Bovine Serum Albumin Bioconjugated Gold Nanoparticles: Synthesis

Apr 10, 2012 - One-Step Synthesis of Stable Colloidal Gold Nanoparticles Through Bioconjugation with Bovine Serum Albumin in Harsh Environments. S. A...
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Bovine Serum Albumin Bioconjugated Gold Nanoparticles: Synthesis, Hemolysis, and Cytotoxicity toward Cancer Cell Lines Poonam Khullar,§ Vijender Singh,§ Aabroo Mahal,§ Pragnesh N. Dave,⊥ Sourbh Thakur,∥ Gurinder Kaur,‡ Jatinder Singh,∥ Sukhdev Singh Kamboj,∥ and Mandeep Singh Bakshi*,† †

Department of Chemistry, Wilfrid Laurier University, Science Building, 75 University Avenue West, Waterloo, ON N2L 3C5, Canada ‡ Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, NL A2V 2K7, Canada § Department of Chemistry, BBK DAV College for Women, Amritsar 143005, Punjab, India ∥ Department of Biochemistry, Guru Nanak Dev University, Amritsar 143005, Punjab, India ⊥ Department of Chemistry, Kachchh University, Mundra Road, Bhuj-370001, Kachchh Gujarat, India S Supporting Information *

ABSTRACT: Bovine serum albumin (BSA) conjugated gold (Au) nanoparticles (NPs) were synthesized to explore their applications as drug delivery vehicles in systemic circulation. They showed little hemolysis and cytotoxic responses essentially required for such applications. This study shows some of the important physiochemical aspects needed for an appropriate synthesis of BSA-conjugated NPs where unfolded BSA is an essential reaction component. Unfolding of BSA was carried out under different experimental conditions in the presence of different ionic/ zwitterionic surfactants and monitored simultaneously by UV−visible studies. Cationic surfactants induced unfolding at relatively lower temperatures than anionic and zwitterionic surfactants due to stronger electrostatic interactions with BSA. TEM analysis revealed the presence of NPs with almost similar shapes and sizes for different samples, and all NPs were stabilized by a coating of unfolded BSA. Isoelectric point of unfolded BSA coating on NP surface was close to 4.7 in all cases, which was similar to that of unconjugated BSA. BSA free and cationic surfactant coated Au NPs were used as controls. They showed high hemolytic activity and very low cell viability under identical conditions. Thus, BSA coated NPs were considered to be the best vehicles for drug release and other possible biomedical applications.

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substantial unfolding and that subsequently promotes fibrillation, while Halas et al.5e have established that fibrillation can be prevented by using polyethylene glycol capped NPs. Thus, unfolding is also related to the NP surface adsorption of protein through electrostatic, close van der Waals, or hydrophobic interactions, which determine the degree of unfolding and consequently other physiochemical aspects like fibrillation. The interfacial adsorption of unfolded protein sometimes works just like that of amphiphilic molecules in controlling the crystal growth of growing nuclei because different crystal planes have different preferential adsorption of unfolded protein as it happens in the case of Au6a and PbS6b NPs due to their identical face centered cubic geometry. In addition, NPs under some special circumstances also act as pseudochaperonins6c−e where they can be involved in the folding of newly developed

INTRODUCTION Ionic surfactants are important components in biochemistry where they have been frequently used in extraction of DNA as well as the denaturation of proteins.1 Water-soluble globular proteins are more prone to denaturation in the presence of ionic surfactants rather than fibrous proteins.2 This behavior stems from the predominantly polar nature of globular proteins, which allows surface amino acids to interact with oppositely charged ionic surfactants through electrostatic interactions and that in turn begins the unfolding of tertiary protein structure.3 Although unfolding impedes the specificity of a protein toward enzyme reactions, it altogether changes the overall physiochemical behavior that departs significantly from its native state as more and more unfolding sets in. The unfolding is also responsible for the fibrillation, which is the consequence of several amyloidosis related protein misfolding diseases.4 It is however established that unfolded protein undergoes seeding and oligomerization before amyloidosis; the exact mechanism of how it happens is still a complicated task to understand. We have recently observed5 that protein−NPs interactions lead to a © 2012 American Chemical Society

Received: January 17, 2012 Revised: March 25, 2012 Published: April 10, 2012 8834

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synthesized as reported elsewhere.12 Double distilled water was used for all preparations. Synthesis of Au NPs in the Presence of BSA. Aqueous mixtures (total 10 mL) of BSA (15 μM), surfactant (0.05−0.25 mM), and HAuCl4 (1 mM) were taken in screw-capped glass bottles. After the components were mixed at room temperature, the reaction mixtures were kept in a water thermostat bath (Julabo F25) at a precise 70 ± 0.1 °C for 6 h under static conditions. The color of the solution changed from colorless to pink-purple. The overall reaction (eq 1) was considered to take place where BSA acts as a weak reducing agent due to the presence of several reducing amino acids like cysteine. No surfactant is involved in the reduction of gold ions because parallel control reactions only in the presence of surfactant did not yield any Au NPs.

proteins from linear amino acids into three-dimensional structures. Herein, we have utilized the unfolded BSA for in vitro synthesis of Au NPs to develop bioconjugated NPs for their possible use as drug delivery vehicles. To explore this feasibility, we systematically determined their hemolytic effect,7 which is considered to be more relevant for their possible transportation in systematic circulation. This allowed us to further explore their potential to determine their cytotoxic8 effect especially toward cancer cell lines. The most important aspect in this study is to achieve a proper conjugation of Au NPs with unfolded protein; otherwise, bare NP surface is expected to show severe hemolysis in systematic circulation, and even known to cross the blood−brain barrier,9 and migrate to various organs10 and tissues, whereby they may cause damage to biological systems through oxidative stress pathways.11 A proper conjugation can only be achieved by complete passivation of all crystal planes of NP by proper capping and stabilization and is only possible through in vitro synthesis. Unfolded protein starts the reduction in the first place to generate Au atoms and then systematically controls their nucleation to achieve appropriate bioconjugated nanogeometries. We have tried different possible environments provided by ionic surfactants to unfold BSA and then to use it simultaneously for the synthesis of Au NPs to generate bioconjugated NPs. Surfactants are expected to have a wide range of polar as well as nonpolar interactions with BSA through surface amino acids, which exist in the form of zwitterionic functional groups. BSA is composed of 580 amino acid residues with 17 interchain disulfide bonds. These functional groups can interact specifically with anionic, cationic, or zwitterionic surfactants of equal hydrophobicity and lead to an unfolding of BSA due to the breaking of disulfide bonds, which could be surfactant specific. The unfolded state of BSA thus produced cannot only reduce Au(III) into Au(0) due to aqueous exposed reducing amino acids like cysteine,5d,6a but it also provides excellent capping/stabilization properties essential for NPs colloidal behavior. Thus, the present study explains a simple comprehensive methodology to develop nontoxic biconjugated NPs, which can be easily used for several biological applications that require their intravenous administration. It is devoted to the physiochemical aspects of the unfolding and denaturalization behaviors of BSA under different environments provided by different ionic surfactants over a wide range of concentrations and temperatures. We have shown that cationic surfactants are the most suitable additives to break disulfide bonds necessary for the unfolding and subsequent capping behavior of BSA to produce bioconjugated Au NPs. Properly capped NPs with unfolded BSA show almost no hemolytic and cytotoxic responses and thus are best suited for drug delivery vehicles.

Au 3 +(aq) + BSA(aq) + 3e− → Au 0(s)

(1)

After 6 h, the samples were cooled to room temperature and kept overnight. They were purified from pure water at least two times to remove unreacted BSA and surfactant. Purification was done by collecting the Au NPs at 10 000−14 000 rpm for 5 min after washing each time with distilled water. Synthesis of Au NPs in the Absence of BSA. Synthesis of 16-2-16 capped Au NPs was carried out by the seed growth (S-G) method essentially similar to that reported by Murphy et al.13 Briefly, 25 mL of seed solution was prepared by mixing [HAuCl4] = 0.5 mM and [Na3Cit] = 0.5 mM, and followed by the addition of 0.6 mL of aqueous NaBH4 ([NaBH4] = 0.1 mol dm−3) solution under constant stirring. Growth solution was prepared by dissolving [16-2-16] = 2−8 mM in total 5 mL of water along with [HAuCl4] = 0.25−1 mM. Twenty-five microliters of previously prepared seed solution was added at the end to start the growth process at 70 °C for at least 6 h. Br− ions act as mild reducing agent toward AuCl4− ions and convert them into nucleating centers, which undergo slow growth over the gold seeds to produce well-defined Au NPs. The samples were purified from pure water as mentioned above. Methods. Each reaction in the presence of BSA was monitored with time as well as with temperature from 20 to 70 °C to determine the influence of denaturation of BSA on the synthesis of Au NPs by simultaneous UV−visible measurements with the help of Shimadzu model no. 2450 (double beam). This instrument was equipped with a TCC 240A thermoelectrically temperature-controlled cell holder that allowed one to measure the spectrum at a constant temperature within ±1 °C. Transmission electron microscopy (TEM) measurements were carried out by mounting a drop of a sample on a carboncoated Cu grid and allowing it to dry in air. They were observed with the help of a Philips CM200 transmission electron microscope operating at 20−200 kV. X-ray diffraction (XRD) patterns were recorded by using Bruker-AXS D8-GADDS with Tsec = 480. Samples were prepared on glass slides by placing a concentrated drop of aqueous suspension and then drying in a vacuum desiccator. The pH of some of the reactions was measured by using a pH-meter to determine the isoelectric point (Ip) of BSA coated NPs. The pH of the as-prepared sample of BSA conjugated NPs was systematically changed. Close to Ip when BSA coated layer acquires no net charge, coagulation happens and NPs settle at the bottom. pH measurements were carried out at regular intervals over a period of 6 h.

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EXPERIMENTAL SECTION Materials. Chloroauric acid (HAuCl4), bovine serum albumin (BSA), sodium dodecyl sulfate (SDS), and dodecyltrimethyl ammonium bromide (DTAB) were purchased from Aldrich. 3-(N,N-Dimethyldodecylammonio) propanesulphonate (DPS), 3-(N,N-dimethyltetradecylammonio) propanesulphonate (TPS), 3-(N,N-dimethylhexadecylammonio) propanesulphonate (HPS), and hexadecyltrimethylammonium bromide (HTAB) were obtained from Flüka. Didodecyldimethylammonium bromide (12-0-12) and dimethylene bis (hexadecyldimethyl-ammonium bromide) (16-2-16) were 8835

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Figure 1. (a) UV−visible scans of BSA+SDS+HAuCl4 mixture with [BSA/residue]/[SDS] mole ratio = 88 at 70 °C. Block arrow shows the absorbance due to tryptophan residues, while dotted indicates the increasing absorbance of Au NPs due to SPR with time. Blank means when no HAuCl4 is added. (b and c) Plots of intensity at 540 nm versus reaction time and temperature, respectively, for different mixtures in the presence and absence of surfactants. (d and e) Plots of intensity at 540 nm versus reaction temperature for BSA+DTAB+HAuCl4 and BSA+12-0-12+HAuCl4 mixtures at different concentrations of DTAB or 12-0-12 and at fixed [BSA] = 15 μM. Dotted line in (e) belongs to a mixture that remained turbid right from 20 °C, and no clear absorbance around 540 nm due to SPR was detected. (f) Plots of intensity at 540 nm versus reaction temperature for BSA+DTAB+HAuCl4 mixtures at different concentrations of BSA and at fixed [DTAB] = 0.1 mM. Inset shows the variation in Td with BSA concentration. See details in text.

streptomycin, and 100 μg/mL gentamycin, and maintained at 37 °C in a humidified atmosphere with 5% CO2. The cytotoxic effect of gold nanoparticles was checked by seeding C6 glial cells at a concentration of 50 000 cells/mL in a 96 well titer plate (100 μL of DMEM containing cells/well). Cells were treated with samples at decreasing concentrations ranging from 100 to 30 μg/mL for 4 h in a 12 well culture plate. The cell viability was checked by MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay, which forms a blue formazan in viable cells, but is unable to do so with dead cells or their debris. MTT dissolved in DMEM at a concentration of 0.5 mg/mL was added at the end of incubation, and then incubated further at 37 °C for 2 h. The resultant formazan product was dissolved by adding dimethylsulfoxide (DMSO, 100 μL/well), and the concentration was read at 550 nm by using a UV−visible spectrophotometer. The greater is the absorbance, the higher is the cell viability and the lower is the toxicity of the sample toward cells. Morphological changes in

Hemolytic assay was performed to evaluate the toxicity of BSA-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 two concentrations (i.e., 50 and 100 μg/mL) of each NPs sample. One milliliter 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 5% RBCs suspension in PBS. PBS as supernatant was discarded, and pellet was resuspended in 4 mL of water. The negative control was PBS. All of the readings were taken at 540 nm, that is, the absorption maxima of hemoglobin. Cytotoxicity assay C6 was performed with glioma cell line, a N-nitrosomethylurea induced cell line from rat brain, obtained from NCCS, Pune, India. It was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 10 U/mL penicillin, 100 μg/mL 8836

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basically related to the greater number density of the NPs produced, which is not much different from that of DTAB. First-order rate constant values (provided by choosing maximum points) for SDS and DTAB are 9.5 × 10−4 and 9.4 × 10−4 s−1, respectively, which are quite close to each other but much greater than DPS (3.5 × 10−4 s−1), while in the absence of surfactant, that is, for BSA only, it is 3.3 × 10−4 s−1. A stronger cationic surfactant like 12-0-1216 starts the reaction instantaneously as soon as gold ions are added in the solution, and the reaction is completed within ∼60 min unlike the other cases where it attains equilibrium (Figure 1b). In contrast, when the reaction is carried out in the absence of any surfactant, that is, in aqueous BSA + HAuCl4 (see dotted line in Figure 1b), although it immediately starts the reduction just like that of 12-0-12, it attains equilibrium within 30 min with much lower magnitude of SPR in comparison to that in the presence of all other surfactants. It means that the reduction is most facilitated in the presence of cationic surfactants, that is, 12-0-12 and DTAB, and least in their absence, that is, aqueous BSA only. The effect of temperature within 20−70 °C is depicted in Figure 1c. The inset magnifies the control reaction with BSA (i.e., in the absence of surfactant) where SPR appears only at 68 °C and indicates the onset of complete unfolding. We termed it as a denaturation temperature (Td) of BSA in the presence of Au NPs because a fully unfolded state triggers an instant reduction of gold ions into atoms. In the presence of SDS, DPS, and DTAB, Td arrives at 65, 60, and 55 °C, respectively, while a marked reduction to 30 °C is observed in the presence of 12-012. Furthermore, the shape of the curve in each case demonstrates the nature of reaction kinetics. A parabolic response in the presence of SDS/DPS/DTAB predicts a slow growth of nucleating centers with temperature contrary to a hyperbolic growth in the presence of 12-0-12 that tends to complete with temperature. Thus, the polarity of the surfactant determines the extent of unfolding, which is most significant for cationic surfactant DTAB3b,c in comparison to anionic SDS and zwitterionic DPS. 12-0-12 with stronger dissociation than DTAB induces instant unfolding17−19 at much lower temperature, that is, 30 °C, and hence it starts a rapid nucleation to produce Au NPs. This is all due to the fact that aqueous BSA with pH ≈ 6.5 at room temperature bears predominantly negative charge because the isoelectric point of BSA is 4.9. In such a situation, DTAB is expected to have strong oppositely charged electrostatic interactions with BSA and is even much stronger for 12-0-12 due to its greater dissociation. Because the mole ratio between BSA and surfactants is [BSA/residue]/ [surfactant] = 88 in all of the above-mentioned experiments, therefore, surfactant with a relative low amount only induces partial unfolding at room temperature, while further unfolding is being carried out by the temperature effect. Thus, the unfolding follows the order of SDS < DPS < DTAB < 12-0-12 with the result being that reduction and subsequent nucleation also follow the same order. That is why 12-0-12 induces instant reduction in Figure 1b, and reaction goes to completion in a much shorter period of time in comparison to all other surfactants. Effect of Surfactant/BSA Concentration. Figure 1d depicts the concentration effect of DTAB on the unfolding of BSA and subsequent effect on the synthesis of Au NPs. Interestingly, there is not much change in the Td within 0.05− 0.25 mM of DTAB (or [BSA/residue]/[surfactant] = 35−176), which remains more or less close to 55 °C, but there is a

these cells were observed with an inverted phase contrast microscope (Olympus CK2, Tokyo, Japan) with a 40× objective and photographed with a digital camera (Coolpix S3000, 12.0 megapixels, Nikon, Japan).

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RESULTS Denaturation of BSA in the presence of different surfactants and its simultaneous use as a reducing agent to convert Au(III) into Au(0) is studied separately with time at fixed 70 °C and with temperature from 20 to 70 °C. Aqueous BSA, in the absence of any additive, remains in its native state up to 40 °C. Between 40 and 50 °C, reversible changes occur in its conformation, which become irreversible between 50 and 60 °C. Above 60 °C, unfolding of BSA with β-aggregation begins that ultimately leads to a gel formation around 70 °C.14 Therefore, at 70 °C, we expect that BSA already exists in its unfolded state and the presence of an ionic surfactant simply blocks oppositely charged electroactive sites, thereby altering the overall physicochemical behavior of BSA essential for its reducing as well as stabilizing abilities toward growing Au nucleating centers. The presence of an ionic surfactant in aqueous BSA at room temperature has the strong ability to interact with folded BSA through both hydrophilic as well as hydrophobic interactions, thereby inducing some degree of unfolding that is related to the concentration of surfactant used. Thus, the temperature effect from 20 to 70 °C helps us to understand the unfolding behavior in the presence of ionic surfactants and its simultaneous effect on the reducing as well as stabilizing abilities of BSA. Effect of Electrostatic Interactions. SDS (anionic surfactant) and DTAB (cationic surfactant) with equal hydrophobic tails of C12 can block the positive and negative sites of BSA, respectively, and hence help us to understand the synthesis of Au NPs under different conditions. Some typical UV−visible scans of a reaction conducted at 70 °C with different intervals of time for BSA+SDS+HAuCl4 mixture are shown in Figure 1a. Aqueous BSA in the presence of SDS (0.1 mM, precmc) gives a peak around 285 nm due to tryptophan residues (indicated by a block arrow), which disappears as soon as 1 mM HAuCl4 is added, and instead a clear peak emerges with time around 540 nm due to surface plasmon resonance (SPR) of Au NPs.15 A decrease in the amount of HAuCl4 to 0.5 or 0.25 mM does not show any clear peak due to SPR (Supporting Information, Figure S1a,b). The same reaction with temperature (Figure S1c) helps us to understand a simultaneous correlation between the onset of denaturation and the initiation of Au NPs formation because denaturation in fact facilitates the reduction by breaking the disulfide bridges and exposing the cysteine amino acids to participate in the reduction reaction.5d,6a In this case, the nucleation among Au nucleating centers does not start until the temperature of the reaction reaches 65 °C; as a result, the absorbance around 540 nm becomes prominent only around this temperature. Similar behavior is observed when reactions were carried out with DTAB = 0.1 mM, precmc (Figure S2a,b,c,d) and DPS = 0.1 mM, precmc (Figure S3a,b,c,d), instead of SDS. Plots of intensity at 540 nm versus time (Figure 1b) indicate the influence of different surfactants on the synthesis of Au NPs carried out by BSA. At constant temperature (i.e., 70 °C, Figure 1b), DTAB starts the nucleation within ∼25 min in comparison to SDS and DPS (both within ∼60 min), although the magnitude for DPS is much lower than that of SDS. The greater magnitude of the curve (i.e., plateau region) for SDS is 8837

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substantial change in the shape of the curve due to SPR of Au NPs with temperature. It is parabolic between 0.05 and 0.1 mM while hyperbolic thereafter, which means that at least [BSA/ residue]/[surfactant] = 88 mol ratio is required for a rapid unfolding of BSA and its subsequent effective participation in the reduction process. On the contrary, stronger interactions between 12-0-12 and BSA lead to phase separation, thereby adversely affecting the synthesis of Au NPs (Figure 1e). As the amount of 12-0-12 reaches 0.15 mM, phase separation sets in with the result of the effective concentration of BSA required for the reduction of Au(III) into Au(0) decreasing, and hence the intensity due to SPR starts decreasing after 44 °C (Figure 1e). Again, Td remains more or less close to 30 °C at different concentrations of 12-0-12, while phase separation occurs close to 44 °C when unfolded BSA is completely saturated with 12-012 and all electroactive sites are neutralized by surfactant molecules. SDS concentration, however, does not show any marked effect on the synthesis of Au NPs (Figure S4) due to relatively much weaker interactions of SDS with BSA. We have intentionally selected a premicellar concentration range for all surfactants (i.e., from 0.05 to 0.25 mM) so that only BSA− monomer interactions should predominate rather than the micelle formation.3b,c,19 The amount of BSA also plays a crucial role in the denaturation process so that surfactant can neutralize available electroactive sites on the protein to make it unfold. The more the unfolded form of BSA is available, the greater is the reduction potential. Figure 1f demonstrates that the synthesis of Au NPs shifts to higher temperature because Td increases likewise (Figure 1f, inset) with the increase in the amount of BSA at constant concentration of DTAB. Because a greater amount of BSA requires a greater amount of the surfactant to unfold and to initiate the reduction process, therefore, one has to keep a low BSA/surfactant mole ratio to achieve greater unfolding that in turn starts the synthesis of Au NPs at relatively low temperature (Figure 1f, inset). Effect of Hydrophobic Interactions. Apart from the electrostatic interactions, we have tried to evaluate the hydrophobic effect of present surfactants on the unfolding of BSA and its subsequent effect on the synthesis of Au NPs. An increase in the hydrocarbon tail length from C12 in DTAB to C16 in HTAB in fact dramatically lowers the reduction potential of unfolded BSA, with the consequence that the curve in the presence of HTAB lies close to that of the control (Figure S5a). It seems that the unfolded BSA is now predominantly interacting with C16 tails through its hydrophobic domains,3b which in turn screen the electrostatic interactions between the polar charged sites of BSA and AuCl4− ions. Similarly, in the case of zwitterionic surfactants, the increased hydrophobicity in the order of DPS < TPS < HPS has little effect on the reducing ability of BSA (Figure S5b). Determination of Isoelectric Point (Ip) of BSAConjugated NPs. Isoelectric focusing of a protein is another useful parameter that helps us to understand the overall charge on the BSA conjugated NPs20,21 in the presence of different conventional surfactants of varying polarities. Figure 2a shows typical UV−visible scans of an as-prepared sample with pH. A prominent absorbance due to SPR of Au NPs around 540 nm appears at all pH values except close to Ip of BSA (i.e., pH = 4.7). A plot of intensity at 540 nm versus pH (Figure 2b) helps us to evaluate Ip of different samples of BSA conjugated NPs. Interestingly, close to Ip, colloidal NPs instantaneously settle at the bottom of the tube due to rapid aggregation rather than at

Figure 2. (a) UV−visible absorbance scans of as-prepared BSA conjugated NPs for BSA+SDS+HAuCl4 mixture at different pH values with uncertainties of ±0.1. A strong absorbance around 540 nm is due to the SPR of Au NPs. (b) Plots of intensity at 540 nm versus pH for as-prepared samples of various BSA coated NPs synthesized in the presence and absence of surfactants. See details in text.

other pH values (see the sample photos at the respective positions). The stability of colloidal particles is best explained on the basis of DLVO theory in terms of flocculation and coagulation.22,23 The potential energy of interacting colloidal particles is the sum of potential energy due to electrostatic (VR) and van der Waals (VA) interactions. Electrical double layer generated by the capping BSA provides necessary VR to colloidal NPs below and above the Ip, and that provides NPs with global positive and negative charge, respectively. However, as Ip approaches, BSA capping acquires no net charge that in turn allows VA to predominate over VR; as a result, coagulation occurs and NPs settle at the bottom with no apparent SPR. Because no specific or covalent interactions of unfolded BSA are expected with surfactants, therefore Ip remains almost constant for different BSA conjugated NPs samples. Microscopic Studies. The size and morphology of Au NPs synthesized in the presence of different surfactants have been determined with the help of TEM. Figure 3a shows a TEM image of a sample prepared in the presence of SDS. Welldefined NPs of 51 ± 13 nm (size distribution histogram is shown in Figure S6a) of different shapes are present. Most of the NPs are hexagonal (Figure 3b) with clear facets bound by {111} planes as shown in the diffractional image in the inset and further supported by the XRD patterns with prominent growth at {111} of fcc geometry (Figure S6b). Interestingly, the NPs produced in the presence of DPS (Figure 3c) as well as 8838

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Figure 3. (a) TEM images of purified Au NPs prepared with BSA+SDS+HAuCl4 mixture at 70 °C. Part (b) shows a single hexagonal NP with diffraction image in inset. (c and d) TEM images of purified Au NPs prepared with BSA+DPS+HAuCl4 and BSA+DTAB+HAuCl4 mixtures, respectively, at 70 °C. Empty block arrows in (c) indicate the presence of thin BSA coating around each NP. (e and f) TEM images of purified Au NPs prepared with BSA+TPS+HAuCl4 at 70 °C. A pearl necklace arrangement of BSA conjugated NPs in (f) is due to the fibrillation of BSA. (g) UV−visible scans of different samples of as-prepared 16-2-16+HAuCl4 mixture at 70 °C with different concentrations of gold salt. Parts (h) and (i) show their respective TEM images with HAuCl4 = 0.25 and 1 mM, respectively. See details in text.

DTAB (Figure 3d) are quite similar to those produced in the presence of SDS. They are almost of the same dimensions (size distribution histogram is shown in Figures S6c and S6d) and morphologies. However, there seems to be a slight change in the morphology in the presence of TPS (Figure 3e). Now many rhombohedral shapes (indicated by arrows) are also visible along with other hexagonal shapes. In addition, increase in the hydrophobicity from DPS to TPS brings the NPs in a typical pearl necklace model (Figure 3f), which happens due to the onset of the fibrillation among the hydrophobic domains of capped BSA.5a,6a A close inspection of NPs of all samples indicates the presence of a thin coating of unfolded BSA (see, for example, the block arrows in Figure 3c), which is usually not clearly visible due to low diffraction. In fact, this coating was helpful in determining the Ip of BSA conjugated NPs for different samples in Figure 2b.

As BSA is involved in the reduction of Au(III) into Au(0), and subsequent formation of NPs, the presence of surfactant does not play any role in the synthesis of Au NPs except in the unfolding mechanism of BSA as is evident from Figure 1b and c. For comparison purposes, we synthesized Au NPs under similar reaction conditions in the absence of BSA by employing another cationic surfactant 16-2-16, which acts as a reducing agent just like BSA (see Experimental Section). All present surfactants used in this study cannot be used as reducing agents to synthesize comparable NPs in vitro because of their virtual inability to convert Au(III) into Au(0). In addition, the unfolding behavior of 16-2-16 is considered to be similar to that of 12-0-12. UV−visible absorbance due to SPR of 16-2-16 coated Au NPs is depicted in Figure 3g, and the TEM images are shown in Figure 3h and i. Both images show Au NPs of almost equal dimensions (i.e., 40−50 nm); however, NPs are 8839

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Figure 4. (a) Percentage hemolysis of purified BSA coated Au NPs prepared with different mixtures, BSA+SDS+HAuCl4 (1); BSA+DPS+HAuCl4 (2); BSA+DTAB+HAuCl4 (3); BSA+12−0−12+HAuCl4 (4), with doses of 50 and 100 μg/mL. (b) Percentage hemolysis of purified 16-2-16 coated Au NPs prepared with 16-2-16+HAuCl4 mixtures at 70 °C at fixed HAuCl4 = 0.25 mM and with different concentrations of 16-2-16 = 2 mM (1); 4 mM (2); 8 mM (3); and at HAuCl4 = 1 mM and 16-2-16 = 4 mM (4), with doses of 50 and 100 μg/mL. (c and d) Bright field optical microscopic images of RBCs without and with 16-2-16 coated Au NPs, respectively, of “sample 2” of (b). Empty block arrows in (d) indicate broken cells with released contents. (e) Percentage cell viability of glioma cell lines with different amounts of BSA and 16-2-16 coated NPs. (f and g) Bright field optical microscopic images of glioma cell lines without and with 16-2-16 coated Au NPs, respectively, of “sample 2” of (b). See details in text.

Hemolysis. Hemolysis is a process in which foreign substances can interact and rupture the RBCs cell membrane

more well-defined with clear facets when prepared with 1 mM of HAuCl4 rather than 0.25 mM. 8840

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Scheme 1. Schematic Representation of the Proposed Mechanism of the Synthesis of BSA and 16-2-16 Coated Au NPs and Their Applications toward the Hemolytic and Cytotoxic Responsesa

a

Note that no marked hemolysis and cytotoxicity occurred in the presence of BSA coated Au NPs in contrast to 16-2-16 coated NPs. See details in text.

glycocalyx on the exterior, protein network on the anterior, and lipid bilayer in between the two. Glycoprotein24 and lipid bilayer25 are highly susceptible to surfactant complexation due to the predominant hydrophobic interactions, and thus are responsible for the rupturing of RBCs. Cytotoxicity. MTT assay was performed on glioma cell lines in the presence of BSA and surfactant conjugated NPs separately to investigate the cytotoxic response, and the results are presented in Figure 4e. No cytotoxic effect is observed for different BSA conjugated NPs as no marked difference in the percentage viability of cells is observed between the control and the sample. On the contrary, surfactant capped NPs demonstrate a huge cytotoxic response26 on the viability of glioma cell lines. We specifically selected “sample 2” for the cytoxicity studies because this sample showed maximum hemolysis in Figure 4b. Cytotoxicity depends on the amount of NPs, and viability reduces to 20% with just 50 μg/mL of the sample. Figure 4f and g shows images of cell lines in the absence and presence of “sample 2”, respectively. In the former case, the cells are neatly connected with each other through dendrites and axons and have arranged themselves in cell lines that transmit information by electrical and chemical signaling.27 Signaling occurs through electrical and chemical gated ion channels, which can be switched on and off, and mainly performed by the electrically sensitive proteins. In the presence of NPs of “sample 2” (Figure 4g), bilayers of surfactant capped NPs interact with membrane proteins and disrupt the signaling process with the result that cells start dying.

and release hemoglobin. A similar mechanism is expected if NPs interact with RBCs7 in the bloodstream when they are planned for use in various biological applications that might require their intravenously administered formulations. All samples discussed above were tested for their hemolytic activities, and the results are presented in percentage hemolysis = (sample absorbance − negative control absorbance)/ (positive control absorbance − negative control absorbance) × 100, and illustrated in Figure 4a and b for the BSA and surfactant (i.e., 16-2-16) conjugated NPs, respectively. BSA conjugated NPs of different samples show almost negligible hemolysis in comparison to surfactant conjugated NPs. Lack of marked hemolysis for these samples is primarily related to the presence of albumin proteins along with the RBCs in blood plasma. Because the NPs surface is completely passivated by BSA coating, therefore, the NP surface never comes in contact with the RBCs. BSA coating has no adverse effect on the cell membrane, and hence practically insignificant hemolysis occurs. On the other hand, when NPs are entirely capped and stabilized by the surfactant, a significant hemolysis occurs, which depends on the amount of NPs as well as 16-2-16 (Figure 4b). An increase in the amount of 16-2-16 while keeping the amount of NPs constant increases the hemolysis, while an increase in the amount of NPs at constant 16-2-16 concentration decreases the hemolysis. Figure 4c shows several disk shaped healthy RBCs with intact cell walls in the absence of NPs, whereas in their presence, many cells have broken membranes and released cell contents (Figure 4d). It means that the hemolysis is primarily carried out by the surfactant capped bilayer when it comes in contact with the cell membrane. RBC membrane consists of three layers with 8841

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DISCUSSION The above results help us to understand a simple systematic way of synthesizing bioconjugated Au NPs. The most significant aspect of the study is the extent of BSA unfolding because that is further related to the extent of reduction reaction to generate the required amount of Au NPs with desired shape and size. Maximum unfolding as has been observed in the presence of cationic surfactant is another important aspect to synthesize Au NPs at relatively low temperatures and low concentrations in comparison to that in the presence of anionic or zwitterionic surfactants. Furthermore, properly capped/stabilized NPs have a much longer shelf life of several months for their use in any kind of formulation, which may not be possible for bioconjugated NPs prepared without in vitro synthesis. Therefore, a proper understanding of an unfolding behavior of BSA is considered to be the essential aspect in the synthesis of BSA conjugated Au NPs. Different surfactants used in this work only lead to a different magnitude of unfolded BSA (Scheme 1), and that subsequently affects its reduction potential. DTAB and 12-0-12 have proved to be stronger surfactants than SDS or DPS due to their stronger electrostatic interactions with BSA and hence induce unfolding at relatively much lower temperatures. Despite a marked difference in the Td in the presence of different surfactants especially between DTAB (Td = 55 °C) and SDS (Td = 65 °C), there is a little morphological difference among the NPs produced (Figure 3b−d). Nonparticipation of different surfactant molecules in the overall growth process of the nucleating centers is considered to be the main reason behind this; otherwise, we would have seen a marked difference in the shapes and sizes of NPs of different samples. The formation of ordered morphologies with clear shapes suggests that the growth process of a nucleating center is fully controlled by an effective blocking of the fcc crystal planes by unfolded BSA (Scheme 1);14,28 otherwise, we would have seen polyhedral shapes without any clear geometry. This is all related to the proper unfolding of BSA, which brings hidden hydrophobic domains in contact with the aqueous phase and hence acquires a much needed amphiphilic character for interfacial adsorption at selective low energy crystal planes like {100} or {110} to control the crystal growth. Therefore, random nucleation that could happen at all crystal planes of a nucleating center in the absence of any selective adsorption of an amphiphilic agent like unfolded BSA can very well be controlled in its presence. Thus, a proper coating of NP surface by an unfolded BSA is achieved only through in vitro synthesis where unfolded BSA instantaneously reduces Au(III) into Au(0) to generate the nucleating centers. Their growth is then selectively controlled by the preferential adsorption of amphiphilic BSA, and that leads to well-defined geometries of bioconjugated NPs. In this way, a proper passivation of NP surface by unfolded BSA acts as a shield to prevent the hemolysis, which however is very significant in the case of surfactant capped NPs. Thus, toxicity of NPs can be significantly reduced if their uncoated surfaces are completely passivated by a coating of suitable stabilizing agent such as BSA, which can easily coexist with surrounding medium in the blood plasma. In contrast, if this coating is formed by a surfactant (Scheme 1) with potential cell membrane rupturing ability, such NPs may not be possible to use for different biological applications, which require their intravenous administration. A non cytotoxic response of BSA coated NPs is also considered to be due to its surface

passivation by unfolded BSA. As the present results demonstrate no marked hemolysis, therefore, they can be easily used for different biological applications, which require them as vehicles for drug release. In addition, they can also be tagged with cancer targeting proteins such as lectins, which have specific ability to destroy cancer cells without affecting the healthy cells.31−33 Such a high affinity of unfolded BSA with NP surface can also be viewed in context of nanotoxicology where nanosized metal pollutants could be subject to strong association if they come in contact with serum albumin in the bloodstream. There are numerous ways through which NPs can enter the systemic circulations and most prominent are through skin and respiratory process.29,30 Once they are in the bloodstream, they are expected to have similar interactions with blood proteins due to the high affinity of the latter for NP surface and hence will result in similar serum albumin coated NPs. Thus, the present BSA-conjugated NPs are considered to be the best models for nanopollutants as well.

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CONCLUSIONS Unfolded BSA has been used to synthesize Au NPs in vitro by using different conventional surfactants. Cationic surfactants like DTAB and 12-0-12 unfold the BSA at much lower temperatures than do anionic SDS and zwitterionic DPS. Unfolded BSA reduces Au(III) into Au(0) and ultimately leads to the formation of Au NPs of almost similar shapes and sizes. All NPs are stabilized by BSA coating, while different surfactants only participated in the unfolding process of BSA. BSA coated NPs do not show any hemolytic response and hence can be used as vehicles for drug release in systemic circulation. In contrast, surfactant capped NPs show significant hemolytic as well as cytotoxic responses. In terms of nanotoxicity, the present results conclude that if nanopollutants find their way into the bloodstream, they are expected to be complexed by the serum albumin and hence will not be toxic. This opens up several possibilities for BSA coated NPs to be used in various biomedical applications, which require their intravenous administration.

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ASSOCIATED CONTENT

* Supporting Information S

UV−visible spectra and size distribution histograms. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*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 23.7.1 of the CAS agreement of WLU, Waterloo, and from UGC [ref no.: 34-323/2008(SR)], New Delhi. Dr. Gurinder Kaur 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. TEM facilities of the DST and the SAIF, IIT Bombay, are thankfully acknowledged. 8842

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