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Article http://pubs.acs.org/journal/acsodf
Auric Chloride Induced Micellization on Fractal Patterned Dicationic Amphiphiles and Stabilization of Gold Nanoparticles Thangavel Muthukumarasamyvel,† Ganapathy Rajendran,§ Devendrapandi Santhana Panneer,† Jayapalan Kasthuri,‡ Krishnan Kathiravan,§ and Nagappan Rajendiran*,† †
Department of Polymer Science, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu, India Department of Chemistry, Quaid-E-Millath Government College for Women (Autonomous), Chennai 600002, Tamil Nadu, India § Department of Biotechnology, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu, India ‡
S Supporting Information *
ABSTRACT: The present article reports the development of sunlight-mediated rapid synthesis of bile acid derived dicationic amphiphiles, namely, dicationic cysteamine-conjugated cholic acid (DCaC), dicationic cysteamine-conjugated deoxycholic acid (DCaDC), and dicationic cysteamineconjugated lithocholic acid (DCaLC) by adopting thiol−yne click chemistry approach. The auric chloride (AuHCl4) induced micellization of amphiphiles from fractal pattern to chainlike aggregates was examined by critical micelle concentration measurements, quenching studies, field emission scanning electron microscopy, and optical microscopy techniques. The micelles thus formed act as ideal templates for the stabilization of gold nanoparticles (AuNPs) and exhibit good stability for more than 6 months. The synthesized AuNPs were characterized using UV−visible spectroscopy, highresolution transmission electron microscopy, DLS, zeta potential, and contact angle measurements. These NPs showed high salt tolerance, and the levels were found to be 420, 460, and 580 mM for DCaC-, DCaDC-, and DCaLC-capped AuNPs, respectively.
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INTRODUCTION Self-assembly of hierarchically ordered structures is a fascinating subject, in which small entities into larger two- or threedimensional (3D) structures exhibiting new features that cannot be achieved by the isolated building blocks.1,2 Nature uses a highly ordered self-assembled structure to construct the functional hybrid materials from inorganic and organic building blocks.3,4 The artificial synthesis of hierarchically self-assembled nanoscale building blocks such as nanoclusters, nanowires, nanobelts, and nanotubes is the versatile technique available for the fabrication of functional electronic and photonic nanodevices.5,6 Fractal structures are extremely familiar in nature with different scales of lengths from self-assembled molecules, to the shapes of coastlines, to the distribution of galaxies, and even to the 3D shapes of clouds.7 They are never-ending patterns and are attained by repeating a simple process over and over in an ongoing reaction loop driven by recursion. Investigation of such hierarchically self-assembled fractal patterns in chemical systems provides a unique size, shape, and chemical functionality, which paves way for new opportunities in the design and fabrication of novel functional nanomaterials.8 Self-assembled amphiphilic molecule-templated noble metal nanostructures have attracted considerable research interest over the past 2 decades because of their striking properties and numerous promising applications in the field of biomedical sciences, catalysis, and optoelectronics.9−13 A variety of © 2017 American Chemical Society
amphiphilic molecules such as surfactants, lipids, glycolipids, biological macromolecules, amphiphilic polymers, and dendrimers could self-assemble under appropriate conditions that they have been used as templates for the synthesis of different metal nanoparticles (MNPs).14−17 Among these nanomaterials, gold NPs (AuNPs) have drawn remarkable interest as they exhibit unique size- and shape-dependent catalytic activity, intense surface plasmon resonance (SPR) in the visible region, electrical properties, high dispersibility in aqueous medium, chemical inertness, and biocompatibility.18−20 Because of these various versatile properties, AuNPs have been used as promising key materials for chemical and biological applications, such as drug delivery and therapeutics, sensing, labeling, imaging, and photodynamic therapy.21−23 Keeping the biological perspective in mind, many efforts have been devoted to develop an environmentally benign synthetic approach using a clean, ecofriendly reducing, and capping agent and an environmentally suitable solvent system for the preparation of AuNPs.24,25 Recent studies have shown that the size, shape, stability of the colloids, and surface functionality of NPs play vital roles in their entry and subsequent access of the NPs into living cells.26−28 Received: February 18, 2017 Accepted: June 28, 2017 Published: July 25, 2017 3539
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Scheme 1. Synthesis of Bile Acid-Based Dicationic Amphiphiles under Sunlight Exposure
Figure 1. UV−vis absorption spectra of AuNP solutions obtained by mixing of HAuCl4 (1 × 10−3 M) with different concentrations of DCAmps under sunlight exposure. (i) DCaC: (a) 3.3 × 10−5, (b) 6.6 × 10−5, (c) 1 × 10−4, (d) 1.6 × 10−4, (e) 2.3 × 10−4, (f) 3.3 × 10−4, (g) 8.3 × 10−4, (h) 1.6 × 10−3, (i) 2.0 × 10−3, and (j) 3.3 × 10−3 M; (ii) DCaDC: (a) 3.3 × 10−5, (b) 6.6 × 10−5, (c) 1 × 10−4, (d) 2 × 10−4, (e) 4 × 10−4, (f) 6.6 × 10−4, (g) 8.3 × 10−4, (h) 1.1 × 10−3, and (i) 2.5 × 10−3; and (iii) DCaLC: (a) 1.6 × 10−5, (b) 2.5 × 10−5, (c) 3.3 × 10−5, (d) 6.6 × 10−5, (e) 8.3 × 10−5, (f) 1.0 × 10−4, (g) 2.0 × 10−4, (h) 3.0 × 10−4, (i) 4.0 × 10−4, (j) 5.0 × 10−4, (k) 6.6 × 10−4, and (i) 7.3 × 10−4 M. The insets show the photographs of the corresponding solutions.
upon varying the concentrations ranging from below to above critical micelle concentration (CMC). In below CMC, these DCAmps exhibited self-assembled fractal patterns such as fractal grass and dendritic and fern leaf-like fractals, which were used as effective templates for the stabilization of AuNPs. Hence, in the present investigation, the preliminary studies of auric chloride (HAuCl4) induced aggregation properties of fractal patterns and the stabilization of AuNPs were extensively studied.
Inspired by the interaction of positively charged NPs with biological systems, in the present investigation, for the first time, we have attempted to synthesize cysteamine-conjugated dicationic bile acid based amphiphiles and to stabilize AuNPs using sunlight. Bile acids are naturally available biosurfactants in mammals, which provide an important function in the digestion of fats and lipids. Unlike conventional surfactant molecules, bile acids consist of special amphiphilicity, chirality, rigid steroidal skeleton, biocompatibility, chemically nonequivalent hydroxyl groups, and easily modifiable carboxylic group, making them ideal building blocks for the stabilization of metal NPs.29,30 The cysteamine-conjugated dicationic bile salts were prepared by adopting our previously reported method with a minor modification. 31 The prepared dicationic amphiphiles (DCAmps) showed hierarchically self-assembled patterns
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RESULTS AND DISCUSSION Effect of Substrate Concentration on the AuNP Formation. DCAmps were synthesized via a simple thiol− yne click reaction between propargylated bile acid and cysteamine hydrochloride under sunlight, as shown in Scheme 3540
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Scheme 2. Plausible DCAmp-Mediated Photochemical Reduction Mechanism for AuNP Formation
trapped in the micellar assemblies that are not assessable for the reduction of gold precursor (Figure S1a,b). To optimize the reaction conditions, NP formation was demonstrated at optimized concentration of HAuCl4 (1 × 10−3 M) with dicationic bile salts (1 × 10−4 M for DCaC; 2 × 10−4 M for DCaDC; and 8.3 × 10−5 M for DCaLC) under ambient conditions in the laboratory and under UV light and sunlight exposure, suggesting that the sunlight exposure produced a narrow SPR peak with high intensity (Figure S2) and the prepared NPs were stable at room temperature for more than 6 months without any noticeable color changes. Previously, Dong et al. have proposed the mechanism of photochemical reduction in the synthesis of spherical shaped AuNPs by using poly(ethylene glycol).32 Also, Pienpinijtham et al. have synthesized micrometer-sized gold nanoplates by starchmediated photochemical reduction of AuCl4.33 Herein, we report to explain the interaction of DCAmps with AuCl4− under sunlight irradiation by adapting their mechanism. In aqueous medium, DCAmp and AuCl4− form the [AuCl4−−DCAmp] ionpair complex through electrostatic interactions. Under sunlight irradiation, [AuCl4−−DCAmp] complex is activated and subsequently the Au3+ ion is reduced to Au(0) by gaining an electron from a chloride ion and releasing a chloride radical from the complex, as shown in Scheme 2. Effect of pH on the AuNP Formation. The effect of pH plays a significant role in the formation and stabilization of NPs because of the availability of pH-sensitive functional groups such as hydroxyl (OH) and ammonium chloride (NH3+). These functional groups act as active sites for the reduction of metal salts and the stabilization of NPs. As the pH of the medium was increased from 2.0 to 4.0 for DCaC-stabilized AuNPs, the intensity of the SPR band decreases significantly along with a small shift toward the lower wavelength region and the resulting band becomes narrow. Above this pH, no shift was observed in λmax and the obtained spectra become slightly broadened (Figure S3a). In the case of DCaDC-stabilized NPs, the intensity of the SPR band slightly increases upon increase in the pH from 2.0 to 4.0 and it reached a maximum when the pH of the medium becomes 4.5. Beyond this pH, the intensity of the peak slightly decreases, which could be due to the decrease in the reducing capability of these functional groups on the NP
1, and the synthesized amphiphiles act as capping and stabilizing agents for the preparation of AuNPs. Ultraviolet−visible (UV− vis) absorption spectroscopy is an important and most commonly used technique to ascertain the formation and stability of MNPs. In aqueous medium, these DCAmps exhibit a hierarchical self-assembled structure upon increase in the concentration from below to above CMC. In below CMC, these amphiphiles exhibit different fractal patterns, which could be attributed to the influence of hydrophobic and hydrophilic interactions.31 Figure 1 shows the UV−vis absorption spectra of AuNP formation in the presence of different concentrations of amphiphiles in aqueous medium under sunlight exposure without any additional reducing agent. In all cases, a broad and weak SPR band was observed when the concentration of these amphiphiles becomes very low. Upon increase in the concentration, the intensity of the SPR band increases along with a small blue shift and it attained maximum when the concentration of the template reached below CMC [1 × 10−4 M for dicationic cysteamine-conjugated cholic acid (DCaC); 2 × 10−4 M for dicationic cysteamine-conjugated deoxycholic acid (DCaDC); and 8.3 × 10−5 M for dicationic cysteamineconjugated lithocholic acid (DCaLC)]. However, further increase in the concentration of CMC results in a decrease in the intensity of the SPR band significantly along with a small shift toward longer wavelength region for all systems. The variation seen in the peak position and the intensity of SPR band with respect to the substrate concentration could be due to the influence of hydrophobic and hydrophilic interactions on the formation of different sized NPs. Accordingly, the color of AuNP solution changed, as shown in the inset of Figure 1. Furthermore, in above CMC, except for DCaLC, no SPR band was observed, which can be explained by the changes observed in the characteristic peak intensity of AuCl4− ions in the absorption spectrum (Figure S1). All systems, up to below CMC, show no evidence for the characteristic peak of AuCl4− ions between 200 and 400 nm, which could be attributed to the complete reduction of Au3+ ions to form AuNPs. However, a broad absorption band was clearly observed in this region, in above CMC for DCaC than the DCaDC and DCaLC; this could be due to the formation of stable ion-pair complex between dicationic bile salts and AuCl4− ions, which were effectively 3541
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Figure 2. Stacked UV−vis spectra of AuNP formation at various time intervals using a fixed concentration of 1 × 10−3 M of HAuCl4 and 1 × 10−4 M of DCaC (a); 2 × 10−4 M of DCaDC (b); and 8.3 × 10−5 M of DCaLC (c) and (d) plot of ln a/(1 − a) vs time using the data obtained from Figure S4.
stabilized NPs, respectively, showing that the reaction was completed. To evaluate the relationship between the formation of NPs and the reaction time, the plot of the optical density at 530 nm is presented for these DCAmp−AuCl4− systems (Figure S4). The sigmoid shape of such a kinetic curve suggests that autocatalysis is involved in the AuNP formation (Figure 2d).35 The apparent rate constant of the autocatalytic process (ka) was calculated by plotting ln(a/(1 − a)) versus time, where a = ODt/OD∞ and ODt and OD∞ are the optical densities at times t and ∞, respectively. The resulting plots deviating from the linearity show that the DCAmp−AuCl4− reaction has both noncatalytic and autocatalytic reaction pathways. From the slopes of these plots, the apparent rate constants (ka) were found to be 71.0 × 10−3, 28.0 × 10−3, and 35.0 × 10−3 s−1 for DCaC-, DCaDC-, and DCaLC-stabilized NPs, respectively. Morphological Studies of DCAmp-Templated AuNPs. The morphological changes of the template and the size of the AuNPs were examined by optical microscopy (OPM), field emission scanning electron microscopy (FE-SEM), and transmission electron microscopy (TEM) analyses. In below CMC, DCaC, DCaDC, and DCaLC exhibited fractal grass (1 × 10−4 M), microdendrites (2 × 10−4 M), and fern leaf-like fractal patterns (8.3 × 10−5 M), respectively, through diffusion-limited aggregation, as evidenced from the OPM and SEM images (Figures 3 and S5). After the stabilization of AuNPs, the morphology of the DCaC changed from fractal grass to discrete chain-like structures, in which the spherical shaped NPs were embedded along the chain with an average size of 24 ± 2 nm (Figures 4 and S5). For DCaDC-stabilized AuNPs, the microdendritic morphology changed to closely associated chainlike structure and the formed spherical shaped NPs were embedded along the chain aggregates with an average size of 25
formation (Figure S3b). Similarly, for DCaLC-stabilized NPs, the SPR peak intensity increases as the pH increased from 2.0 to 4.5. Above this, there was a steady decrease in the SPR intensity and the resulting spectra become broadened (Figure S3c). To check the stability of the synthesized NPs, the solutions were kept at room temperature for more than 6 months, in which the DCaC-capped NPs show good stability only at pH 4.5 in comparison to other pHs (inset of Figure S3a). The DCaDCstabilized NPs were highly stable in the range of pH 3.0−4.5 (inset of Figure S3b). In the case of DCaLC-stabilized NPs, the pink color of the solution remains the same only at pH 3.5, and below and above this pH, the color of the solution slightly changed to purple, indicating the formation of aggregates (inset of Figure S3c). In all cases, stable NP dispersions were observed only in the mid acidic pH ranging from 3.5 to 4.5, which could be due to the formation of stable ion-pair adduct, which facilitating the formation of AuNPs. Kinetics of the AuNP Formation. To examine the reaction completion period, the kinetics of NP formation was monitored at various time intervals using optimized concentration of DCAmps in below CMC at mid acidic pH under sunlight exposure (Figure 2). Initially, no characteristic SPR peak was observed. As the reaction proceeded, a weak and broad absorption band at around 550−600 nm appeared and shifted gradually to a constant value of 530 nm with an increased optical intensity. This could be due to the formation of large fluffy particles that appear to be composed of smaller clusters. As the time elapsed, the large agglomerates broke into small-sized particles, giving rise to a gradual increase in the number density of NPs.34 Soon after, there was no significant change in the intensity of the SPR band when the exposure time was extended to 90, 180, and 160 min for DCaC-, DCaDC-, and DCaLC3542
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Figure 3. Microscopic images of DCAmps in the absence and presence of AuNPs. (a) DCaC and (a′) DCaC-AuNPs; (b) DCaDC and (b′) DCaDCAuNPs; and (c) DCaLC and (c′) DCaLC-AuNPs.
± 3 nm (Figures 5 and S5). In the case of DCaLC, the fern leaflike fractal pattern changed to gel-like morphology, wherein the spherical shaped NPs embedded over the gel network and the average size of the NPs was found to be 22 ± 3 nm (Figures 6 and S5). To measure the hydrodynamic radius and charge of the amphiphile-stabilized AuNPs, dynamic light scattering (DLS) and zeta potential experiments were performed, as shown in Figure S6, and the obtained results are presented in Table 2. The difference in the TEM and DLS data is due to the fact that DLS measurements record higher values because the light scatters from both the core particle and the layer on the surface of the NPs. Whereas in TEM measurements, only the metallic particle core is measured. Further, the value obtained from the zeta potential measurements confirms the existence of positive charge on the surface of AuNPs. Recently, we have reported that the charge interactions and hydrophobic and hydrophilic character of amphiphiles are the driving forces for the formation of fractal patterns.31 The disturbance observed in the fractal patterns of these amphiphiles upon the interaction of AuCl4− ions could be due to the aggregation or micellization at lower
concentrations. Besides, these metal ions were reduced by the hydrophilicity of the amphiphiles and were subsequently stabilized by the head-to-head interaction of micelles (Scheme 3), where they follow the chainlike aggregation process driven by the hydrophobicity of the amphiphiles. Fluorescence Measurements of AuNPs. The aggregation behavior of DCAmp-templated AuNPs was studied by a spectrofluorometer using pyrene as a fluorescent probe. The intensity ratio of I3/I1 values of pyrene as a function of the concentrations of DCaC, DCaDC, and DCaLC both in the absence (curve i) and presence (curve ii) of AuNPs showed a sigmoid variation (Figure 7). The point of intersection of the horizontal line and the line of inflation represents the onset of the first and second CMC of amphiphile-templated AuNPs and pure amphiphiles, and the results are listed in Table 1. The significant decrease in the CMC of the amphiphile-templated AuNPs could be related to charge screening,36 where the charge neutralization takes place during the adduct formation between DCAmps and AuCl4− through electrostatic interaction, enabling the aggregation or micellization even at lower concentrations. In 3543
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Figure 4. FESEM (a−c) and the corresponding HRTEM images (d−f) of DCaC-stabilized AuNP solutions at different magnifications.
Figure 5. FESEM (a−c) and the corresponding HRTEM images (d−f) of DCaDC-stabilized AuNP solutions at different magnifications.
I0 = 1 + KSV[CPC] I
recent studies, AuNPs have been used as an efficient quencher in a wider range of wavelengths for many fluorophores in the field of biological and chemical sensors.37−39 To study the protection efficiency of micelle-solubilized pyrene, the quenching experiment was demonstrated at various concentrations of AuNPs. At low concentration of AuNPs, the intensity of fluorescence increases as compared to that of control, indicating that more number of pyrene molecules were solubilized into the AuCl4−induced micelles. Upon further increase in the concentration of AuNPs, the intensity of fluorescence decreases, which could be due to the quenching of pyrene molecules through energytransfer process or alteration of polarity around the pyrene molecules,40 and the obtained data were analyzed using the following Stern−Volmer equation
where I0 and I are the maximum fluorescence intensity ratio of pyrene (I1/I3) in the absence and presence of a quencher (Q), respectively, KSV is the Stern−Volmer constant (M−1), which provides a direct measure of the quenching sensitivity, and [Q] is the quencher concentration. The plots of I0/I versus concentration of NPs show a linearity up to the concentrations of 4.9 × 10−4, 5.0 × 10−4, and 6.1 × 10−4 M; the KSV values were found to be 820, 840, and 810 M−1 for DCaC, DCaDC, and DCaLC, respectively (inset of Figure 8). However, above these concentrations, the plots were nonlinear with significant upward curvature, suggesting that the extent of quenching is large (Figure 8). At higher concentrations, micellization or 3544
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Figure 6. FESEM (a−c) and the corresponding HRTEM images (d−f) of DCaLC-stabilized AuNP solutions at different magnifications.
Table 1. Fluorescence Measurements of AuNPs at 25 °C in Aqueous Medium species
DCaC DCaDC DCaLC
CMC (M)
Normal saline (0.154 M NaCl) solution is commonly used as intravenous drips for patients with danger of developing dehydration or hypervolemia. Hence, it is important to evaluate the salt tolerance of the as-synthesized AuNPs, wherein they were treated with various concentrations of NaCl and their stabilities were monitored by absorption spectroscopy. Upon increase in the concentration of NaCl to 100, 150, and 120 mM for DCaC-, DCaDC-, and DCaLC-stabilized AuNPs, respectively, the intensity of the initial band at around 520 nm slightly decreases without any noticeable color changes, suggesting that these NPs were highly stable up to these concentrations (Figure 9). However, above these concentrations, the intensity of the SPR band slowly decreased with a slight red shift. At the same time, a highly intense band developed between 600 and 700 nm with a gradual shift toward a longer wavelength region, indicating that the aggregation of AuNPs was accompanied by a visual color change, as shown in the insets of Figure 9. The stability of the charged NPs depends on the interaction of the electrostatic repulsive potential and the van der Waals attractive potential.41 In the presence of high concentration of salt, the positively charged gold surfaces are electrostatically screened by Cl− ions, thereby decreasing the electrostatic repulsion, favoring the aggregation of AuNPs, and correspondingly changing the color of the solution from red to blue. The aggregation of NPs was further confirmed by TEM analysis, showing that the spherical shaped particles were closely associated with each other (Figure 9a′−c′). The aggregation (close association) of colloids generally termed “flocculation” can be qualitatively distinguished by color changes of the colloids followed by precipitation. The optimized concentration of NaCl used for the salt-induced aggregation of NaCl-capped AuNPs reported in our earlier report was 400 mM.21 Whereas, in the present study, color change of the DCaC-, DCaDC-, and DCaLC-capped AuNPs was observed at 420, 460, and 580 mM of NaCl concentrations, respectively. Thus, the high salt tolerance of these AuNPs could be used in biological experiments without
quenching constant (K)
in the absence of AuNPs
in the presence of AuNPs
3.5 × 10−3 to 1.0 × 10−2 1.0 × 10−3 to 5.3 × 10−3 3.7 × 10−4 to 7.5 × 10−4
9.8 × 10−4 to 7.1 × 10−3 3.0 × 10−4 to 3.7 × 10−3 2.4 × 10−4 to 5.2 × 10−4
in the presence of AuNPs (M−1 s−1) 245.19 × 104 182.51 × 104 330.04 × 104
Table 2. Size, Charge, and Contact Angle Measurements of Amphiphile-Stabilized AuNPs species
DCaC DCaDC DCaLC
contact angle
AuNPs size (nm)
in the absence of AuNPs (deg)
in the presence of AuNPs (deg)
TEM
DLS
51.8 53.6 58.3
53.6 56.4 61.8
22 ± 0.5 23 ± 0.8 20 ± 3
47.12 44.43 45.58
zeta potential (mV)
+20.30 +31.54 +25.29
aggregation becomes more obvious, where the distance between micelle-solubilized pyrene and AuNPs becomes close enough for energy transfer and/or electron transfer, leading to high degree of quenching.41 Effect of Salt on the AuNP Aggregation. The color change properties and the stability of colloidal dispersions in the presence of an electrolyte are very much sensitive to the degree of aggregation of AuNPs in suspension.21 In biomedical applications, preventing the aggregation of AuNPs is a very challenging task. The media in which the cell grows, the cytoplasm of the cell, and the blood stream are complex aqueous mixtures of nutrients, proteins, electrolytes, and so forth. 3545
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Scheme 3. Schematic Representation of HAuCl4-Induced Hydrophobically Derived Chainlike Aggregate-Stabilized AuNPs
Figure 7. Variation of the I3/I1 ratio of pyrene fluorescence as a function of different concentrations of DCAmps in the absence (curve i) and presence (curve ii) of AuNPs: (a) DCaC, (b) DCaDC, and (c) DCaLC.
any further stabilization. Besides, flocculation parameter (FP) studies can be used to gain semiquantitative information about the nanostructure stability by measuring the integrated
extinction between 600 and 800 nm (I) in the absorbance spectra at various concentrations of NaCl (Figure 9). The obtained FPs were plotted as a function of the concentration of 3546
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Figure 8. Steady-state fluorescence spectra and the corresponding quenching plots of pyrene-solubilized DCaC (a and a′), DCaDC (b and b′), and DCaLC (c and c′) with increased concentrations of AuNPs as a quencher ranging from 2.3 × 10−4 to 8.3 × 10−4 M. The insets show the linear range in the low-quencher-concentration regime.
dendrites, and fern leaf-like fractals for DCaC, DCaDC, and DCaLC, respectively. Upon the interaction with HAuCl4, these fractal patterned amphiphiles change to chainlike aggregates because of micellization, which was examined by CMC plots and quenching studies. Although the NPs were formed in all systems from lower to higher amphiphile concentrations, only the micelles formed from the fractal pattern concentration produced well-stabilized AuNPs in which these patterns changed into discrete chainlike structures, closely associated chainlike structure, and gel-like morphology for DCaC-, DCaDC-, and DCaLC-stabilized AuNPs, respectively. To gain semiquantitative information about the nanostructure stability, FPs were calculated at various concentrations of NaCl and the values were found to be 45.2, 77.9, and 29.8 for DCaC-, DCaDC-, and DCaLC-stabilized AuNPs, respectively. We believe that the cationic AuNPs with various degrees of surface hydrophobicity presented in this paper represent fascinating entities for their
NaCl (Figure 10). The FPs gradually increased as the concentration of salt increased and attained maximum values of 45.2, 77.9, and 29.8 for DCaC-, DCaDC-, and DCaLCstabilized AuNPs, respectively. These findings demonstrate the possibility of regulating the aggregation rate of the AuNPs in the presence of NaCl. Also, the changes in the hydrodynamics radius in the presence of NaCl were measured for DCaC-, DCaDC-, and DCaLC-stabilized AuNPs and were found to be 897, 914, and 1200 nm, respectively. The results further support the formation of aggregation of AuNPs.
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CONCLUSIONS In summary, we have demonstrated a facile and single-step approach of sunlight-mediated thiol−yne click reaction for the preparation of highly water-soluble cysteamine-conjugated bile acid derived DCAmps. In below CMC, these amphiphiles exhibit different fractal patterns such as fractal grass, micro3547
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Figure 9. UV−vis spectra of optimized concentration of (a) 1 × 10−4 M of DCaC, (b) 2 × 10−4 M of DCaDC, and (c) 8.3 × 10−5 M of DCaLCstabilized AuNPs with different concentrations of NaCl ranging from 20 to 600 mM. The TEM images show the corresponding aggregated AuNPs at the highest concentration of NaCl.
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EXPERIMENTAL SECTION
Materials. Cholic acid (CA, 98%), deoxycholic acid (DCA, 98%), lithocholic acid (LCA, 98%), propargyl bromide, pyrene (99.99%), cysteamine·HCl, and 2,2-dimethoxy-2-phenylacetophenone (DMPA) (99.99%) were purchased from SigmaAldrich (India). Potassium bromide (KBr), methanol (MeOH), dimethylformamide (DMF), ethyl acetate (EtoAC), dimethylsulfoxide (DMSO), potassium hydrogen sulfate (KHSO4), sodium sulfate (Na2SO4), cesium carbonate, diethyl ether, and sodium chloride were obtained from Merck (India). All materials and solvents were used as received from the suppliers with no further purification. The glass containers were washed with aqua regia (HCl/HNO3 3:1, v/v) and then rinsed with double-distilled water. All solutions for the CMC measurements were prepared using deionized double-distilled water. Instrumentation and Methods. 1H NMR and 13C NMR spectra were recorded on a Bruker ADVANCE 300 NMR MHZ spectrometer in DMSO-d6 or CDCl3 solution using tetramethylsilane (TMS) as an internal reference, and 13C NMR spectra were recorded at 75 MHz. The Fourier transform infrared (FTIR) measurements were recorded using a PerkinElmer FT-IR
Figure 10. Plot of FP of DCaC- (1 × 10−4 M), DCaDC- (2 × 10−4 M), and DCaLC (8.3 × 10−5 M)-stabilized AuNPs with different concentrations of NaCl.
entry and subsequent access into living cells in view of biomedical applications. 3548
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For optical microscopic studies, the glass slides were initially washed with alcohol and then with double-distilled water and were dried in a hot air oven. The amphiphile-templated AuNPs were dropped onto a glass slide using a micropipette and were allowed for slow evaporation at ambient temperature. The resulting dried drops were imaged on a Leica DM 2500 optical microscope. DLS and Zeta Potential Measurements. The DLS and zeta potential of amphiphile-stabilized AuNPs were measured in aqueous medium using the Malvern Zetasizer Nano ZS system.
spectrometer. Mass spectra were recorded on Bruker Daltonics FlexAnalysis. Synthesis of Propargylated Bile Acid Derivatives. Propargylated bile acids were prepared by adopting previously reported procedure,27,42 and the NMR and FT-IR results were consistent with the reported values. Synthesis of Dicationic Cysteamine-Conjugated Bile Acid. Propargylated bile acid (0.5 g, 0.00116 mol), 5 wt% 2,2′dimethoxy-2-phenylacetophenone (DMPA), 5 mL of MeOH, and finally cysteamine hydrochloride (0.26 g, 0.00228 mol) were added into 50 mL three-neck round-bottom flask containing a magnetic bar. The reaction mixture was purged with N2 for 20 min and then kept under the bright sunlight exposure for 20 min. After completion of reaction, the solvent was concentrated under reduced pressure and subsequently precipitated in diethyl ether three times to obtain a waxy solid, which was further subjected under vacuum to get colored solid compounds (1a, 1b, 1c) with good yield. The compound was stored for further analysis (see the Supporting Information for NMR details S1− S3). Sample Preparation. Stock solution of 3 × 10−5 M aqueous pyrene was prepared by gentle evaporation of pyrene-solubilized MeOH solution, followed by the addition of an appropriate volume of double-distilled water in a volumetric flask, and was sonicated for 1 h. The synthesized hygroscopic dicationic bile salts were lyophilized and used to prepare exact concentration of stock solutions. The stock solutions were prepared by adding appropriate amount of DCaC (0.05 M), DCaDC (0.05 M), and DCaLC (0.005 M) using double-distilled water, and the subsequent dilutions were made from this stock solution to get the desired final concentration. Aliquots of 300 μL of stock solutions of pyrene were added into 4 mL of various concentrations of DCAmp-templated AuNPs to obtain a final concentration of 2.25 × 10−6 M of pyrene to measure the CMC. The solutions were kept for few hours for the encapsulation of pyrene into the microenvironment of amphiphile-templated AuNPs. Owing to their self-assembly behavior in aqueous solution, these DCAmps exhibit strong scattering between 350 and 600 nm, upon excitation at 336 nm, which was nullified by making a blank subtraction file (ACI file) for each solution to examine the salt-induced CMC. Steady-State Fluorescence Measurements. Fluorescence measurements of pyrene were recorded using a FluoroMax 4 (Horiba Jobin Yvon) spectrofluorometer equipped with a Xe-150 W lamp at an excitation wavelength of 336 nm. The emission spectrum was recorded from 350 to 500 nm at a scan rate of 30 nm/s. The slit widths of excitation and emission were 20 and 1.5 nm, respectively. A band-pass filter (10BPF10340) was used to avoid white light from the monochromators. Microscopic Studies. The morphologies of self-assembled amphiphiles before and after the interaction of HAuCl4 were obtained from SEM (Hitachi S-3000N, Japan) and FESEM (Hitachi High Tech SU6600), and high-resolution transmission electron microscopic (HRTEM) images were recorded with a JEOL JEM 2100 microscope equipped with a Gatan imaging filter. Samples for SEM and FESEM imaging were prepared by casting 5−10 μL of amphiphile-templated AuNPs on aluminum foils and then were dried at ambient temperature. The HRTEM analysis was conducted by placing a drop of the NP solution on a carbon-coated copper grid and followed by solvent evaporation under ambient temperature. The average size of the NPs was analyzed using ImageJ software.
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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/acsomega.7b00192. NMR data, UV−vis absorption spectra, kinetic plot of absorbance and wavelength, contact angle and zeta potential measurements, and SEM images (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected] (N.R.). ORCID
Thangavel Muthukumarasamyvel: 0000-0002-5582-3480 Nagappan Rajendiran: 0000-0002-8903-5132 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank the Department of Science and Technology (DST), Government of India, for awarding Inspire fellowship. N.R. thanks DST-EMEQ (NO SB/EMEQ-133/2013) for financial support. The National Centre for Nanoscience and Nanotechnology, University of Madras, is thanked for the FESEM and TEM analyses. The authors thank Dr. D. Mohan, Department of Chemical Engineering, Anna University, for providing contact angle instrument facility.
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REFERENCES
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