contact area and the mechanical force due to the inserting effect can highly enhance the stability of BSA corona on AP-AuNPs, making sense of the resistance to GE.

Figure 7. The schematic illustration of the mortise-tenon joint mediated formation of BSAn-AP-AuNP. The BSA crystal structure A) and AP-AuNP B) were modelled to the prism structure C) and the sphere structure D), respectively. In B), the inorganic gold core, the protecting surfactants (dodecanethiol) and the AP (amphiphilic polymer) were depicted as the purple sphere, the yellow sticks and the blue-green comb-like structures (blue backbone mainly consists of carboxylic groups, and the green sticks are dodecylamine molecules). The yellow regions of both BSA crystal A) and its modelled prism structures in C) and E) were the epitope peptide as the tenon tongue, and the mortise holes were also modelled by the grooves on the surface of AP-AuNP’s sphere structure in D) and E). This hypothesized model was just used for better understanding the robust protein-NP conjugate/corona without considering the dynamic binding procedure. All of the sketches are plausible idealized models for the conformation of the


22

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conjugates and are not drawn to scale.

CONCLUSIONS In conclusion, we discovered a 105-peptide as the possible binding epitope of the robust physisorption mediated BSAn-AP-AuNP conjugate/corona by using protease digestion coupled with both PAGE and MS analyses. And with the help of a NSET-based NP-protein distance estimation, we tried proposing a hypothesized mortise-tenon joint model to better understand the protein-NP binding mode with such a long peptide epitope. Different inter-/intra-molecular interaction mechanisms have been discovered via studying the interface between molecules and NPs up to now. Some of them had been debated and testified for decades, for example, the existence of catch bonds was debated for many years until the strong evidence of their existence in bacteria 50 and the definite proof of their existence in leukocytes 51. The depletion force which was originally discovered in colloidal NPs systems

52-54

, has not been applied/discovered in

bio-systems so far. The concept of mortise-tenon joint found in proteins

55

has been cranked up

to nanofabrication applications via DNA origami 56. In order to show more interesting results and applications of this specific protein-NP joint, other serum proteins and some engineered fusion proteins by P-e with other functional proteins like antibodies, streptavidin, fluorescent proteins (e.g. GFP) and so on are under investigations in our lab, and the results including affinity comparison of different segmented sequences derived from P-e and the corresponding theoretical simulations will be published elsewhere in the near future. Regarding to the inorganic NPs as protein mimics

57

, we envision that the mortise-tenon joint model might allow a deeper

understanding of the structural features responsible for protein corona formation and also open


23


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Page 24 of 33

up the possibility of better modeling and predicating the interaction of protein and other biomolecules to NPs. ASSOCIATED CONTENT Supporting Information. The corresponding reaction tables for physisorption, enzyme digestion assay, and ibuprofen/SDS competition assay; Proteinase digestion assay of pepsin and trypsin; the amino acid sequence homology and the structural conformation comparison between BSA and HSA; the PAGE image and MS/MS analysis results of epitope peptides. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Email: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. # These authors contribute equally. ACKNOWLEDGMENT We thank Mr. Jun Guo and Mr. Zhijun Bai for their help and valuable discussions. This work was supported by grants from the National Natural Science Foundation of China (No. 21171086, 81160213), and Inner Mongolia Grassland Talent (No. 108-108038), Inner Mongolia Autonomous Region Natural Science Foundation (No.2013MS1121), Inner Mongolia


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Department of Science and Technology (No. 211-202077) and the Inner Mongolia Agricultural University (No. 109-108040, 211-109003 and 211-206038). REFERENCES 1.

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Table of Contents


33

Understanding the Robust Physisorption between Bovine Serum

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