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Surface Adsorption and Molecular Modeling of Biofunctional Gold Nanoparticles for Systemic Circulation and Biological Sustainability Manoj Kumar Goshisht,§,∥ Lovika Moudgil,⊥ Poonam Khullar,*,§ Gurinder Singh,⊥ Aman Kaura,⊥ Harsh Kumar,∥ Gurinder Kaur,‡ and Mandeep Singh Bakshi*,† †

Department of Chemistry and Biochemistry, Wilfrid Laurier University, Science Building, 75 University Ave. W., Waterloo, Ontario N2L 3C5, Canada ‡ Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, Newfoundland A2 V 2K7, Canada § Department of Chemistry, B.B.K. D.A.V. College for Women, Amritsar 143005, Punjab, India ∥ Department of Chemistry, Dr. B. R. Ambedkar National Institute of Technology, Jalandhar-144011, Punjab, India ⊥ Faculty of Applied Sciences, UIET Panjab University Regional Centre, Hoshiarpur 146001, Punjab, India S Supporting Information *

ABSTRACT: Fundamental aspects of protein complex coated gold nanoparticles (Au NPs) were presented for their possible use in systemic circulation in terms of pharmaceutical formulations. For this purpose, protein complexes of bovine serum albumin (BSA), lysozyme (Lys), and zein with an industrial important bioactive polymer diethylaminoethyl dextran (DEAE) were studied in the presence of Au NPs. Surface adsorption of such complexes magnified DEAE−protein interactions which were easily monitored spectroscopically under the effect of temperature and reaction time. In vitro synthesis of Au NPs allowed a simultaneous adsorption of the DEAE−protein complex on the NP surface to achieve colloidal stability, which was dramatically influenced by the nature of the DEAE−protein complex. The DEAE−BSA complex demonstrated strong favorable mainly electrostatic interactions followed by DEAE−Lys, while DEAE−zein interactions were predominantly influenced by the hydrophobic nature of zein. At the molecular level, the interactions were evaluated from the molecular dynamics (MD) studies which focused on the protein surface charge, dihedral angle variations, and protein unfolding upon dextran−protein complexation as well as its surface adsorption. MD studies further helped us to identify specific amino acid residues which promoted such interactions among the DEAE and protein as well as the surface adsorption of DEAE−protein complexes and allowed the synthesis of suitable biofunctional Au NPs with interesting bioapplicability with blood cells. Biofunctional NPs coated with DEAE−BSA and DEAE−Lys complexes over the entire mixing range proved to be the best suited vehicles for biomedical applications in systemic circulation. KEYWORDS: Biofunctional nanoparticles, Dextran−protein complexes, Hemolysis, Molecular dynamics studies

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INTRODUCTION In order to extract the full potential of biofunctional nanoparticles (NPs) in systemic circulation, understanding of their fundamental aspects at the molecular level is essential. Protein coated biofunctional NPs are promising vehicles for drug release in systemic circulation, but a fundamental understanding of protein adsorption and its ability to interact with the blood cells is an important aspect to be studied for their effective use. Herein, we try to explore the potential of protein−dextran complex coated NPs as drug carriers in systemic circulation and present their fundamental properties essential to mark them suitable biological nanomaterials for their applications in pharmaceutical formulations. Protein−dextran complexes play a significant role in tissue morphogenesis, cell proliferation, signal transduction, infection, and therapeutics.1,2 Since such complexes are the result of weak intermolecular interactions, therefore, it is always difficult © XXXX American Chemical Society

to detect them through complicated spectroscopic measurements. Carbohydrates usually interact through weak amphiphilic or van der Waal’s interactions with proteins. Such interactions can be magnified if an ionic polysaccharide such as diethylaminoethyl dextran chloride (DEAE) is used, which possesses greater ability to interact with water-soluble proteins.3,4 DEAE is one of the most versatile polysaccharides with promising applications.5,6 It has a high affinity for negatively charged DNA, while its cellulose counterpart is used in ion exchange chromatography, protein and nucleic acid purification, as well as separation.7,8 It is also used as an adjuvant in vaccine production,9,10 enhancement of protein and nucleic acid uptake,11,12 gene therapy,13 Received: July 23, 2015 Revised: October 9, 2015

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DOI: 10.1021/acssuschemeng.5b00747 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering protein stabilizers,14 and flocculating agents.15,16 Its nontoxic nature allows it to be used in oral formulations especially designed to decrease serum cholesterol and triglycerides. These applications are primarily related to its water-soluble nature based on amphiphilic behavior that arises from a neutral sugar backbone and charged quaternary amine side chains (Scheme 1). In this

understood model protein with specific applications, their complexes with DEAE allow us to produce biofunctional NPs with even better biological applications. We use both presynthesized Au NPs as well as those prepared by following the in vitro synthesis carried out by the weak reducing ability of DEAE20 and protein17−19,21 to produce biomolecule coated Au NPs. Both protocols lead to an inherent surface adsorption of the DEAE−protein complex on Au NPs which is simultaneously monitored by spectroscopic studies in terms of DEAE−protein interactions and the extent of their surface adsorption to produce bioactive NPs. Bioapplicability is evaluated from the hemolytic analysis and a possible use of such biofunctional NPs in systemic circulation and their potential role as drug release vehicles. To understand the site specific interactions at the atomic level among DEAE and protein as well as their surface adsorption on the NPs, we simultaneously performed the molecular dynamic (MD) studies. These studies allowed us to identify the specific amino acid residues which determine DEAE−protein interactions as well as the surface adsorption of their complexes. The adsorption of protein on inorganic surfaces is a potentially important field in biotechnology22 and biomaterials23 synthesis and can very well be characterized by MD studies at the microscopic level.

Scheme 1. Molecular Structure of Diethylaminoethyl Dextran Chloride (red, O; blue, N; white, H; black, C)

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EXPERIMENTAL SECTION

Materials. Diethylaminoethyl dextran chloride (DEAE), white powder, average molar mass = 500 000, hygroscopic, lot # 39 H1323; bovine serum albumin (BSA); lysozyme (Lys) chicken egg white; zein protein; chloroauric acid (HAuCl4); and sodium dodecyl sulfate (SDS) were purchased from Aldrich. Double distilled water was used for all preparations. Preparation of DEAE−Protein Binary Mixtures and Synthesis of Biofunctional Au NPs. Stock solutions of each component of binary mixtures of DEAE + BSA, DEAE + Lys, and DEAE + zein were made by dissolving 10 mg/10 mL in pure water (zein was aqueous solubilized by taking 24 mM SDS solution). This was followed by the mixing of the components to produce respective binary mixtures covering the entire weight fraction (wDEAE) range by keeping the total amount constant. Then, 1 mM of HAuCl4 was added in 10 mL of each weight fraction in screw-capped glass bottles and kept in a water thermostat bath (Julabo F25) at a precise 70 ± 0.1 °C for 6 h under static conditions. Each mixture initiated the reduction of Au(III) into Au(0) due to the weak reducing ability of both DEAE and protein that resulted in the color change from colorless to bright pink or pink-purple. After 6 h, the samples were cooled to room temperature and kept overnight. They were purified from pure water at least three times to remove unreacted protein. Purification was done by collecting the Au NPs at 8000− 10 000 rpm for 5 min after washing each time with distilled water. In the second method, calculated amounts of presynthesized citrate stabilized Au NPs were taken in small UV−visible cuvettes. The size of the Au NPs was 11 ± 3 nm with a zeta potential of −18.5 ± 1.7 mV. They were titrated with a freshly prepared DEAE−protein mixture by keeping constant the amount of Au NPs at 20 and 70 °C. At 20 °C, the protein was considered to be in the folded state when it interacted with DEAE, while at 70 °C it acquired maximum unfolding. The DEAE−protein complex demonstrated dramatic surface adsorption on NPs and was simultaneously monitored by the spectroscopic analysis. Methods. Spectroscopic Analysis. UV−visible (Shimadzu-Model No. 2450, double beam) measurements in the wavelength range of 200−900 nm were carried out to simultaneously monitor the reactions under the effect of temperature as well as reaction time to observe the influence of the DEAE−protein complex on the synthesis of Au NPs in terms of protein unfolding and subsequent DEAE−protein interactions as well as their surface adsorption. This instrument was equipped with a TCC 240A thermoelectrically temperature controlled cell holder that allowed the measurement of the spectrum at a constant temperature within ±1 °C.

study, we explore the interactions of DEAE with different kinds of model proteins such as bovine serum albumen (BSA), lysozyme (Lys), and zein, in the presence of Au NPs. Usually such interactions are very weak and complex, and it is not easy to detect and properly understand them. However, the presence of Au NPs magnifies them during the process of surface adsorption and allows us to precisely understand them because protein and its complexes are highly prone to the surface adsorption, and the magnitude of the surface adsorption is further related to the DEAE−protein interactions. Such surface activity also leads to the formation of bifunctional NPs with interesting biological applications.17 Lys and BSA are well-known model proteins, while zein is corn storage protein with several industrial applications. Lys coated NPs demonstrate wonderful antimicrobial activities,18 while BSA coated NPs are considered to be excellent vehicles for drug release in systemic circulation. Since zein is an edible protein, zein coated NPs can be easily used for various pharmaceutical formulations.19 In combination with DEAE, DEAE−protein complex coated NPs are expected to provide even better applicability in order to understand the bioactive role of DEAE in various biological applications. Our objective in this work is to understand the DEAE interactions with these proteins over the entire mixing range and to identify a suitable DEAE−protein mixture which best suits the synthesis of biofunctional Au NPs. Since each protein is a well B


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Figure 1. (a) Plots of UV−visible absorbance of presynthesized Au NPs upon the systematic addition of a DEAE−BSA mixture with wDEAE = 0.4 at 20 °C. Notice the red shift as well as decrease in the intensity with the increase in the [Total DEAE−BSA]. (b and c) Variation in the intensity and wavelength of the absorbance of Au NPs at 20 and 70 °C, respectively. (d) Series of photographs of color change of Au NPs suspension in UV−visible cuvette upon increasing the amount of DEAE−BSA. The color change from the first cuvette to the last one occurs over a period of few hours. (e) Plots of size/nm and (f) zeta potential of DEAE−BSA coated Au NPs with concentration from DLS measurements for wDEAE = 0.4, DEAE, and BSA at 20 °C. See details in the text. structures were obtained from the Protein Data Bank at Brookhaven,26 while a peptide chain was selected for zein. The entry codes were 4F5S and 2LYZ for BSA and Lys, respectively. All simulations used parallelepiped simulation boxes with periodic conditions. The box size was selected in such a way so that DEAE, protein/peptide, and its periodic images were separated by at least 1.2 nm, and the protein did not interact with its periodic images via van der Waals potentials. Peptide chains were initialized in the form of α-helices. Each system is subjected to a steepest descent energy minimization until a tolerance of 1000 kJ/mol was achieved. First, all water molecules and ions (NaCl), with the whole protein and DEAE fixed, were energy minimized, followed by the minimization of protein and DEAE by fixing the mainchain and Cα atoms. Finally, the whole system was minimized. NVT (constant Number of atoms, Volume, and Temperature) simulations were performed in order to bring the system to the target temperature, and NPT (constant Number of atoms, Pressure, and Temperature) simulations allowed the system to find the correct density. Temperature coupling was performed using the Nose−Hoover thermostat method. For the interactions of the protein with gold, we used the recently developed GolP (Gold−Protein Force Field) force field,27 which was parametrized on the basis of density functional calculations and

Microscopy and Dynamic Light Scattering (DLS). Protein coated Au NPs were characterized by transmission electron microscopic (TEM) analysis on a JEOL 2010F at an operating voltage of 200 kV. The samples were prepared by mounting a drop of a solution on a carbon coated Cu grid and allowed to dry in the air. DLS measurements were performed using a light scattering apparatus (Zetasizer, Nano series, Nano-ZS, Malvern Instruments) equipped with a built-in temperature controller with an accuracy of ±0.1 °C. The measurements were made using a quartz cuvette with a path length of 1 cm. Averages of 10 measurements were analyzed using the standard algorithms with an uncertainty of less than 7%. Molecular Dynamics (MD) Simulations. We employed an all-atom MD simulation using the GROMACS (GROningen Machine for Chemical Simulations) program (version 4.6.5)24 with the OPLS-AA force field. The molecular topology file and force field parameters for the DEAE were generated by using the Automated Topology Builder (ATB) program.25 The structure of DEAE was prepared by simplified molecular-input line-entry system (SMILES) fragments (Scheme 1) with two configurations of four and 10 monomers. We employed 5.0 ns conventional molecular dynamics simulation of DEAE with BSA, Lys, and zein proteins at temperatures of 310 and 350 K. BSA and Lys C


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scheme using cut-offs 1.0/1.0 nm and a pair list update frequency of once every five steps was used. The electrostatic interactions were calculated by using the Particle-mesh Ewald (PME) algorithm,29 and simulations were performed in explicit simple point charge solvent. The time step of 2 fs was used to study the interactions of DEAE with proteins, whereas a time step of 1 fs was used for DEAE and peptide in the presence of a gold surface. Simulations were performed in physiological ion concentration (150 mM NaCl) with a neutralized box to avoid effects from PME background charge on the potential of mean force (PMF) calculations. The lengths of bonds are constrained with the LINCS (Linear Constraint Solver) algorithm. Surface gold atoms and bulk gold atoms were frozen during all simulations, but gold dipole charges were left free. Hemolysis. A hemolytic assay was performed to evaluate the response of DEAE−protein complex conjugated NPs on blood group B of red blood cells (RBCs) from a healthy human donor. Briefly, a 5% suspension of RBCs was used for this purpose after giving three washings along with three concentrations (i.e., 25, 50, and 100 μg/mL) of each purified NPs sample. A 1 mL packed cell volume (i.e., hematocrit) was suspended in 20 mL of 0.01 M phosphate buffered saline (PBS). The positive control was RBCs in water, and it was prepared by spinning 4 mL of a 5% RBC suspension in PBS. PBS as a supernatant was discarded, and the pellet was resuspended in 4 mL of water. The negative control was PBS. All the readings were taken at 540 nm, i.e., the absorption maxima of hemoglobin.

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RESULTS AND DISCUSSION DEAE−Protein Interactions Using Presynthesized Au NPs. The interactions were determined by titrating fixed weight fractions (wDEAE) of DEAE−protein mixtures with 1 mM citrate stabilized Au NPs (average size = 11 ± 3 nm and zeta potential = −18.5 ± 1.7 mV). Figure 1a demonstrates a typical UV−visible scan of such a titration of the DEAE−BSA mixture with wDEAE = 0.4 at 20 °C. Presynthesized Au NPs give a sharp absorbance close to 515 nm30 due to the surface plasmon resonance (SPR). A systematic addition of wDEAE = 0.4 initially leads to an increase in this absorbance with a regular blue shift due to the greater

Figure 2. (a) Variation in the absorbance of Au NPs upon the addition of DEAE−BSA mixtures with different wDEAE’s. (b) Plots of Conc,m of DEAE−BSA, DEAE−Lys, and DEAE−zein mixtures at 20 and 70 °C. See details in the text. experimental data, including a term describing metal polarizability. GolP has already been used to model adsorption free energy of the natural amino acids on Au(111), yielding results coherent with available experimental data.28 A package was designed to study proteins in bulk water, and the introduction of a solid surface required some modifications. As an approximation, the solid was rigid and bulk-like, i.e. not distorted at the surface. In the simulations, the twin range cutoff

Figure 3. (a) Plots of UV−visible absorbance of in vitro synthesis of Au NPs in the presence of DEAE−BSA (wDEAE = 0.5). Peaks at 280, 310, and 550 nm belong to the absorbance due to tryptophan of BSA, LMCT, and surface plasmon resonance of Au NPs, respectively. (b, c, and d) Plots of intensity versus temperature of the pure components and wDEAE = 0.5 of DEAE−BSA, DEAE−Lys, and DEAE−zein mixtures, respectively. See details in the text. D


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ACS Sustainable Chemistry & Engineering colloidal stabilization achieved by the Au NPs upon the adsorption of a DEAE−BSA complex on the NP surface.31 After a certain concentration, the intensity of the absorbance starts decreasing with no more blue shift. Figure 1b explains the variation of both intensity as well as wavelength with respect to the total concentration of the DEAE−BSA complex at 20 °C. Intensity passes through a strong maximum where wavelength reaches a constant value. The maximum indicates the “minimum concentration of the complex” (Conc,m) required to fully stabilize the 1 mM Au NPs. Beyond Conc,m, self-aggregation among the coated NPs diminishes the intensity. The same reaction at 70 °C induces an instant reduction in the intensity with a blue shift (Figure 1c) due to the self-aggregation of Au NPs in view of the instant surface adsorption of the DEAE−BSA complex because BSA is in its unfolded state at 70 °C,21 and hence the complex is highly surface active. A break in the intensity profile with a larger slope coincides with the wavelength profile where no more blue shift is observed. This break point provides the “Conc,m” value at 70 °C. This causes a dramatic color change of Au NPs from bright red to purple in each sample at both 20 and 70 °C, which eventually leads to a complete coagulation of Au NPs as depicted in Figure 1d. Similar titrations with 1 mM Au NPs for other wDEAE’s of DEAE−BSA, DEAE−Lys, and DEAE−zein at 20 and 70 °C are shown in the Supporting Information (Figure S1−S6). DLS measurements indicate a dramatic size increase of Au NPs upon the DEAE−protein complex adsorption (Figure 1e). BSA and DEAE alone as well as their mixtures instantaneously lead to a size increase of Au NPs initially, which is relatively very prominent for the DEAE−BSA mixture rather than for BSA or DEAE alone. This demonstrates that the complex is clearly more surface active than either of the components. Likewise, zeta potential measurements indicate that an initial DEAE−protein complex adsorption is driven by strong electrostatic interactions. Citrate stabilized Au NPs are negatively charged with a zeta potential of −18.5 ± 1.7 mV. The addition of the DEAE−BSA complex converts the negative zeta potential value into a large positive value which subsequently shows a small decrease with the increase in the amount of DEAE−BSA due to subsequent deposition of more layers which may cause the screening of charged sites. There is also little difference between the zeta potential values for the DEAE and DEAE−BSA mixture, suggesting the fact that the positive charge of the complex was mainly contributed by the cationic DEAE. Figure 2a depicts a collective comparison of the intensity profiles of some of the mixing ratios along with that of pure components of DEAE−BSA where the maximum in each case represents the “minimum concentration of the complex” (Conc,m) required to fully stabilize the colloidal NPs. The Conc,m values for DEAE−BSA, DEAE−Lys, and DEAE−zein mixtures are presented in Figure 2b at both 20 and 70 °C. All mixtures show significant nonideal deviations with much larger values than corresponding linear ideal mixing (solid line joining the pure components) particularly in the protein rich region of each mixture. A larger amount of the deposition of the DEAE− protein complex on the NP surface in the protein rich region can be attributed to the seeding process where adsorbed protein further invites and complexes with the aqueous solubilized folded protein through predominantly hydrophobic interactions31 among their hydrophobic domains since the adsorbed protein is usually in an unfolded state. That is why the nonideal deviations are more prominent at 20 rather than at 70 °C, because a greater amount of the folded protein is present in the aqueous phase at 20 °C, which readily complexes through the seeding process.

Figure 4. (a) Plots of nucleation temperature (Ntemp) and (b) nucleation time (Ntime) versus the wDEAE of DEAE−BSA, DEAE−Lys, and DEAE− zein mixtures. TEM micrographs of Au NPs along with their selected area diffraction patters of these mixtures are shown in c−e, f−h, and i−k, respectively. See details in the text.

At 70 °C, instant surface adsorption (Figure 1c) leaves little aqueous solubilized protein for the seeding process, and hence, conc,m values are higher at 20 °C than 70 °C. In addition, the conc,m value is much greater for pure Lys and zein in comparison to that of BSA, which is due to the greater surface active nature of Lys and zein in comparison to BSA.32,19 DEAE−Protein Interactions in in Vitro Synthesis of Au NPs. In this section, the surface adsorption of the DEAE− protein complex is studied under in vitro synthesis of Au NPs by varying the temperature from 20 to 70 °C. Figure 3a shows the UV−visible scans of a typical reaction of DEAE−BSA with wDEAE = 0.5. A blank scan (dotted line) refers to the spectrum of DEAE−BSA in the absence of gold salt. The absorbance close to 285 nm is due to the tryptophan residues of BSA, while DEAE does not show any absorbance.31 The addition of gold salt eliminates the 285 nm absorbance and produces a strong absorbance at 310 nm due to the ligand to metal charge transfer band (LMCT)32 between the electron donating DEAE−BSA complex and the metal center. The intensity of the 310 nm peak shows a regular decrease with the increase in temperature due to the conversion of LMCT into the nucleating centers to produce tiny Au NPs which generate weak absorbance around 550 nm (indicated by a black arrow) due to the SPR. Similar plots of pure BSA, DEAE, and their mixtures at other mixing ratios have been shown in Figure S7. The variation of 550 nm intensity of E


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Figure 5. (a) Total energy of the DEAE and BSA complex as a function of simulation time for 5 ns MD simulation at 310 K. (b) The van der Waals energy (LJ) and electrostatic energy (Coulomb) between DEAE and BSA during the simulation at 310 K. (c and d) The number of hydrogen bonds formed between DEAE and BSA and between DEAE and Lys, respectively, at 310 K. (e) Changes in α-carbon dihedral angles of chain of amino acids of zein. The change is calculated as the difference between the average dihedral in the first and last 500 ps. Dihedral n corresponds to dihedral angle formed by α carbons n, n + 1, n + 2, and n + 3. See details in the text.

and the onset of nucleation is referred to as Ntime. Both Ntemp and Ntime are plotted in Figure 4a and b, respectively, with almost identical variation. DEAE−BSA and DEAE−Lys show far lower and DEAE−zein complexes show higher values than their respective pure components. The lower value indicates a higher reduction potential (to convert Au(III) into Au(0)) of DEAE− BSA than BSA−Lys complexes in contrast to that of DEAE−zein due to its predominantly hydrophobic nature. All complexes are involved in the colloidal stabilization (Figure 2b) as well as shape control effects of Au NPs as depicted in Figure 4c−e, f−h, and i−k for DEAE−BSA, DEAE−Lys, and DEAE−zein mixtures, respectively, where faceted NPs of mainly 15−35 nm with predominant growth on {111} crystal planes are obtained (Figure 4e, h, and k). Molecular Dynamics (MD) Simulations. DEAE−Protein Interactions. In this section, we present the results of atomistic simulations on the interactions of DEAE with BSA, Lys, and zein, as well as their complexes with the Au NP surface. The stability of DEAE−BSA and DEAE−Lys complexes is tested by simulating it

wDEAE = 0.5 is compared with that for pure BSA and DEAE in Figure 3b, while Figure 3c and d show a similar comparison for DEAE−Lys and DEAE−zein mixtures, respectively. In the case of DEAE−BSA (Figure 3b), all plots show an initial slight change in the SPR up to a certain temperature; after that it increases instantaneously, indicating the onset of nucleation among the nucleating centers to produce Au NPs. This temperature is known as nucleating temperature (Ntemp) and is indicated by black arrows. It is interesting to note that Ntemp for wDEAE = 0.5 is much lower than that for BSA or DEAE because nucleation is facilitated by their complex. An almost similar situation is observed for DEAE− Lys (Figure 3c), though no nucleation is observed for pure Lys within the temperature range. However, in the case of DEAE−zein (Figure 3d), the Ntemp for wDEAE = 0.5 is higher than either of the pure components, suggesting a delayed nucleation. Thus, the nucleation of Au NPs is clearly facilitated in the mixtures of DEAE−BSA and DEAE−Lys in comparison to DEAE−zein. Similar results are obtained if the reactions are carried out at a constant temperature (70 °C) with reaction time (see Figure S8), F


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and white regions represent the positive, negative, and neutral regions, respectively, on the surface of the protein. For comparison, the hydrophilic and hydrophobic regions of each protein are shown in their skeletal structures in panels “b” and “d.” Contrasting results are observed for BSA and Lys proteins in the presence of DEAE. Most of the region around BSA is surrounded by negative electrostatic potential (Figure 6a), whereas the region around Lys has positive potential (Figure 6c). The negative potential region around BSA makes favorable contact with DEAE. To bring DEAE in contact with the surface, it is necessary to desolvate electronegative patches on the surface of BSA by overcoming the dewetting barrier. Thus, the interactions between DEAE and the charged amino acids set in with the entropic gain obtained from freeing the water molecules bound to the surface of the protein in lieu of the loss of salvation enthalpy. Such interactions are in line with the site specific nature of protein− protein interactions and can be regarded as model systems for protein−protein interactions.35 Thus, stronger electrostatic interactions in the DEAE−BSA complex make it highly amphiphilic because unfolding of BSA upon interacting with DEAE exposes the hidden hydrophobic domains to an aqueous environment. A combination of both hydrophilic and hydrophobic interactions in the DEAE−BSA complex makes it highly surface active, as depicted in Figure 1e. Such behavior further permits the reducing amino acids like cysteine to effectively reduce Au(III) into Au(0), and that is why DEAE−BSA mixtures have a lower Ntemp (Figure 4a) as well as Ntime (Figure 4b) in comparison to DEAE− Lys mixtures. For DEAE and zein, no electrostatic interactions are observed among them. We compared the dihedral angles derived from four consecutive α-carbons of the peptide chain of zein (mentioned above) between the initial and final configurations (Figure 5e) upon its interactions with DEAE. The difference in dihedral angles indicates that there is a change in the configuration in terms of conformational stability when zein interacts with DEAE. DEAE interacts with −OH and −NH2 groups of SER and GLN, respectively, through H-bonding because the dihedral angle change is highest in the vicinity of these residues. Both SER and GLN are the amino acids with polar uncharged side chains. In addition, MET and LEU with hydrophobic side chains are also pulled toward DEAE during the simulation because they are near SER. It demonstrates that nonpolar interactions driven by the hydrophobic domains of zein are responsible for its interactions with DEAE, and hence, the DEAE−zein complex remains predominantly hydrophobic. That is why this complex exhibits its poor reducing ability to convert Au(III) into Au(0) with the result that both Ntemp (Figure 4a) as well as Ntime (Figure 4b) end up with much higher values in comparison to DEAE−BSA and DEAE−Lys complexes. Temperature Effect. As observed in Figure 2b, the surface adsorption of the DEAE−protein complex diminishes at 70 °C in comparison to 20 °C. We try to understand it from the shape and charge complementarity of the protein. It has been observed that the most large-scale changes in the overall root-mean-square (RMS) α-carbon displacement occurred within 5 ns. The average displacements are high in the case of BSA (Figure 7a) compared to those of Lys (Figure 7b) where large fluctuations occur in the α-carbon positions in short periods of time. Although the α-carbon displacement stabilizes with time, the simulations at high temperatures are quite mobile and exhibit large fluctuations about their mean conformations due to the unfolding with many distinct conformations (Figures S12,S13). This reduces the favorable electrostatic interactions between DEAE and BSA

for 5 ns at 310 K. Molecular dynamic simulation depends on the orientation of the DEAE relative to the protein, yet computational limitations prevent significant rotation of the constituents during the simulation time. To overcome this difficulty, we have considered interactions starting from different orientations of DEAE with respect to protein. Visual inspection of the trajectory has confirmed that DEAE makes favorable interactions with BSA, while the contact with Lys does not last up to the end in all the simulations. In order to understand the interactions of DEAE with zein, we had to restrict our choice to the following chain (ILE-ILE-PRO-GLN-CYS-SER-LEU-ALA-PRO-SER-SER-ILEILE-PRO-GLN-PHE-LEU-PRO-VAL-THR-SER-MET-ALAPHE-GLU-HIS-PRO-ALA-VAL-GLN-GLN-ALA-TYR-ARGSER) in view of the absence of any appropriate molecular structure of zein due to its highly complex nature.33 The total energy of the simulation model of the DEAE−BSA complex versus simulation time at 310 K is shown in Figure 5a, which gives an indication of the overall stability of the MD trajectory. Similar plots for other systems at 310 and 350 K are shown in Figures S9−S11. The interaction energy between DEAE and BSA is described by two main terms, i.e., van der Waals energy and electrostatic energy, which are shown in Figure 5b during the simulation. It is clear that the electrostatic interactions play a more important role than the van der Waals interactions, which allow DEAE to interact strongly with predominantly negatively charged BSA. Furthermore, the number of hydrogen bonds formed increases between DEAE and BSA (Figure 5c), whereas it remains almost the same with Lys (Figure 5d). These interactions are explained well from the electrostatic potential map by using an adaptive Poisson−Boltzmann34 approach on the solvent accessible surface of this system. Figure 6a and c show the electrostatic potential map around BSA and Lys, respectively. Blue, red,

Figure 6. (a) Electrostatic surface potential on the solvent accessible surface of BSA with DEAE simulation system (310 K) from explicit water simulations. Image did not show the entire simulation box to facilitate display of the protein and DEAE. Water molecules are omitted for clarity. Simulations are performed at physiological ion concentration (150 mM NaCl). (b) Representative secondary structure of BSA. Hydrophobic surface (orange) and hydrophilic surface (green) regions. (c and d) Similar images of Lys with DEAE at 310 K. See details in the text. G


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Figure 7. (a and b) Average RMS α-carbon displacements for BSA and Lys, respectively, at 310 K. (c) The electrostatic surface potential on the solvent accessible surface of BSA with DEAE simulation system (350 K) from explicit water simulations. Image did not show the entire simulation box to facilitate display of the protein and DEAE. Water molecules are omitted for clarity. Simulations are performed in physiological ion concentration (150 mM NaCl) and (d) for Lys and DEAE at 350 K. Water molecules and ions have been omitted for the sake of clarity. See details in the text.

primarily driven by the exposed hydrophobic domains of unfolded BSA and, hence, result in a complex with poor surface adsorption behavior (Figure 2b). However, strong electrostatic interactions at 20 °C result in the formation of a stable complex with better electron donating ability, amphiphilicity, and surface activity (Figure 2b). Protein−Gold Surface (NP) Interactions. Interactions between the protein and solid surface are at the heart of many potential applications in bionanotechnology36 and medicine,37 where we need to understand which amino acid, peptide, or protein binds favorably to a given solid surface. MD studies help us to locate specific amino acid residues which drive such

(Figure 7c) as well as DEAE and Lys (Figure 7d) at 350 K with the consequence that their smaller amount is surface adsorbed, as depicted in Figure 2b. To elaborate further, we focused our attention on the Defined Secondary Structure of Protein (DSSP) at the start and end of the simulation for BSA. BSA undergoes a dramatic change from turn conformation (Figure 8a) to α-helix conformational (Figure 8b) during the simulation, while Lys shows relatively much fewer conformational changes (Figured S14,S15). The yellow areas in Figure 8a referring to the turn configuration in the beginning of the simulation disappears into gray lines related to the α-helix in Figure 8b. Thus, relatively weaker electrostatic interactions at 70 °C are H


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Thus, a comparison between Figure 9d and e clearly highlights the role of ASP in helping the DEAE−protein complex to bind the Au surface, and hence, ASP facilitates electrostatic interactions between DEAE and BSA/Lys (Figure 6), leading to unfolding in the protein (Figure 8), which permits different amino acid residues mentioned above to interact with the Au surface as depicted in Figure 2b. In the case of the DEAE−zein complex, where nonpolar interactions play a predominant role, MD simulations show large changes in the dihedral angles of a peptide chain of zein (mentioned above) leading to a significant conformational change in the vicinity of the SER residue (Figure 9f). In fact, SER is included in the part of one of the three polar surfaces on the helical wheel of zein40 with high affinity to undergo hydrogen bonding due to its nonpolar side chain with DEAE to form the DEAE−zein complex. In this way, SER plays a governing role in allowing the zein chain to interact with the gold surface in the presence of DEAE. Even SER residues of the DEAE−zein complex come in contact with the Au surface due to its side group interactions with water molecules that are adsorbed on the Au surface.41 Such a predominant nonpolar nature of the DEAE− zein complex driven by SER residues allows its significant surface adsorption almost over the entire mixing range (as shown in Figure 2b), which is quite different from that of DEAE−BSA and DEAE−Lys complexes. The latter complexes demonstrate greater surface adsorption only in the protein rich region of the mixtures (Figure 2b) and which is mainly driven by Au selective amino acid residues mentioned above. Hemolysis and Possible Biological Applications. Hemolysis is the first step toward the understanding of the biological applicability of present DEAE−protein coated Au NPs in pharmaceutical formulations for their administration in systemic circulation.31 Figures 1 and 9 demonstrate that the Au NPs are fully coated by the surface adsorption of the DEAE−protein complex, and hence, the toxicity of the nanometallic surface can be easily passivated.42−44 The naked NP surface has a high surface energy to interact with the cell wall of a blood cell, leading to the deformation and eventually hemolysis.21,45 It can be tested by performing the hemolytic assay. We carried out the hemolysis of coated NPs by DEAE−BSA, DEAE−Lys, and DEAE−zein complexes over the entire mixing range by using purified samples of comparable shape and size. Although hemolysis depends on the morphology and size of the NPs, naked NPs have far more potential to interact with the blood cell wall and, consequently, demonstrate pronounced effects. Figure 10 shows some of the representative UV−visible plots of heme absorption within 500 to 600 nm of wavelength range with positive and negative controls (see Experimental Section). Figure 10a−d show the spectra of heme absorption of blood cells in the presence of DEAE−BSA coated NPs, while similar spectra for DEAE−Lys coated NPs have been shown in Figure S16. All spectra are quite similar, and no appreciable hemolysis occurred when Au NPs coated with DEAE−BSA and DEAE−Lys are mixed with blood cells (see sample photos in Figure 10d). However, when DEAE− zein coated NPs are used, the hemolysis is quite prominent in the zein rich region of the mixtures only (Figure 10e−h). The percentage hemolysis = [(sample absorbance − negative control absorbance)/(positive control absorbance − negative control absorbance) × 100] for samples of all mixtures at three dose concentrations is plotted in Figure 10i. It is quite insignificant for the NP coated DEAE−BSA and DEAE−Lys complexes; however, relatively large hemolysis is observed for the NPs coated with the complexes of the zein rich region. The latter is due to the

Figure 8. (a) Secondary structure change of BSA protein during the start of simulation and (b) at the end of simulation. See details in the text.

interactions. We have selected peptide chains, i.e., peptide 1 for BSA (GLU-LYS-LYS-PHE-TRP-GLY-LYS-TYR-LEUTYR-GLU-ILE-ALA-ARG-ARG-HIS) and peptide 2 for Lys (VAL-SER-ASP-GLY-ASN-GLY-MET-ASN-ALA-TRP-VALALA-TRP-ARG-ASN-ARG-CYS-LYS) to understand their interactions with the Au surface in the presence of DEAE. These chains along with DEAE are placed on the top of the gold slab mimicking the surface of a NP. Peptide 2 interacts with the Au surface through MET, TRP, CYS, GLY, SER, ARG, and ASP, while peptide 1 interacts via LYS, HIS, TRP, ARG, and TYR.37,38 Generally, CYS shows strong binding with the Au surface while other residues obey the following order: His ≈ Trp > Met > (Tyr, Lys, Arg). ASP, which contributes to the maximum negative potential for BSA (Figure 6a), does not have strong potential to bind the Au surface.38,39 However, in the presence of DEAE, ASP plays a governing role to help both BSA as well as Lys interact with the Au surface because ASP undergoes electrostatic interactions with cationic DEAE. Figure 9a and b show this specifically for Lys where ASP helps in anchoring peptide 2 to the gold surface, and its approach to the gold surface is plotted in Figure 9c. It is further explained on the basis of a change in the dihedral angles of peptide 1 (Figure 9d) and peptide 2 (Figure 9e). A change in the dihedral angles indicates a conformational change in the peptide upon its binding to the Au surface. The maximum change in dihedral angle occurs in the region where ASP is located in peptide 2 (Figure 9e), whereas Figure 9d which lacks ASP for peptide 1 (especially chosen to show the effect of ASP) does not show this drastic change. I


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Figure 9. (a) Snapshot of a DEAE and peptide 2 complex on the surface of gold slab. (red, O; blue, N; white, H; yellow, S; black, C). (b) Specific location of ASP. In both figures, water molecules and ions have been omitted for the sake of clarity. (c) Distance of amino acid residue ASP with time from the gold slab. (d) Changes in α-carbon dihedral angles of peptide 1, (e) peptide 2, and (f) zein chain. The change is calculated as the difference between the average dihedral in the first and last 500 ps. Dihedral n corresponds to dihedral angle formed by α carbons n, n + 1, n + 2, and n + 3. See details in the text.

and lipid bilayers47 are highly susceptible to complexation due to the predominant hydrophobic interactions of hydrophobic domains of unfolded zein, and hence, they are responsible for the rupturing of the blood cell. However, when the amount of DEAE

interactions of aqueous exposed hydrophobic domains of unfolded zein with the blood cell wall. The cell membrane consists of three layers with glycocalyx on the exterior, a protein network on the anterior, and a lipid bilayer in between the two. Glycoprotein46 J


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Figure 10. (a) UV−visible plots of heme absorption of different doses of Au NPs coated with DEAE−BSA (a−d) and DEAE−zein (e−h) mixtures at various wDEAE’s along with positive and negative controls. (i) Plots of hemolysis % versus wDEAE for 25 μg/mL (1), 50 μg/mL (2), and 100 μg/mL (3) Au NPs coated with DEAE−protein complex. See details in the text.

systemic circulation. The biological applicability is demonstrated from the hemolysis measurements where both DEAE−BSA as well as DEAE−Lys coated Au NPs do not show any marked hemolysis, thus proving to be the best suited vehicles for drug release in systemic circulation. DEAE−zein coated NPs, on the other hand, show this behavior only in the DEAE rich region of the mixture. MD simulations further support these results and demonstrate that the negative surface potential of BSA is mainly provided by the ASP residues. The negative surface potential drives the electrostatic interactions of BSA with cationic DEAE. The lower number of ASP residues makes the Lys surface almost neutral, and DEAE has to search for the active sites with negative potential to interact electrostatically. Hence, it reduces the magnitude of such interactions in the DEAE−Lys complex in comparison to the DEAE−BSA complex. In addition, ASP also demonstrates its active role in the surface adsorption of the

increases in the DEAE rich region of the DEAE−zein mixture, the effect of hydrophobic interactions is minimized with the predominance of amphiphilic character of the DEAE−zein complex. It consequently reduces the hemolysis. Thus, NPs coated with all mixing ratios of DEAE−BSA and DEAE−Lys complexes and those of the DEAE rich region of DEAE−zein mixtures can very well be used as drug delivery vehicles in the systemic circulation.

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CONCLUSIONS The above results show remarkable DEAE−protein interactions which are predominantly amphiphilic in the case of DEAE−BSA and DEAE−Lys mixtures, whereas they are nonpolar in the case of DEAE−zein mixtures. All different complexes demonstrate strong surface adsorption on both presynthesized Au NPs as well as in vitro synthesis of Au NPs, which leads to the formation of biofunctional Au NPs best suited for biological applications in K


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(8) Larive, C. K.; Lunte, S. M.; Zhong, M.; Perkins, M. D.; Wilson, G. S.; Gokulrangan, G.; Williams, T.; Afroz, F.; Schoneich, C.; Derrick, T. S.; Middaugh, C. R.; Bogdanowich-Knipp, S. Separation and analysis of peptides and proteins. Anal. Chem. 1999, 71, 389R−423R. (9) Westbrook, S. I.; McDowell, G.H. Immunization of lambs against somatotropin release inhibiting factor to improve productivity: comparison of adjuvants. Aust. J. Agric. Res. 1994, 45, 1693−1700. (10) Joo, I.; Emod, J. Adjuvant effect of DEAE- dextran on cholera vaccines. Vaccine 1988, 6, 233−237. (11) Fox, R. M.; Mynderse, J. F.; Goulian, M. Incorporation of deoxynucleotides into DNA by diethylaminoethyldextran-treated lymphocytes. Biochemistry 1977, 16, 4470−4477. (12) Rigby, P. Prologation of surivival of tumour − bearing animals by transfer of immune RNA with DEAE-dextran. Nature 1969, 221, 968. (13) Liptay, S.; Weidenbach, H.; Adler, G.; Schmid, R. M. Colon epithelium can be transiently transfected with liposomes, calcium phosphate precipitation and DEAE dextran in vivo. Digestion 1998, 59, 142−147. (14) Gavalas, V. G.; Chaniotakis, N. A. Polyelectrolyte stabilized oxidase based biosensors: effect of diethylaminoethyl-dextran on the stabilization of glucose and lactate oxidases into porous conductive carbon. Anal. Chim. Acta 2000, 404, 67−73. (15) Ghimici, L.; Constantin, M.; Fundueanu, G. Novel biodegradable flocculanting agents based on pullulan. J. Hazard. Mater. 2010, 181, 351−358. (16) Ghimici, L.; Morariu, S.; Nichifor, M. Separation of clay suspension by ionic dextran derivatives. Sep. Purif. Technol. 2009, 68, 165−171. (17) Goshisht, M. K.; Moudgil, L.; Rani, M.; Khullar, P.; Singh, G.; Kumar, H.; Singh, N.; Kaur, G.; Bakshi, M. S. Lysozyme complexes for the synthesis of functionalized biomaterials to understand protein − protein interactions and their biological applications. J. Phys. Chem. C 2014, 118, 28207−28219. (18) Mahal, A.; Goshisht, M. K.; Khullar, P.; Kumar, H.; Singh, N.; Kaur, G.; Bakshi, M. S. Protein Mixtures of Environmental Friendly Zein to understand Protein − Protein Interactions through Biomaterials Synthesis, Hemolysis, and their Antimicrobial Activities. Phys. Chem. Chem. Phys. 2014, 16, 14257−14270. (19) Mahal, A.; Khullar, P.; Kumar, H.; Kaur, G.; Singh, N.; JelokhaniNiaraki, M.; Bakshi, M. S. Green chemistry of zein protein towards the synthesis of bioconjugated nanoparticles: understanding unfolding, fusogenic behavior, and hemolysis. ACS Sustainable Chem. Eng. 2013, 1, 627−639. (20) Singh, V.; Khullar, P.; Dave, P. N.; Kaur, G.; Bakshi, M. S. Ecofriendly route to synthesize nanomaterials for biomedical applications; bioactive polymers on the shape control effects of nanomaterials under different reaction conditions. ACS Sustainable Chem. Eng. 2013, 1, 1417−1431. (21) Khullar, P.; Singh, V.; Mahal, A.; Dave, P. N.; Thakur, S.; Kaur, G.; Singh, J.; Singh Kamboj, S.; Singh Bakshi, M. Bovine serum albumin bioconjugated gold nanoparticles; synthesis, hemolysis, and cytotoxicity towards cancer cell lines. J. Phys. Chem. C 2012, 116, 8834−8843. (22) Zhdanov, V. P.; Kasemo, B. Bistability in Catalytic Reactions on the nm Scale. Surf. Sci. 2002, 496, 251−263. (23) Ratner, B. D.; Bryant, S. J. Biomaterials: where we have been and where we are going. Annu. Rev. Biomed. Eng. 2004, 6, 41−75. (24) Berendsen, H. J. C.; Van der Spoel, D.; Van Drunen, R. Gromacs: a message-passing parallel molecular dynamics implementation. Comput. Phys. Commun. 1995, 91, 43−56. (25) Malde, A. K.; Zuo, L.; Breeze, M.; Stroet, M.; Poger, D.; Nair, P. C.; Oostenbrink, C.; Mark, A. E. An automated force field topology builder (ATB) and repository: version 1.0. J. Chem. Theory Comput. 2011, 7, 4026−4037. (26) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J. B.; Meyer, E. F.; Brice, M. D.; Rodgers, J. R. The protein data bank: a computer based archival file for macromolecular structure. J. Mol. Biol. 1977, 112, 535− 542.

DEAE−protein complex on the gold surface. Electrostatic interactions between DEAE−BSA/Lys induce the unfolding in the protein with the result that several Au selective amino acid residues get a chance to interact with the Au surface and, hence, facilitate the surface adsorption of the DEAE−protein complex on Au NPs. Interactions between DEAE and zein are mainly triggered by the nonpolar amino acid residues like SER and GLN through hydrogen bonding, which consequently produce a predominantly nonpolar DEAE−zein complex. SER even drives the surface adsorption of the DEAE−zein complex on Au NPs mainly through hydrogen bonding with surface adsorbed water molecules. Thus, a combination of both experimental and theoretical aspects helped us to understand DEAE−protein interactions up to the molecular level and also demonstrated their versatile bioapplicability.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.5b00747. UV−visible and molecular dynamics profiles (PDF)

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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 These studies were partially supported by financial assistance under Article 28.8 of the CAS agreement of WLU, Waterloo, and from CSIR [ref no: 01(2683)/12, ref no: 01(2700)/12/EMR-II] and DST [ref no: SR/FT/CS-28/2011], New Delhi. G.K. thankfully acknowledges the financial support provided by the Research and Development Council (RDC) of Newfoundland and Labrador, NSERC, and the Office of Applied Research at CNA.

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