Hierarchical Self-Assembly of Bile-Acid-Derived ... - ACS Publications

Research Article www.acsami.org

Hierarchical Self-Assembly of Bile-Acid-Derived Dicationic Amphiphiles and Their Toxicity Assessment on Microbial and Mammalian Systems Thangavel Muthukumarasamyvel,† Raju Baskar,‡ Shanmugam Chandirasekar,† Kesavachandran Umamaheswari,‡ and Nagappan Rajendiran*,† †

Department of Polymer Science, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu, India Department of Biotechnology, University of Madras, Guindy Campus, Chennai 600025, Tamil Nadu, India

‡

S Supporting Information *

ABSTRACT: A thiol-yne click chemistry approach was adopted for the first time to prepare highly water-soluble bile acid derived dicationic amphiphiles. The synthesized amphiphiles dicationic cysteamine conjugated cholic acid (DCaC), dicationic cysteamine conjugated deoxycholic acid (DCaDC), and dicationic cysteamine conjugated lithocholic acid (DCaLC) exhibited hierarchically self-assembled microstructures at various concentrations in an aqueous medium. Interestingly at below critical micellar concentration (CMC) the amphiphiles showed distinct fractal patterns such as fractal grass, microdendrites and fern leaf like fractals for DCaC, DCaDC and DCaLC respectively. The fractal dimension (Df) analysis indicated that the formation of fractal like aggregates is a diffusion limited aggregation (DLA) process. The preliminary aggregation studies such as determination of CMC, fluorescence quenching, wettability and contact angle measurements were elaborately investigated. The morphology of the aggregates were analyzed by SEM and OPM techniques. Further, we demonstrated the antimicrobial and hemolytic activity for the cationic amphiphiles. DCaC had potent antimicrobial activity and showed no toxicity on human RBCs indicating that DCaC could be used in biomedical applications, in addition to their industrial and laboratory applications such as detergency, surface cleaning, and disinfection agent. KEYWORDS: hierarchical self-assembly, fractals, dicationic bile salts, cysteamine hydrochloride, amphiphiles, thiol-yne, wettability, surfactant

■

INTRODUCTION Self-assembly has emerged as a tailorable process for the construction of well-ordered supramolecular structures through bottom-up approach at the different length scales.1 Fabrication of hierarchical structures through self-assembly is a fascinating subject, in which small entities into larger two or threedimensional structures exhibiting new features that cannot be achieved by the isolated building blocks.2,3 The hierarchical selfassembly occurs naturally in all living systems such as proteins, peptides, DNA and lipids where it plays a vital role either to © XXXX American Chemical Society

achieve its biological function or as a part of pathogenic process.4−6 Hence, designing self-assembled hierarchical structures in synthetic materials has become significant research interest in the past decade, and has been evaluated in drug and gene delivery, diagnostics, regenerative medicine, and tissue engineering applications.7,8 The creation of self-assembly in Received: July 1, 2016 Accepted: September 1, 2016

A

DOI: 10.1021/acsami.6b08018 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

hierarchical microstructures, wettability and their biological activity have been investigated in the present study.

aqueous medium depends on their molecular interactions such as hydrogen bonding, hydrophobic and electrostatic interactions under experimental conditions, thereby the switching and responsive properties of self-assembly can be tuned.9,10 There are many reports available on artificial self-assembled systems, such as graft polymers, copolymers, peptide polymers, amino acid derivatives, and various amphiphiles through template free self-assembled approaches.11−13 Bile acids are a class of naturally occurring steroidal biosurfactant containing fused aliphatic tetracyclic rings with one or more hydroxyl groups, connected to a short chiral aliphatic chain with a carboxylic end group.14 Unlike conventional surfactant molecules, bile acids consist of special structure, chirality, rigid steroidal skeleton, biocompatibility with chemically nonequivalent hydroxyl groups and easily modifiable carboxylic group enabling them to function as building blocks for the construction of self-assembled amphiphiles.15,16 Because of these versatile properties and easy availability, several research groups have made significant efforts to synthesize conjugated bile acids and evaluate their applicability in the fields of pharmacology, supramolecular chemistry, and material science.17−23 In view of abovementioned applications, these biosurfactants are directly related to their amphiphilicity, which could be further improved by chemical modification of various hydrophobic and hydrophilic groups either in the facial or side chain through different linkages.24,25 Over the last few decades, researchers have worked on the synthesis and studies of cationic bile acid derivatives owing to their wide range of applications as cancerostatic, cholesterol dissolution agents, gallstone dissolution enhancers, and DNA transfection agents.26−30 Moreover, these derivatives were found to be potent antibacterial agents in biomedical applications.31,32 Many reports are available on the synthesis of cationic bile salts through ether, ester, and amide linkages in both facial and side chain of the bile acids and the level of difficulty varies widely in these syntheses.33−37 In most of the cases, bile salts has been synthesized with a cationic group at the side chain by introducing a bis (amine) through amide conjugation chemistry and converting the second amine to a quaternary ammonium salt.34,38 On the other hand, Maitra and co-workers has reported cationic bile salts without amide linkage at the side chain and studied their aggregation and gelation behavior.39 Recently, Galantini and co-workers has reported the self-assembly of a tryptophan and phenylalanine substituted bile acids.40 The present study is the first report on the synthesis of highly water-soluble cysteamine conjugated dicationic bile salts by adopting thiol−yne click reaction. The advantages of this approach is simple, very fast and cost-effective. Recently, Noponen et al. has reported on the synthesis of cysteamine conjugated bile acids by amidation reaction and their gelation and toxicity studies.41 Cysteamine is a simplest stable aminothiol compound which was biologically derived from cysteine metabolism. It has been used to suppress HIV replication in vitro, used as antioxidants, potent antimalarial drug and in the treatment of neurodegenerative disorders such as Huntington’s disease.42−45 The synthesized cysteamine conjugated dicationic bile acid derivatives can hierarchically assemble into different patterns upon varying the concentration ranging from below to above CMC. To understand the properties of these derivatives, the aggregation behavior,

■

EXPERIMENTAL SECTION

Materials. Cholic acid (CA, 98%), deoxycholic acid (DCA, 98%), lithocholic acid (LCA, 98%), propargyl bromide, pyrene (99.99%), cetylpyridinum chloride (CPC), cysteamine·HCl, and 2,2-dimethoxy2-phenylacetophenone (DMPA) (99.99%) were purchased from Sigma-Aldrich, India. Potassium nitrate (KNO3), potassium bromide (KBr), methanol (MeOH), dimethylformamide (DMF), ethyl acetate (EtoAC), potassium hydrogen sulfate (KHSO4), Sodium sulfate (Na2SO4) were obtained from Merck, India. Cesium carbonate and diethyl ether were obtained from Loba-chemie Ltd., India. All materials and solvents were used as received from the suppliers with no further purification. The glass containers were washed with aquaregia (HCl: HNO3 3:1, v/v) and then rinsed with double distilled water. All solutions for the critical micelle concentration (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 TMS as an internal reference and 13 C NMR were recorded at 75 MHz. The FT-IR measurements were carried out using a PerkinElmer FT-IR spectrometer. Mass spectra were recorded on Bruker Daltonics flex analysis. Synthesis of Propargylated Bile Acid Derivatives. Propargylated bile acids were prepared by adopting previously reported procedure.46 Briefly to a solution of bile acid (1.0 g, 2.45 mM) in anhydrous DMF (6 mL), cesium carbonate (0.87 g, 2.45 mM) was added at room temperature under inert atmosphere and the reaction mixture was stirred for 1 h before adding propargyl bromide (1.48 mL, 17.1 mM). The reaction was monitored by TLC. After completion of reaction, double distilled water (40 mL) was added, and the solution was acidified with 2 M KHSO4. The extraction of oily residue was carried out with 16 mL of ethyl acetate thrice and the organic layer was washed with brine, dried with Na2SO4, filtered and evaporated to dryness under reduced pressure. The propargyl ester was purified by column chromatography on silica-gel using ethyl acetate in hexane (70−100%) as the eluent and separated as white solid. The NMR and FT-IR data’s of the prepared compounds were consistent with the previous literature reports. Synthesis of Dicationic Cysteamine Conjugated Bile Acid. Propargylated bile acid (0.5 g, 1.16 mM), 5% weight 2,2′-dimethoxy-2phenylacetophenone (DMPA), 5 mL of MeOH and finally cysteamine.HCl (0.26g, 2.28 mM) were all charged into 20 mL vial containing a magnetic bar. The reaction mixture was then purged with N2 for 20 min before irradiation with UV-light at 365 nm for 1h. The solvent was then concentrated under pressure and subsequently precipitated in diethyl ether for 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 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 methanol solution, followed by addition of an appropriate volume of double distilled water in a volumetric flask, and sonicated for 1 h. The synthesized dicationic bile salts were hygroscopic and lyophilized salts were used to prepare defined concentration of stock solutions. The stock solutions of dicationic bile salts were prepared by adding appropriate amount of DCaC (50 mM), DCaDC (50 mM), and DCaLC (5.0 mM) using double distilled water and the actual pH of the solutions were found to be 4.3, 4.5, and 4.6, respectively. These dicationic amphiphiles were highly soluble only in acidic pH, and the solution got precipitated when the pH of the medium was increased to neutral and basic. Subsequent dilutions were made from this stock solution to obtain the desired final concentration. Aliquots of 300 μL stock solutions of pyrene were added to 4 mL of dicationic bile salts 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 B


Research Article

ACS Applied Materials & Interfaces Scheme 1. Synthesis of Dicationic Bile-Acid-Based Amphiphiles

Zeta Potential Measurements. The zeta potential of DCaC, DCaDC, and DCaLC were measured at various concentrations in aqueous medium using Malvern zetasizer nano ZS system. The obtained zeta potentials were plotted for different concentration of amphiphiles from below to above CMC. In Vitro Antimicrobial Activity of Dicationic Amphiphiles. The in vitro antimicrobial activities of three dicationic amphiphiles were tested qualitatively by agar well diffusion method and quantitatively by micro broth dilution assay following the guidelines of Clinical Laboratory Standards Institute (CLSI). The standard strains of Enterobacter aerogenes (ATCC 13048), Proteus mirabilis (ATCC 25933), Shigella f lexneri (ATCC 12022), Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Candida albicans (IFM 40009), Candida krusei (IFM 55058), Candida tropicalis (IFM 46521), Candida glabrata (ATCC 2001), and Candida parapsilosis (ATCC 22019) were included in the study for testing the antimicrobial activity. Each dicationic amphiphiles were tested at five different concentrations from below to above CMC, that is, DCaC [26.9 μg/mL (1 mM); 67.3 μg/mL (2.5 mM); 99.7 μg/mL (3.7 mM); 202.1 μg/mL (7.5 mM) and 296.4 μg/mL (11.0 mM)], DCaDC [9.7 μg/mL (0.37 mM); 19.7 μg/mL (0.75 mM); 28.9 μg/mL (1.0 mM); 97.3 μg/mL (3.7 mM) and 131.5 μg/mL (5.0 mM)] and DCaLC [3.0 μg/mL (0.12 mM); 6.4 μg/mL (0.25 mM); 9.4 μg/mL (0.37 mM); 12.8 μg/mL (0.5 mM), and 17.9 μg/mL (0.7 mM)]. The agar well diffusion test was carried out using Mueller Hinton agar and the inoculum suspension of the test organisms was made in sterile saline (0.85%) and the turbidity was adjusted to match 0.5 McFarland standard (0.5−2.5 × 103 CFU/mL). The surface of the agar plate was inoculated with the test organism and well was made using sterile cork borer. A volume of 100 μL of three dicationic amphiphiles at different concentrations were added onto the wells and plates were incubated at 37 °C for 24 h. The zones of inhibition were measured and recorded. In micro broth dilution assay, Mueller Hinton broth and RPMI 1640 were used as a test medium for bacterial and fungal species, respectively. Each well of the sterile disposable microtiter plates (96wells) was loaded with 100 μL of dicationic amphiphiles at different concentrations and 100 μL of inoculum (0.5−2.5 × 103 CFU/mL) and the plates were sealed and incubated at 37 °C for 24 h. The absorbance at 495 nm for fungal species and at 630 nm for bacterial species was recorded in a microtiter plate reader and MIC80 values were calculated. MIC80 is defined as the lowest concentration of the dicationic amphiphiles that inhibited 80% growth of microbial cells in comparison to growth control. Further, the effect of dicationic amphiphiles on bacterial cell morphology was analyzed by SEM. Log−phase culture of Staphylococcus aureus was treated with each dicationic amphiphiles at their MIC concentration and incubated at 37 °C for 18 h. After incubation, the cells were centrifuged and fixed with glutaraldehyde (2.5%) for 1 h at room temperature and washed thrice with phosphate buffer saline. The fixed bacterial cells were then dehydrated in an alcoholic solutions (30%, 50%, 70%, 90% and 100%), dried, gold sputtered and then

pyrene into the microenvironment of the aggregates of dicationic bile salts. Because of their self-assembly behavior in aqueous solution, these dicationic bile salts exhibits strong scattering between 350 and 600 nm, upon excited at 336 nm, which could be nullified by making as a blank subtraction file (ACI file) for each solution to examine the appropriate CMC. In addition, the CMC of dicationic bile salts were also demonstrated by conductometric technique as per standard protocol. The required mole fraction was prepared by mixing precalculated volumes of stock solutions with double distilled water. The conductivity was recorded by successive addition of concentrated stock solution in water. The primary and secondary CMC values were measured at the break points of conductivity versus total surfactant concentration curve. Steady State Fluorescence Measurements. Fluorescence measurements of pyrene was recorded using a Fluoromax 4 (Horiba Jobin Yvon) spectrofluorimeter equipped with a Xe-150 W lamp with an excitation wavelength of 336 nm. The emission spectrum was recorded from 350 to 500 nm at a scan rate of 30 nm/sec. The slit widths of excitation and emission were 20 and 1.5 nm, respectively. Band pass filter (10BPF10-340) was used to avoid white light from the monochromators. Conductivity Measurements. Conductivity values were measured using Metrohm 912 conductometer using conductivity cell with cell constant value of 0.5 cm−1 and the conductance measuring range of 0.1 μS to 500 mS. Scanning Electron Microscopy (SEM). The morphologies of the assemblies were obtained from SEM (Hitachi S-3000N, Japan). Samples for SEM imaging were prepared by casting a 5−10 μL of micellar solution on aluminum foil and then dried at ambient temperature. An ultrathin layer of gold was deposited onto the samples before SEM imaging. Optical Microscopy. For optical microscopic studies, the glass slides were initially washed with alcohol and then with double distilled water, and dried in hot air oven. The dicationic amphiphiles were dropped onto a glass slide using a micropipette and the droplets were allowed for slow evaporation at ambient temperature. The resulting dried drops were imaged on a Leica DM LP 2500 optical microscope. Measurements of Fractal Dimensions. Fractal dimensions was determined from SEM images using the ImageJ image processing program. The box counting algorithm was used to estimate the fractal dimension as previously described.47 Wettability Measurements. For contact angle measurements a good-quality glass plate was selected and diced to specific sizes (25 mm × 25 mm). The glass slide was cleaned using piranha solution (3:1 mixture of H2SO4 and H2O2) and subsequently dried using nitrogen gas. The same protocol was repeated for each surfactants and all the surfactant solutions were prepared freshly before the experiments. Dynamic contact angle measurements (θA, θR) was carried out via the dynamic sessile drop method, using surface tensiometer, Data Physics (DCAT 11EC) equipped with a Hamilton Syringe. Four microliters of liquid droplets was used in each measurements. The average advancing (θA) and receding (θR) contact angles were measured and images were recorded at room temperature. C


Research Article


Figure 1. Changes of I3/I1 ratio of pyrene monomer fluorescence as a function of various concentration of dicationic bile salts. (a) DCaC, (b) DCaDC, and (c) DCaLC, The inset show corresponding emission spectra.

Scheme 2. Schematic Representation of Micelle Formation of Dicationic Bile Salts

In Vitro Hemolysis Assay. The hemolytic activities of dicationic

examined using SEM (Hitachi S-3000N, Japan) at an accelerating voltage of 15 kV.

amphiphiles were determined on Human Red Blood Cells (RBCs). D


Research Article


Figure 2. CMC detection plots from specific conductance (κ) versus various concentration of dicationic bile salts: DCaC (a), DCaDC (b), and DCaLC (c). Fresh human RBCs were isolated from 2 mL of citrated blood by centrifugation at 1000g for 10 min. The cells were washed thrice with phosphate-buffered saline (pH 7.4) to remove plasma and resuspended in 20 mL of PBS. 100 μL of diluted RBCs suspension was treated with equal volume of each dicationic bile salts at various concentrations and incubated at 25 °C for 30 min. After incubation, the mixture was centrifuged at 1000g for 5 min and the absorbance of the supernatant was measured at 540 nm using UV−visible spectrophotometer (Thermo Scientific, Evolution 201, USA). RBCs treated with Triton X-100 (2%) and PBS was used as the positive and negative controls, respectively. Hemolysis was expressed as the level of hemoglobin content (Absorbance at 540 nm) and the percentage of hemolysis of RBCs was calculated using the formula:

Table 1. Critical Micellar Concentration Values of Dicationic Amphiphiles at 25 °C in Aqueous Medium techniques fluorescence

%hemolysis = 100 × (ODsample − ODPBS)/(ODTriton X − ODPBS)

sample no.

compounds

primary CMC (mM)

1 2 3

DCaC DCaDC DCaLC

3.5 1.0 0.37

conductivity

secondary CMC (mM)

primary CMC (mM)

secondary CMC (mM)

10.0 5.1 0.75

3.85 1.5 0.39

11.6 5.1 0.78

study the properties of dicationic surfactant, it is necessary to investigate the aggregation parameters such as critical micellar concentrations (CMC), interaction constant (K) and selfassembling nature in aqueous medium. The CMC for dicationic bile salts were determined by fluorescence technique using pyrene as a probe by measuring the intensity ratio of the third and first (I3/I1) highest vibronic bands in the emission spectra which is sensitive to polarity of the microenvironment.48,49 A very low concentration of pyrene was used in this study, hence only negligible effect on the micellization process was noticed. Figure 1a shows the changes in the I3/I1 ratio of emission spectra plotted against DCaC concentration where the obtained ratio at the initial concentration is about 0.59, which

■

RESULTS AND DISCUSSION Determination of Critical Micellar Concentration (CMC). Dicationic amphiphiles were synthesized via a simple thiol-yne click reaction between propargylated bile acid and cysteamine hydrochloride as shown in Scheme 1. Many reports are available on the synthesis of bile acid based cationic amphiphiles, in which the protocol involves multiple steps, time-consuming and expensive. In the present study, the thiolyne click chemistry was employed to design highly watersoluble dicationic amphiphiles in a single step manner. To E


Research Article


Figure 3. Direct nonlinear Stern−Volmer plots and the corresponding modified plots of various concentrations of quencher for different concentration of dicationic amphiphiles: DCaC (a, b), DCaDC (c, d), and DCaLC (e, f).

is comparable to that of pyrene in pure water. As the concentration is increased to 1.5 mM, no significant changes in the I3/I1 was noted owing to the lack of assembled DCaC, thus showing negligible changes in the polarity of the microenvironment around the pyrene molecules. However, above this concentration the intensity ratio increases remarkably indicating that pyrene molecules are moving into the assembled DCaC. The values of primary and secondary CMC were determined from the extrapolation of the first and second inflection points, which are found to be 3.5 and 10.0 mM, respectively. Similarly, the emission spectra was recorded for DCaDC, where the intensity ratios increased steadily upon

increasing the concentration up to 1.0 mM, demonstrating the transfer of pyrene molecules from the aqueous phase to the hydrophobic environment of primary binding sites (Figure 1b). Above this concentration, the intensity ratio increases gradually with an increase in the concentration of DCaDC. The values of primary and secondary CMC were determined from the extrapolation of the first and second inflection points which are found to be 1.0 and 5.1 mM respectively. In the case of DCaLC, slight change in the intensity ratio was observed below the CMC and then steeply increased on increasing the concentration to second CMC, which could be due to the micellization (Figure 1c). The intersections of the two lines F


Research Article


Schematic representation on the micelle formation of dicationic amphiphiles is shown in Scheme 2. In addition, the CMC values were determined by conductometric method, by measuring the specific conductivity (κ) as a function of total concentration of the surfactant in aqueous medium. The results showed that conductivity increased linearly on increasing the concentration of cationic surfactants with two break points that could be attributed to the primary and secondary CMC of the respective surfactants (Figure 2a−c). The CMC values obtained by this method were found to be comparable with the values obtained in the fluorescence studies which are summarized in Table 1. From the above studies, the CMC values of these surfactants were found to be in this order: DCaC > DCaDC > DCaLC. Fluorescence Quenching Studies. Steady-state fluorescence quenching experiments were performed to understand the interaction of probe molecules in the primary and secondary aggregates of dicationic amphiphiles and their protection efficiency. The quenching studies of pyrene were carried out both in water and in the presence of various concentrations of dicationic amphiphiles as a function of quencher concentration using cetylpyridinium chloride (CPC) as cationic quencher. The quenching data’s were analyzed according to the classical Stern−Volmer equation, which is given below

Table 2. Quenching Constant Values (K) and Fraction of Quencher Assessable Probe (fa) Obtained from the Modified Stern−Volmer Plots of Dicationic Amphiphiles at Various Concentrations sample no.

system

1

DCaC

2

DCaDC

3

DCaLC

concentration (mM) 2.5 3.75 5.0 6.25 0.75 1.0 2.0 3.12 0.25 0.37 0.5 0.62

quenching constant K (M−1 s−1) 14.9 1.65 0.699 0.611 2.42 3.78 2.89 2.56 4.11 1.09 1.68 2.95

× × × × × × × × × × × ×

104 104 104 104 104 104 104 104 104 104 104 104

fa (%) 30.33 73.58 55.96 27.56 46.51 55.00 40.77 57.00 19.12 25.43 22.84 19.57

were taken as the primary and secondary CMC and were found to be 0.37 and 0.75 mM respectively. Compared to the previous report on the synthesis of bile salts and its side chain conjugates, the synthesized dicationic surfactants showed lowest value of CMC which could be due to the enhanced hydrophobicity and dicationic charged head groups and the results were compared as shown in (Table S1). Mysels et al. and Wang et al. also showed that the hydrophobicity and number of charged groups affect the CMC of the bile salts.50,51

I0/I = 1 + KSV[CPC] where I0 and I are fluorescence intensity ratio of pyrene (I1/I3) in the absence and presence of quencher, respectively, and Ksv

Figure 4. SEM micrographs of DCaC at (a) 2.5 (fractal grass), (b) 3.75, (bundled grass), (c) 5.0 (nano flower), and (d) 6.25 mM (aggregated nano flower) surfactant concentrations. Inset shows the contact angle and high magnifications of the corresponding SEM images. G


Research Article


Figure 5. SEM micrographs of DCaDC at (a) 0.75 (microdendrites), (b) 1.0 (defected dendrites), (c) 2.0 (cubic assembles), and (d) 3.12 mM (gellike morphology) surfactant concentrations. Inset shows the contact angle and high magnifications of the corresponding SEM images.

is the Stern−Volmer constant. In the absence of cationic amphiphiles a linear relationship was obtained with the Ksv value of 75.94 M−1. However, the quenching plots obtained in direct SV-plots were nonlinear and exhibited slightly downward curvatures when the concentration of amphiphiles were at below CMC for all the systems (Figure 3a, 3c, and 3e). The curvature becomes more significant with an increase in the concentration of amphiphiles from primary to near secondary CMC, suggests that the existence of two different environment around the pyrene molecules are quenched by the CPC cations with different efficiency. Furthermore, the SV-plot shows linear response at low concentration of quencher which implies that considerable amount of pyrene molecules are quenched by the CPC, whereas at higher quencher concentrations, the quenching efficiency was found to be less significant indicating that the species were more protected from the quencher. Therefore, the modified SV-plot is applied to determine the actual quenching process.

fa =

Here, I0a and I0i are the fluorescence intensities accessible and inaccessible to the quencher, respectively; fa is the fraction of micelle solubilized accessible fluorophore to the CPC and K is the Stern−Volmer quenching constant. The linear plots between I0/(I0 − I) against 1/[CPC] yields fa−1 as an intercept, and ( faK)−1 as a slope. These values were used to calculate the quenching constant (K) and fraction of quencher accessible probe in the bile salt aggregates (fa) which are shown in Table 2. From the results, it was noted that the K values decreases from primary to above CMC for DCaC and DCaDC, which could be attributed to the solubilization of hydrophobic probes in the bile salt aggregates, thereby increasing the protection efficiency with an increasing amphiphiles concentration. In contrast, for DCaLC the K values increases from primary to above CMC and this may be due to the less number of amphiphiles required to form micelles which are unable to repel cationic CPC effectively. Considering the fa values at below CMC, the accessibility becomes less for all the amphiphiles which may be due to the existence of free micelles in which the quencher have a little effect on the micelle solubilized probe. The increase in the concentration of amphiphiles to primary CMC, the fa values becomes high and this could be due to the solubilization of considerable amount of pyrene molecules into the primary micelles which are more accessible to the quencher. However, the fa values decreases significantly except DCaDC (3.12 mM) when the concentrations of amphiphiles reached to the above CMC, this could be due to the different selfassembled nature of secondary micelles that protect the pyrene

I0 = I0a + I0i

I=

I0a {1 + K[CPC]} + I0i

ΔI = I0 − I = I0a·

I0a I0a + I0i

k[CPC] {1 + K[CPC]}

I0 1 1 1 = + · ΔI fa fa ·K [CPC]

Where, H


Research Article


Figure 6. SEM micrographs of DCaLC at (a) 0.25 (fern leaf like fractal morphology), (b) 0.37 (gel-like morphology), (c) 0.5 (intermediate morphology of gel to diamond), and (d) 0.62 mM (damond shape) surfactant concentrations. Inset shows the contact angle and high magnifications of the corresponding SEM images.

counting method and the linear regression were found to be 1.948, 1.914, and 1.936 for DCaC, DCaDC and DCaLC respectively (Figure S1). Upon further increasing the DCaC concentration to primary CMC, the fractal grass pattern transformed into bundled grass morphology in which the grasses were fused together as shown in the Figure 4b. In above primary CMC, spherical shaped self-assembled flower like morphology was obtained which could be attributed to the aggregation of primary micelles through hydrophobic and hydrophilic interactions. In near secondary CMC, the secondary force of interactions could be governing forces leading to the formation of aggregated spherical shapes with matured flower like morphology. The magnification at the edges of spherical aggregates revealed the presence of two different axial orientations of tightly arranged grasses centered with highly matured flower like morphologies (Figure 4d). In case of DCaDC, defected dendritic structures were obtained at primary CMC as shown in Figure 5b, and this could be attributed to the similar charged amphiphiles leading to strong electrostatic repulsion between the aggregates and as a result, the structure of fractal microdendrites dissociated into defected microdendrites. However, at above primary CMC the repulsion becomes more significant in which the defected microdendrites fragmented into the shorter cubic segments and formed as a group of cubic assemblies (Figure 5c). In near secondary CMC, gel like morphology was obtained, this may be the result of the secondary force of interactions between the micelles (Figure 5d). As evidenced from the SEM images (Figure 6b), DCaLC showed gel like morphology even at primary CMC, and it may be due to the enhanced hydrophobicity of DCaLC. This

molecules from the quencher and the repulsive forces existing between cationic micelles and CPC could be another reason. Hence the fa values for different dicationic amphiphiles are as follows: DCaC > DCaDC > DCaLC. Morphological Studies of Dicationic Bile Salts. The OPM and SEM studies revealed that the self-assembled dicationic amphiphiles exhibited fascinating morphological changes in an aqueous medium at different concentrations. In below CMC, these dicationic amphiphiles exist as small aggregates, since different fractal patterns and dimensions were obtained upon drying at ambient temperature that were influenced by hydrophobic nature of the amphiphiles. Here, the small aggregate undergoes a random walk (diffusive motion) attach to the growth of fractal assembly through diffusion limited aggregation (DLA).52 Only few reports are available on the fractal growth mechanism. In particular, Chau and Wang have reported the formation of fractal patterns from a simple self-assembled dipeptide derivative.53 Li and co-workers reported the pH-induced hierarchical assembly using amphiphilic polymer nanotubes as building blocks through template free self-assembled approach.54 Besides, fractal growth mechanism of silk protein nanostructures on two-dimensional surfaces through DLA has been reported by Yadavalli et al.55 In the present study, three different fractal patterns were obtained based on the individual structural features and characteristics as evidenced in SEM images. In below CMC, the morphologies of DCaC, DCaDC, and DCaLC were found to be fractal grass, microdendrites and fern leaf like fractal patterns respectively (Figures 4a, 5a, and 6a). Further, the dimension of the fractal patterns (Df) was calculated by boxI


Research Article


Figure 7. Advancing and receding contact angles of (a) DCaC, (b) DCaDC, and (c) DCaLC solutions on glass surface.

microscopic glass surface. For all the amphiphiles, the contact angle increased with an increase in the surfactant concentration to the above primary CMC as shown in the insets of SEM images (Figure 4−6), showing an increased hydrophobic nature of the amphiphiles (Figure S3), and a slight decrease in the contact angle was noted on further increase in concentration and this could be due to the secondary forces of interaction between the micelles. The optical microscope images (OPM) of hierarchical self-assembled dicationic bile amphiphiles were recorded at selective concentrations and are shown in Figure S4. Effect of Surfactants Concentration on Contact Angle. The wettability studies on hydrophilic solid surfaces in the presence of surfactant solutions has crucial importance in many industrial applications such as detergency, surface cleaning, coating, adhesion, printing, colloidal stability and ore flotation.56 The study of the wetting behavior on glass surface using contact angle measurements is one of the important parameter to determine the surfactant properties of a newly synthesized amphiphiles. The changes in advancing and receding contact angles (θA and θR) of DCaC, DCaDC, and DCaLC with different concentrations are shown in Figure 7a− c. The θA and θR on glass surface in the presence of pure water are 43.68° and 21.16° respectively. Upon increasing the concentration of DCaC, the θA increases gradually (47.55°) up to the primary CMC, after which the increase in θA becomes less significant and attains a maximum value 49.0° at secondary CMC. However, θR increases sharply (37.7°) up to the primary CMC and above which it decreases slightly until a secondary CMC is reached (Figure 7a). In presence of DCaDC, θA increases significantly to a value of 49.05° at primary CMC,

Table 3. Minimum Inhibitory Concentration (MIC) of Dicationic Amphiphiles in μg/mL against Tested Species minimum inhibitory concentration (MIC) (μg/ mL) sample no.

species tested

DCaC

DCaDC

DCaLC

1 2 3 4 5 6 7 8 9 10

E. aerogenes (ATCC 2001) P. mirabilis (ATCC 2001) S. f lexneri (ATCC 2001) S. aureus (ATCC 25923) E. faecalis (ATCC 2001) C. albicans (IFM 40009) C. krusei (IFM 55058) C. tropicalis (IFM 46521) C. glabrata (ATCC 2001) C. parapsilosis (ATCC 22019)

202.1 202.1 202.1 99.7 99.7 99.7 202.1 202.1 202.1 202.1

97.3 97.3 97.3 28.9 28.9 28.9 97.3 97.3 97.3 97.3

>17.9 >17.9 >17.9 >17.9 >17.9 >17.9 >17.9 >17.9 >17.9 >17.9

observation is well supported by the CMC measurements in which the CMC of DCaLC is lower when compared to the other amphiphiles. In the above primary CMC, mixture of gel and diamond shaped morphology were obtained (Figure 6c and d). In order to study the exposure of surface charges on the selfassembled structures, zeta potential measurements were carried out from below to above CMC for all the systems as shown in (Figure S2). The obtained values show that the surface potential plays an important role in the formation of different self-assembled structure with various concentration of amphiphiles. The morphological changes of the hierarchical self-assembled structures were further supported by contact angle measurement, which was carried out on hydrophilic J


Research Article


Figure 8. Scanning electron micrograph of untreated Staphylococcus aureus (a), Staphylococcus aureus treated with DCaC (b), DCaDC (c), and DCaLC (d).

interaction between the micelles, thereby changing the surface properties from hydrophobic to less hydrophilic nature. Hence, the changes in the wettability of dicationic amphiphiles was found to be in this order: DCaC > DCaDC> DCaLC. In Vitro Antimicrobial Activity of Dicationic Bile Salts. The antimicrobial activities of dicationic amphiphiles tested by Agar well diffusion method confirmed that all the tested pathogens were sensitive to DCaC and DCaDC at various concentrations and resistant to DCaLC (Figure S5). The results were further confirmed by MIC studies and the MIC values showed that the dicationic amphiphiles, DCaC and DCaDC exhibited potent antibacterial and antifungal activity against all the tested species while DCaLC showed no activity (Table 3). The Gram-positive bacterial species and C. albicans were found to be more susceptible to DCaC and DCaDC with a MIC value of 99.7 and 28.9 μg/mL while Gram-negative and non albicans Candida species had a MIC value of 202.1 and 97.3 μg/mL respectively. The MIC values noted in the present study suggested the relation between CMC and antimicrobial activity. The CMC and above CMC of DCaC and DCaDC

then it increases slowly (51.64°) up to near secondary CMC and beyond this the value slightly decreases. θR sharply increases (40.85°) up to primary CMC and the values becomes plateau when the concentration of DCaDC is reached to near the secondary CMC, above which it shows a slight decrease (Figure 7b). In the case of DCaLC, the changes in the contact angles were more, θA (54.23°) increases gradually up to primary CMC and then it increases slowly up to secondary CMC, similarly the θR (50. 81°) increases sharply at primary CMC and increases gradually until it reaches the secondary CMC (Figure 7c). For all these cases, the θA and θR values increasing up to primary CMC may be due to the adsorption of cationic surfactants on the negatively charged glass surface through electrostatic interactions, thereby the hydrophobic character increases on the glass surface. The increase in contact angle values becomes less significant on further increase in the concentration may be due to the saturation of adsorbed surfactant on the glass surface. Except for DCaLC, the contact angle values decreased slightly at above secondary CMC and that could be due to the existence of weak secondary forces of K


Research Article


Figure 9. Hemolytic activity of dicationic amphiphiles (a) DCaC, (b) DCaDC, and (c) DCaLC on human RBC and the inset shows the photographs of the corresponding color changes: i and ii are the positive and negative controls, and iii−vii are the concentrations ranging from below to above CMC of each amphiphiles.

resulting in cell death.57 The dicationic amphiphiles DCaC and DCaDC synthesized in the present study, possessed high levels of cationic residues that might have resulted in stronger interaction with bacterial membranes and also the insertion of hydrophobic moieties of the amphiphiles into lipid bilayer of the cell wall could be responsible for cell membrane disruption, thus eventually leading to the cell death. It is well-known that charge density and hydrophobicity exhibits stronger effects on the antibacterial properties in case of cationic amphiphiles. Therefore, the amphiphiles with moderate hydrophobicity should hold great potential for use as promising antibacterial agents in combating the microbial pathogens.58 In Vitro Hemolysis Assay. Hemolysis occurs as a result of the tested compounds incorporation into the membrane and can be used as a measure of these compounds ability to damage the membrane structure and increase its permeability59 The impact of dicationic amphiphiles on human erythrocytes (RBC) at various concentrations ranging from below to above CMC were demonstrated by measuring the release of hemoglobin content and expressing the result as percent of hemolysis. The results showed that among the three dicationic amphiphiles tested, DCaC exhibited concentration dependent hemolysis with lowest activity, whereas immediate onset and variable degrees of hemolysis were observed for both DCaDC and DCaLC and these dicationic amphiphiles were found to be the most potent hemolytic. DCaC showed 4.3% hemolysis, whereas DCaDC and DCaLC exhibited 100% hemolysis at the

formed micelles that might allow the synergistic cooperation of surface charges and hierarchical architecture for the amphiphiles to interact and to exhibit antimicrobial effect on both bacterial and fungal species than the below CMC. SEM analysis was carried out to further understand the structural alterations and cell membrane damage of the microbial cells, when treated with dicationic amphiphiles. The micrograph of untreated and dicationic amphiphiles treated cells of S. aureus is shown in Figure 8. The untreated S. aureus showed characteristics of spherical shaped cells with smooth surface, while DCaC and DCaDC treated cells showed significant alterations in their cell membrane morphology. The external surface of the cells marked by ruptures with pores on the cell membrane showed the loss of cellular integrity. The DCaLC showed no antimicrobial activity in the susceptibility testing and the same was evidenced in SEM analysis as the microbial cells remained intact and unaltered. The DCaLC treated cells did not show any structural alterations, as DCaLC forms micelles at lower concentration and the charges for the microbial interaction could not have been sufficient to damage microbial cell membrane. Both bacterial and fungal cells are composed of large proportion of anionic phospholipids in their cytoplasmic membrane and it is widely believed that the drugs with cationic charges selectively interact with the anionic components of cytoplasmic membrane through electrostatic and hydrophobic interactions that leads to the dissolution of the proton motive force and leakage of essential molecules L


Research Article


Figure 10. Photomicrograph of RBC by light microscopy: (a) Control RBC, (b) RBC treated with DCaC (below CMC), (c) DCaC (CMC), (d) DCaC (above CMC), (e) DCaDC (below CMC), (f) DCaDC (CMC), (g) DCaDC (above CMC), (h) DCaLC (below CMC), (i) DCaLC (CMC), and (j) DCaLC (above CMC).

bile acids are able to pass through the membrane and interact with DNA and intracellular molecules60−63 Since, the DCaC was less hydrophobic in nature and had no membrane permeable potency, while the more hydrophobic DCaDC and DCaLC showed higher membrane perturbing activities.

CMC and above CMC values (Figure 9). These results are in good agreement with the earlier report by Beata et al.59 The light microscopic observations of RBCs treated with dicationic amphiphiles further supported the results of hemolysis assay (Figure 10). Hydrophobicity has long been considered as a marker of the biological activity of bile acids.59 The mammalian cell membrane exhibits neutral charges while the bacterial cell membrane is negatively charged.58 It is hypothesized that bile acids with increased hydrophobicity have a greater capacity to perturb the structure of or partly digest, the cell membranes. In addition, it has also been postulated that highly hydrophobic

■

CONCLUSION In conclusion, a simple and efficient strategies for the preparation of highly water-soluble cysteamine conjugated bile acid based dicationic amphiphiles in a single step manner by adopting thiol−yne click reaction. The CMCs of these M



■

amphiphiles were determined by both fluorescence and conductometric methods, and the obtained results were comparable and followed the order: DCaC > DCaDC > DCaLC. The SEM and OPM techniques revealed the amphiphiles exhibiting fascinating hierarchically self-assembled microstructures at various concentrations ranging from below to above CMC and the morphological changes were further supported by contact angle measurements. Moreover in below CMC each amphiphiles showed distinct fractal patterns such as fractal grass, microdendrites and fern leaf like fractals. The dimension of the fractal patterns (Df) were calculated by using box-counting method and the linear regression were found to be 1.948, 1.914, and 1.936 for DCaC, DCaDC, and DCaLC, respectively. The modified SV-plots provides quenching constant and fraction of quencher accessible probe in bile salt aggregates which could be correlated with the hydrophobicity of hierarchically self-assembled amphiphiles. The wettability of these amphiphiles were measured by taking advancing and receding contact angles showing that DCaC had good wettability when compared to DCaDC and DCaLC. Further, we demonstrated the antimicrobial and hemolytic activity of these cationic amphiphiles. The DCaC and DCaDC exhibited potent antibacterial and antifungal activity against all the tested species whereas, no activity was observed for DCaLC and these results were in comparable to SEM analysis. SEM studies showed changes in the cell membrane integrity of the microbial cells treated with the amphiphiles. The hemolysis assay of DCaC, failed to lyse the human erythrocytes, due to less hydrophobic nature compared to DCaDC and DCaLC exhibiting more hydrophobicity that were responsible for higher membrane perturbing activities. The results of the present study showed that, DCaC could be used in biomedical applications in addition to their industrial and laboratory applications such as detergency, surface cleaning, and disinfection agent.

■

REFERENCES

(1) Kaplan, D. L.; Fossey, S.; Viney, C.; Muller, W. Self-Organization (Assembly) in Biosynthesis of Silk Fibers−A Hierarchical Broblem. MRS Online Proc. Libr. 1991, 255, 19−29. (2) Ikkala, O.; ten Brinke, G. Hierarchical Self-Assembly in Polymeric Complexes: Towards Functional Materials. Chem. Commun. 2004, 2131−2137. (3) Sun, G.; Chu, C.-C. Self-Assembly of Chemically Engineered Hydrophilic Dextran into Microscopic Tubules. ACS Nano 2009, 3, 1176−1182. (4) Koley, P.; Pramanik, A. Nanostructures from Single Amino AcidBased Molecules: Stability, Fibrillation, Encapsulation, and Fabrication of Silver Nanoparticles. Adv. Funct. Mater. 2011, 21, 4126−4136. (5) Baccile, N.; Nassif, N.; Malfatti, L.; VanBogaert, I. N. A.; Soetaert, W.; Pehau-Arnaudet, G.; Babonneau, F. Sophorolipids: A YeastDerived Glycolipid as Greener Structure Directing Agents for SelfAssembled Nanomaterials. Green Chem. 2010, 12, 1564−1567. (6) Goodman, R. P.; Schaap, I. A. T.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J. Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication. Science 2005, 310, 1661−1665. (7) Barnard, A.; Posocco, P.; Pricl, S.; Calderon, M.; Haag, R.; Hwang, M. E.; Shum, V. W. T.; Pack, D. W.; Smith, D. K. Degradable Self-Assembling Dendrons for Gene Delivery: Experimental and Theoretical Insights into the Barriers to Cellular Uptake. J. Am. Chem. Soc. 2011, 133, 20288−20300. (8) Lee, K. Y.; Mooney, D. J. Hydrogels for Tissue Engineering. Chem. Rev. 2001, 101, 1869−1880. (9) Zhang, S. Fabrication of Novel Biomaterials Through Molecular Self-Assembly. Nat. Biotechnol. 2003, 21, 1171−1178. (10) Zhao, X.; Pan, F.; Xu, H.; Yaseen, M.; Shan, H.; Hauser, C. A. E.; Zhang, S.; Lu, J. R. Molecular Self-Assembly and Applications of Designer Peptide Amphiphiles. Chem. Soc. Rev. 2010, 39, 3480−3498. (11) Oh, J. K. Polylactide (PLA)-Based Amphiphilic Block Copolymers: Synthesis, Self-Assembly, and Biomedical Applications. Soft Matter 2011, 7, 5096−5108. (12) Honglawan, A.; Yang, S. Evaporative Assembly of Ordered Microporous Films and Their Hierarchical Structures From Amphiphilic Random Copolymers. Soft Matter 2012, 8, 11897−11904. (13) Jin, H.-E.; Jang, J.; Chung, J.; Lee, H.-J.; Wang, E.; Lee, S.-W.; Chung, W.-J. Biomimetic Self-Templated Hierarchical Structures of Collagen-Like Peptide Amphiphiles. Nano Lett. 2015, 15, 7138−7145. (14) Small, D. M. The Physical Chemistry of Cholanic Acids. In The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Publishing Corp.: New York, 1971; Vol. 1, pp 249−356. (15) Mukhopadhyay, S.; Maitra, U. Chemistry and Biology of Bile Acids. Curr. Sci. 2004, 87, 1666−1683. (16) Jia, Y.-G.; Zhu, X. X. Thermo- and pH-Responsive Copolymers Bearing Cholic Acid and Oligo(ethylene glycol) Pendants: SelfAssembly and pH-Controlled Release. ACS Appl. Mater. Interfaces 2015, 7, 24649−24655. (17) Virtanen, E.; Kolehmainen, E. Use of Bile Acids in Pharmacological and Supramolecular Applications. Eur. J. Org. Chem. 2004, 2004, 3385−3399. (18) Berlati, F.; Ceschel, G.; Clerici, C.; Pellicciari, R.; Roda, A.; Ronchi, C. The Use of Bile Acids as Antiviral Agents. WO 9400126, 1994. (19) Campazzi, E.; Cattabriga, M.; Marvelli, L.; Marchi, A.; Rossi, R.; Pieragnoli, M. R.; Fogagnolo, M. Organometallic Radiopharmaceuticals: Rhenium(I) Carbonyl Complexes of Natural Bile Acids and Derivatives. Inorg. Chim. Acta 1999, 286, 46−54. (20) Swaan, P. W.; Hillgren, K. M.; Szoka, F. C., Jr.; Øie, S. Enhanced Transepithelial Transport of Peptides by Conjugation to Cholic Acid. Bioconjugate Chem. 1997, 8, 520−525. (21) Sliedregt, L. A. J. M.; Rensen, P. C. N.; Rump, E. T.; van Santbrink, P. J.; Bijsterbosch, M. K.; Valentijn, A. R. P. M.; van der Marel, G. A.; van Boom, J. H.; van Berkel, T. J. C.; Biessen, E. A. L. Design and Synthesis of Novel Amphiphilic Dendritic Galactosides for

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b08018. NMR data, comparison table, calculation of fractal dimension, contact angle, zeta potential, OPM, and images of agar well diffusion plate (PDF)

■

Research Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS 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 SEM analysis. We thank Dr. D. Mohan, Department of Chemical Engineering, Anna University for providing contact angle instrument facility. N


Research Article


(39) Sangeetha, N. M.; Balasubramanian, R.; Maitra, U.; Ghosh, S.; Raju, A. R. Novel Cationic and Neutral Analogues of Bile Acids: Synthesis and Preliminary Study of Their Aggregation Properties. Langmuir 2002, 18, 7154−7157. (40) Travaglini, L.; D'Annibale, A.; Di Gregorio, M. C.; Schillén, K.; Olsson, U.; Sennato, S.; Pavel, N. V.; Galantini, L. Between Peptides and Bile Acids: Self-Assembly of Phenylalanine Substituted Cholic Acids. J. Phys. Chem. B 2013, 117, 9248−9257. (41) Noponen, V.; Belt, H.; Lahtinen, M.; Valkonen, A.; Salo, H.; Ulrichová, J.; Galandáková, A.; Sievänen, E. Bile Acid−Cysteamine Conjugates: Structural Properties, Gelation, and Toxicity Evaluation. Steroids 2012, 77, 193−203. (42) Charrier, C.; Rodger, C.; Robertson, J.; Kowalczuk, A.; Shand, N.; Fraser-Pitt, D. F.; Mercer, D.; O'Neil, D. Cysteamine (Lynovex) A Novel Mucoactive Antimicrobial and Antibiofilm Agent for the Treatment of Cystic Fibrosis. Orphanet J. Rare Dis 2014, 9, 189. (43) Anderson, R. A.; Feathergill, J.R. K.; Kirkpatrick, R.; Zaneveld, L. J. D.; Coleman, K. T.; Spear, P. G.; Cooper, M. D.; Waller, D. P.; Thoene, J. G. Characterization of Cysteamine as a Potential Contraceptive Anti-HIV Agent. J. Androl. 1998, 19, 37−49. (44) Omran, Z.; Kay, G.; Hector, E. E.; Knott, R. M.; Cairns, D. Folate Pro-Drug of Cystamine as an Enhanced Treatment For Nephropathic Cystinosis. Bioorg. Med. Chem. Lett. 2011, 21, 2502− 2504. (45) Sun, L.; Xu, S.; Zhou, M.; Wang, C.; Wu, Y.; Chan, P. Effects of Cysteamine on MPTP-Induced Dopaminergic Neurodegeneration in Mice. Brain Res. 2010, 1335, 74−82. (46) Nonappa; Maitra, U. Simple Esters of Cholic Acid as Potent Organogelators: Direct Imaging of the Collapse of SAFINs. Soft Matter 2007, 3, 1428−1433. (47) Wang, W.; Chau, Y. Self-Assembled Peptide Nanorods as Building Blocks of Fractal Patterns. Soft Matter 2009, 5, 4893−4898. (48) Kalyanasundaram, K.; Thomas, J. K. Environmental Effects on Vibronic Band Intensities in Pyrene Monomer Fluorescence and Their Application in Studies of Micellar Systems. J. Am. Chem. Soc. 1977, 99, 2039−2044. (49) Hashimoto, S.; Thomas, J. K. Photophysical Studies of Pyrene in Micellar Sodium Taurocholate at High Salt Concentrations. J. Colloid Interface Sci. 1984, 102, 152−163. (50) Rodas, A.; Hofmann, A. F.; Mysels, K. J. The Influence of Bile Salt Structure on Self-Association in Aqueous Solutions. J. Biol. Chem. 1983, 258, 6362−6370. (51) Ye, W.; Li, Y.; Zhou, Z.; Wang, X.; Yao, J.; Liu, J.; Wang, C. Synthesis and Antibacterial Activity of New Long-Chain-Alkyl Bile Acid-Based Amphiphiles. Bioorg. Chem. 2013, 51, 1−7. (52) Niinivaara, E.; Kontturi, E. 2D Dendritic Fractal Patterns From an Amphiphilic Polysaccharide. Soft Matter 2014, 10, 1801−1805. (53) Wang, W.; Chau, Y. Self-Assembled Peptide Nanorods as Building Blocks of Fractal Patterns. Soft Matter 2009, 5, 4893−4898. (54) Lee, C. H.; Li, P. pH-Induced Formation of Various Hierarchical Structures From Amphiphilic Core−Shell Nanotubes. RSC Adv. 2012, 2, 1303−1306. (55) Kurland, N. E.; Kundu, J.; Pal, S.; Kundu, S. C.; Yadavalli, V. K. Self-Assembly Mechanisms of Silk Protein Nanostructures on TwoDimensional Surfaces. Soft Matter 2012, 8, 4952−4959. (56) Ghosh Chaudhuri, R.; Paria, S. Effect of Electrolytes on Wettability of Glass Surface Using Anionic and Cationic Surfactant Solutions. J. Colloid Interface Sci. 2014, 413, 24−30. (57) Friedrich, C. L.; Moyles, D.; Beveridge, T. J.; Hancock, R. E. W. Antibacterial Action of Structurally Diverse Cationic Peptides on Gram-Positive Bacteria. Antimicrob. Agents Chemother. 2000, 44 (8), 2086−2092. (58) Wang, H.; Shi, X.; Yu, D.; Zhang, J.; Yang, G.; Cui, Y.; Sun, K.; Wang, J.; Yan, H. Antibacterial Activity of Geminized Amphiphilic Cationic Homopolymers. Langmuir 2015, 31, 13469−13477. (59) Jasiewicz, B.; Mrówczyńska, L.; Malczewska-Jaskóła, K. Synthesis and Haemolytic Activity of Novel Salts Made of Nicotine Alkaloids and Bile Acids. Bioorg. Med. Chem. Lett. 2014, 24, 1104− 1107.

Selective Targeting of Liposomes to the Hepatic Asialoglycoprotein Receptor. J. Med. Chem. 1999, 42, 609−618. (22) Davis, A. P.; Menzer, S.; Walsh, J. J.; Williams, D. J. SteroidBased Receptors with Tunable Cavities; A Series of Polyhydroxylated Macrocycles of Varying Size and Flexibility. Chem. Commun. 1996, 453−455. (23) Annadhasan, M.; Sankar Babu, V. R.; Naresh, R.; Umamaheswari, K.; Rajendiran, N. A Sunlight-Induced Rapid Synthesis of Silver Nanoparticles Using Sodium Salt of N-Cholyl Amino Acids and Its Antimicrobial Applications. Colloids Surf., B 2012, 96, 14−21. (24) Zhao, Y. Facial Amphiphiles in Molecular Recognition: From Unusual Aggregates to Solvophobically Driven Foldamers. Curr. Opin. Colloid Interface Sci. 2007, 12, 92−97. (25) Taotafa, U.; McMullin, D. B.; Lee, S. C.; Hansen, L. D.; Savage, P. B. Anionic Facial Amphiphiles From Cholic Acid. Org. Lett. 2000, 2, 4117−4120. (26) Nguyen, T. T. H.; Protiva, J.; Klinotova, E.; Urban, J.; Protiva, M. Synthesis of 24-(Piperidin-1-yl, Morpholin-4-yl and 4-Methylpiperazin-1-yl)-5β-Cholan-3α-ols and Four Hydroxylated 23-(4, 5Dihydroimidazol-2-yl)-24-nor-5β-cholanes. Collect. Czech. Chem. Commun. 1997, 62, 471−478. (27) Fears, R.; Brown, R.; Ferres, H.; Grenier, F.; Tyrrell, A. W. R. Effects of Novel Bile Salts on Cholesterol Metabolism in Rats and Guinea-Pigs. Biochem. Pharmacol. 1990, 40, 2029−2037. (28) Kwan, K. H.; Higuchi, W. I.; Molokhia, A. M.; Hofmann, A. F. Cholesterol Gallstone Dissolution Rate Accelerators I: Exploratory Investigations. J. Pharm. Sci. 1977, 66, 1105−1108. (29) Reid, D. G.; Gajjar, K.; Robinson, S. P.; Hickey, D. M. B.; Benson, G. M.; Haynes, C.; Leeson, P. D.; Whittaker, C. M. Precipitation and 13C-NMR Relaxation Enhancement Measurements of the Interactions of Bile Acids With Synthetic Cationic Bile Acid Derivatives, and With Spin Labelled Fatty Acids. Chem. Phys. Lipids 1991, 60, 143−151. (30) Borgström, B. The Action of Bile Salts and Other Detergents on Pancreatic Lipase and the Interaction with Colipase. Biochim. Biophys. Acta, Lipids Lipid Metab. 1977, 488, 381−391. (31) Bernheim, F.; Lack, L. The Effect of Quaternary Ammonium Derivatives of Bile Acids on the Rate of Swelling of Pseudomonas Aeruginosa in Solutions of Sodium and Potassium Salts. Can. J. Microbiol. 1971, 17, 323−7. (32) Lai, X.-Z.; Feng, Y.; Pollard, J.; Chin, J. N.; Rybak, M. J.; Bucki, R.; Epand, R. F.; Epand, R. M.; Savage, P. B. Ceragenins: Cholic AcidBased Mimics of Antimicrobial Peptides. Acc. Chem. Res. 2008, 41, 1233−1240. (33) Bhat, S.; Leikin-Gobbi, D.; Konikoff, F. M.; Maitra, U. Use of Novel Cationic Bile Salts in Cholesterol Crystallization and Solubilization In Vitro. Biochim. Biophys. Acta, Gen. Subj. 2006, 1760, 1489−1496. (34) Araki, Y.-I.; Lee, S.; Sugihara, G.; Furuichi, M.; Yamashita, S.; Ohseto, F. New Cationic Surfactants Derived From Bile Acids: Synthesis and Thermodynamic and Biophysicochemical Properties Such as Membrane Perturbation and Protein Solubilizing Abilities. Colloids Surf., B 1996, 8, 81−92. (35) Willemen, H. M.; Marcelis, A. T. M.; Sudhölter, E. J. R. Thermodynamics of Micellization of Cholic Acid Based Facial Amphiphiles Carrying Three Permanent Ionic Head Groups. Langmuir 2003, 19, 2588−2591. (36) Zhong, Z.; Yan, J.; Zhao, Y. Cholic Acid-Derived Facial Amphiphiles with Different Ionic Characteristics. Langmuir 2005, 21, 6235−6239. (37) Ye, W.; Li, Y.; Zhou, Z.; Wang, X.; Yao, J.; Liu, J.; Wang, C. Synthesis and antibacterial activity of new long-chain-alkyl bile acidbased amphiphiles. Bioorg. Chem. 2013, 51, 1−7. (38) Nguyen, T. H.; Protiva, J.; Klinotova, E.; Urban, J.; Protiva, M. Synthesis of 24-(Piperidin-1-yl, Morpholin-4-yl and 4-Methylpiperazin-1-yl)-5β-cholan-3α-ols and Four Hydroxylated 23-(4,5-Dihydroimidazol-2-yl)-24-nor-5β-cholanes. Collect. Czech. Chem. Commun. 1997, 62, 471−478. O


Research Article

ACS Applied Materials & Interfaces (60) Sagawa, H.; Tazuma, S.; Kajiyama, G. Protection Against Hydrophobic Bile Salt-Induced Cell Membrane Damage by Liposomes and Hydrophilic Bile Salts. Am. J. Physiol. 1993, 264, G835−G839. (61) Vyvoda, O. S.; Coleman, R.; Holdsworth, G. Effects of Different Bile Salts upon the Composition and Morphology of a Liver Plasma Membrane Preparation. Deoxycholate is More Membrane Damaging than Cholate and Its Conjugates. Biochim. Biophys. Acta, Biomembr. 1977, 465, 68−76. (62) Coleman, R.; Iqbal, S.; Godfrey, P. P.; Billington, D. Membranes and Bile Formation. Composition of Several Mammalian Biles and Their Membrane-Damaging Properties. Biochem. J. 1979, 178, 201− 208. (63) Aldini, R.; Roda, A.; Montagnani, M.; Roda, E. Bile Acid Structure and Intestinal Absorption in the Animal Model. Ital. J. Gastroenterol. 1995, 27, 141−144.

P


Hierarchical Self-Assembly of Bile-Acid-Derived ... - ACS Publications

Recommend Documents