Solvent Induced Morphological Evolution of Cholesterol Based

Silvia Bartocci , José Augusto Berrocal , Paola Guarracino , Maxime Grillaud , Lorenzo Franco , Miriam Mba. Chemistry - A European Journal 2018 24 (1...
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Solvent Induced Morphological Evolution of Cholesterol Based Glucose Tailored Amphiphiles: Transformation from Vesicles to Nanoribbons Deep Mandal, Soumik Dinda, Pritam Choudhury, and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata − 700 032, India S Supporting Information *

ABSTRACT: Supramolecular self-assembly of low molecular mass amphiphiles is of topical interest with the urge to achieve precise control over the formation of various self-aggregated structures. Particularly, fabrication of multifarious nanostructures from single molecular backbone would be highly advantageous for task specific applications of the selfaggregates. To this end, the present study reports the solvent triggered evolution of hierarchical self-assembled structures of cholesterol based glucose appended amphiphiles and the pathway of structural transition. The amphiphiles formed bilayered vesicles in water and gels in different organic solvents. In DMSO−water solvent mixture, it showed gradual transition in the morphology of self-aggregates from vesicle-to-fiber and intermediate morphologies depending on the solvent compositions. Microscopic and spectroscopic investigations showed that morphological transformation took place through fusion, elongation and twisting of self-aggregates owing to the reorganization of the amphiphiles (H-type to J-type) in varied solvent polarity. Moreover, sheetlike molecular organization originating from hydrogen bonding and solvophobic interaction played a vital role in the formation of nanoribbons that led to the formation of gel fibril network. This study endows a new strategy to develop solvent induced multistructured self-aggregates from a single molecular scaffold, unraveling the route of forming hierarchical self-assembly. micelle to fiber transformation.14 In most cases, external stimuli like light, heat, sound, enzyme have been used as driving force for the supramolecular structural transformation.15−20 Rajamalli et al. had reported naphthalene-containing poly(aryl ether) based dendrons that formed diverse self-assemblies (vesicles, sheet, elongated fibers, etc.) in different polar and nonpolar solvents.3 To this end, Meijer and co-workers reported the kinetic modeling of the multifarious self-assemblies (discotic, helical, columnar, one-dimensional supramolecular polymers) generated by benzene-1,3,5-tricarboxamide based derivatives.21,22 They also showed solvent dependent coil-to-globule transition of single-chain polymeric nanoparticles particularly the influence of cosolvent on the molecular folding. However, reports on solvent triggered stepwise morphological transition through direct visualization of intermediate topologies with proper mechanistic view is really scarce. Herein, we aim to develop smart amphiphiles that have the ability to form solvent induced multifaceted supramolecular nanostructures from single molecular backbone and to study the mechanism of their structural transformation. The present study describes the synthesis of cholesterol based glucose tailored amphiphiles that formed vesicular self-assembly in

1. INTRODUCTION The inspiration to study the supramolecular self-association of low molecular mass amphiphiles is gaining interest in particular to achieve precise control over the formation of diverse welldefined nanoarchitectures. Vesicles, liposomes, micelles, low molecular weight gels, and other noncovalent aggregates are beautiful manifestations of supramolecular self-assembly that are of topical interest across the scientific disciplines.1−3 These supramolecular aggregates are finding notable applications in different research domain including material science, chemical science, biomedicine, biocatalysis, electronic devices, and others.4−8 In many instances, it was perceived that task specific applications of self-assemblies depend on their morphological identities. 9−11 Thus, tuning the morphology of these nanostructures is of great demand, and in particular, fabrication of multistructured self-assemblies from single molecular backbone is highly important. Stimuli sensitive structural transition of supramolecular self-assemblies from single molecular backbone is finding notable importance for developing multishaped nanoarchitectures. To this end, Yao and co-workers had reported ultrasound and heat induced reversible transformations of vesicles and nanofibers.12 Similarly, Kim and co-workers had designed photoresponsive dendritic vesicular building blocks that undergo a structural transformation upon UV irradiation.13 Ulijn and co-workers had also designed phenylacetyl-peptide based amphiphiles, which exhibited enzyme (MMP-9) triggered © XXXX American Chemical Society

Received: June 9, 2016 Revised: August 10, 2016

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Langmuir water and gradually transformed to fibrillar nanoribbon network in DMSO (dimethyl sulfoxide)−water (4:1 v/v) binary solvent mixture. We have chosen cholesteryl unit in the amphiphilic backbone because of its ability to assist self-organization process through hydrophobic interactions in aqueous milieu. In addition, cholesterol is one of the most ubiquitous natural components that were exploited in nanoscience, to influence self-assembly process while eliciting specific biological responses.23,24 The amphiphiles also exhibited gelation ability in different organic solvents. The influence of noncovalent forces in the morphological transition including the various intermediate morphologies formed during the solvent induced vesicle-to-nanoribbon transformation was investigated by microscopic and spectroscopic techniques. This structural transition of self-aggregates is solely influenced by the solvent compositions and not by the amphiphile concentration or aging of the material. The reorganization of amphiphiles from plane-to-plane stacked H-aggregation to head-to-tail fashioned J-aggregation initiated vesicle-to-nanoribbon transition. Moreover, sheetlike molecular arrangement was found to be responsible for the twisted nanoribbon structure that led to the formation of a gel fibril network. The present work delineates a facile approach to develop solvent induced vesicle to fiber transformation of cholesterol based glucose functionalized amphiphiles which offers direct visualization of intermediate topologies.

and then worked up with water and brine to remove excess base and diamine. The dichloromethane part was vacuum evaporated and the product obtained was purified using column chromatography 100−200 mesh silica gel and methanol-chloroform (7% v/v) as the eluent. The cholesteryl coupled amine was then refluxed with δ-D(+)glucono-1,5-lactone in dry methanol for 16 h. The product obtained was further column purified using methanol−chloroform (10% v/v) as the eluent. The product obtained was characterized thoroughly by HRMS and 1H NMR spectra. 1 H NMR of Amphiphile-1. Amphiphile-1 (500 MHz, methanol-d4, 25 °C): δ/ppm 0.78−1.67 (m, 39H, cholesteryl), 1.85−1.95 and 2.36−2.45 (m, 4H, allylic cholesteryl protons), 2.04−2.15 (m, 5H, glucose-OH), 3.25−3.81 (m, 12H, oxyethelene proton), 3.50−3.59 (m, 3H, C-3, C-4 and C-5 protons of glucose), 3.68−3.71 (m, 2H, C-6 proton of glucose). 4.09−4.11 (m, 1H, CH-O-(CO) of cholesteryl proton), 4.21−4.23 (d, 1H, C-2 proton of glucose), 5.38−5.41 (t, 1H, vinylic proton of cholesteryl group). Elemental analysis calcd (%) for C40H70N2O10: C, 65.01; H, 9.55; N, 3.79. Found: C, 65.01; H, 9.59; N, 3.77. MS (ESI): m/z calcd for C40H70N2O10: 738.50. Found: 761.59 [M + Na+]+. 1 H NMR of Amphiphile-2. Amphiphile-2 (500 MHz, methanol-d4, 25 °C): δ /ppm 0.81−1.69 (m, 39H, cholesteryl), 1.28−1.45(m, 4H, −CH2−CH2−), 1.41−1.60 (m, 4H, −NH−CH2−CH2−), 1.80−1.96 and 2.26−2.34 (m, 4H, allylic cholesteryl protons), 2.03−2.10 (m, 5H, glucose-OH), 3.02−3.12 (m, 2H, Chol−CONH−CH2−), 3.22−3.29 (m, 2H,−CH2−CONH−glucose), 3.52−3.65 (m, 3H, C-3, C-4 and C-5 protons of glucose), 3.69−3.74 (m, 2H, C-6 proton of glucose). 4.08−4.11 (m, 1H, −CH−O−(CO)− of cholesteryl proton), 4.32− 4.40 (d, 1H, C-2 proton of glucose), 5.38−5.41 (t, 1H, vinylic proton of cholesteryl group). Elemental analysis calcd (%) for C40H70N2O8: C, 67.95; H, 9.98; N, 3.96. Found: C, 67.91; H, 9.96; N, 3.91. MS (ESI): m/z calcd for C40H70N2O8: 706.51. Found: 729.50 [M + Na+]+. Critical Aggregation Concentration (CAC) Measurement. The critical aggregation concentration (CAC) of vesicular solutions of 1 and 2 in water were measured using a tensiometer (Jencon, India) by applying the Du Noüy ring method at 25 °C in water. A plot of surface tension (γ) versus concentration was drawn to obtain the inflection point which indicates the CAC. Preparation of Vesicle and Gel. Requisite amount of 1 and 2 was taken in Milli-Q water and bath sonicated to obtain opaque and translucent vesicular solution upon formation of self-assembly. On the other hand, requisite amount of amphiphile-1 and -2 was taken in a screw capped vial having an internal diameter (i.d.) of 10 mm and slowly heated to dissolve in 1 mL of desired solvent such as DMSO− water, DMF−water, benzene, toluene etc. The solution was then allowed to cool slowly (undisturbed) to room temperature. The gelation was checked by “stable to inversion” of the aggregated material in the glass vial. High Resolution Transmission Electron Microscopy (HRTEM). For HRTEM studies, 4 μL of sample solution of 1 (0.9−8.4 mg mL−1) and 2 (0.65−6.5 mg mL−1) taken in water, DMSO−water mixtures, and/or benzene was deposited on a 300-mesh carbon coated copper grid and dried. The copper grid was negatively stained with freshly

2. EXPERIMENTAL SECTION Materials and Methods. Silica gel of 100−200 mesh, triethylamine (Et3N), solvents, and all other reagents were procured from SRL, India. δ-D(+)-Glucono-1,5-lactone was procured from Alfa AesarJohnson Matthey company. Water used throughout the study was Milli-Q water. Thin layer chromatography was performed on Merck precoated silica gel 60-F254 plates. CDCl3 and other deuteriated solvents for NMR and FTIR, uranyl acetate, cholesteryl chloroformate, 2,2′-(ethylenedioxy)bis(ethylamine), hexan-1,6-diamine, sephadex-G50, calcein, 1,6-diphenyl-1,3,5-hexatriene (DPH) dye, and 8-anilino-1naphthalenesulfonic acid (ANS) were purchased from Sigma-Aldrich. Bath sonication was performed with a Telsonic Ultrasonics bath sonicator. Synthesis of Amphiphile-1 and -2. Cholesterol-based glucose appended amphiphiles (Figure 1) were synthesized following the methods mentioned below (Scheme S1, Supporting Information). To synthesize amphiphile-1, one end of diamine (2,2′-(ethylenedioxy) bis(ethylamine)) was coupled with cholesteryl chloroformate in dry dichloromethane (DCM) using equivalent amount of dry triethylamine. In case of amphiphile-2 the spacer used was hexan-1,6-diamine and one end was coupled with cholesteryl chloroformate in a similar way. The cholesteryl chloroformate was added dropwise to the DCM solution of diamine under cold conditions (0−5 οC) over a period of 4−5 h. After complete addition, the solution was stirred for further 8 h

Figure 1. Structures of amphiphile-1 and -2. B

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Langmuir prepared uranyl acetate solution (2 μL, 1% w/v) and the excess solution was immediately blotted with a filter paper. The sample was then dried for 4 h in vacuum before taking the image. The HRTEM images were obtained in a JEOL JEM 2010 microscope. Field-emission Scanning Electron Microscopy (FESEM) Study. FESEM images were obtained on a JEOL-6700F microscope. A 6 μL solution of 2 (0.65 mg mL−1) taken in water or DMSO−water mixture was placed separately on a piece of coverslip and dried for overnight. Then it was kept few hours under vacuum before imaging. Fluorescence Microscopic Study. Amphiphile-2 (0.65 mg) was mixed with 20 μL of calcein (2 mM) solution in water, and the volume was made up to 1 mL of water. The final concentrations of 2 and calcein were 0.65 mg mL−1 (5 times higher than CAC) and 40 μM, respectively, and they was kept overnight under stirring condition. The solution was then loaded into a Sephadex G-50 column (15 cm height and 1.2 cm diameter) pre-equilibrated with water and eluted with the water. Vesicular solutions were eluted right after the void volume. The filtration was carried out until free calcein was gel-filtrated and removed completely. The eluent was collected in 2 mL fraction each. The absorbance for all the fractions was taken at 490 nm to confirm the presence of calcein in each fraction. To one part of the calcein loaded aqueous solution of 2, DMSO was added to maintain the DMSO−water content 4:1 v/v. Now the aqueous and DMSO−water solution of amphiphile-2 having entrapped calcein was placed on a glass microscope slide, dried and observed under a fluorescence microscope (Olympus BX- 61) at 40× magnification. FTIR Study. FTIR measurements were performed with amphiphile-2 in nonself-assembled state using KBr pellets and in the self-assembled state using 1 mm CaF2 cell (for vesicle-2 in D2O and organogel-2 in CHCl3). All experiments were carried out in a Perkin−Elmer Spectrum 100 FTIR spectrometer. Solvent and Temperature Dependent 1H NMR Measurement. Solvent dependent and temperature dependent 1H NMR spectra of 2 was recorded in AVANCE 300 MHz (Bruker) spectrometer. The solvents were varied from methanol-d4 (nonself-aggregating) to D2O and 4:1 DMSO-d6−D2O (v/v) (self-aggregating solvents) for 2. In the case of temperature dependent 1H NMR, temperature was varied from 25 to 80 and 95 °C in benzene-d6 and DMSO-d6−D2O, respectively. Dynamic Light Scattering (DLS) Measurements. Mean hydrodynamic diameter of the aggregates of 2 in water and DMSO−water mixtures was determined by DLS using a fixed-angle apparatus (Zen 3690 Zetasizer Nano ZS instrument (Malvern Instruments Ltd.)). The scattering intensity was measured at an angle of 175°. Measurement of Complex Viscosity (η*). The rheological experiments were carried out in cone and plate geometry (diameter = 40 mm) on the rheometer plate by using an Anton Paar MCR 302 instrument. The solution of 2 (6.5 mg mL−1 taken in water and DMSO−water) was placed on the rheometer plate so that there was no air gap with the cone. A frequency sweep experiment was done as a function of angular frequency (0.001−10 rad s−1) at a fixed strain of 1% at 25 °C, and the complex viscosity was plotted against the angular frequency (ω). Circular Dichroism (CD) Study. CD spectra of amphiphile-2 taken in water and DMSO−water mixtures were recorded in a quartz cuvette (1 mm path length) on a JASCO J-815 spectropolarimeter. X-ray Diffraction (XRD). XRD spectra of the various selfassemblies formed by 2 were obtained on a Bruker D8 Advance diffractometer and the source used was Cu Kα radiation (λ = 0.15406 nm) with a voltage of 40 kV and current of 30 mA. UV−Vis Study. To investigate the molecular level aggregation of 2, UV−vis spectroscopic study was carried out using 8-anilino-1naphthalenesulfonic acid (ANS) as the probe. The solvent dependent UV−vis spectra of ANS doped 2 were recorded on a PerkinElmer Lambda 25 spectrophotometer. We have taken the UV−vis spectra of ANS in the presence of 2 (0.65 mg mL−1, 5 times higher than CAC), [ANS] = 1 × 10−6 M) in varying solvents from methanol (non-selfaggregating solvent) to water and DMSO−water (self-aggregating solvents). Fluorescence Anisotropy. Fluorescence spectra were recorded with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene

(DPH) in a Varian Cary Eclipse fluorescence spectrophotometer. The steady-state fluorescence anisotropy (r) of DPH was measured in individual solutions of 2 in water with varying concentrations. A stock solution of DPH (0.2 mM) was prepared in tetrahydrofuran (THF), and the final concentration of DPH was maintained at 1 μM in each solution (500 μL of each sample solution). DPH including 2 in water was excited at 370 nm. The emission intensity was measured at 450 nm using an emission cutoff filter at 430 nm to avoid any scattering due to turbidity of the solution. The excitation and emission slit widths were kept at 5 nm. In one set of experiments, the concentration of 2 was varied from 0.05 to 7.5 mg mL−1. In another set of experiments, the solvent was varied from water to DMSO−water keeping a fixed concentration of 2 (1.9 mg mL−1). The fluorescence anisotropy value (r) was calculated via the instrumental software using the following eq 1.

r = (IVV − GIVH)/(IVV + 2GIVH)

(1)

where IVV and IVH are the intensities of the emission spectra obtained with vertical and horizontal polarization (for vertically polarized light), respectively, and G = IHV/IHH is the instrumental correction factor, where IHV and IHH are the emission intensities obtained with vertical and horizontal polarization (for horizontally polarized light), respectively. The fluorescence measurements were performed at least five times for each sample at 25 °C and averaged.

3. RESULTS AND DISCUSSION Designing Amphiphiles and Their Solvent Induced Self-Assembling Behavior. Low molecular mass amphiphiles in general comprises of a hydrophilic head and a hydrophobic tail.25,26 An optimal hydrophilic−liphophilic balance (HLB) in the structural backbone of amphiphile plays a key role in the formation of well-defined nanostructures through self-aggregation.27 In this study, we have chosen two biologically relevant moieties, cholesterol and D(+)-gluconic acid, as the hydrophobic and hydrophilic units of the amphiphile, respectively.27,28 In particular, the cholesterol unit was chosen because of its influential role in the formation of diverse self-assembly commonly used in drug encapsulation and intracellular delivery.23,24 Now, these two components (cholesterol and glucose) were coupled through a 2,2′-(ethylenedioxy)bis(ethylamine) linker (1, Figure 1 and Scheme S1 (Supporting Information)). Amphiphile-1 formed translucent solution in water. Gradual addition of DMSO to this aqueous solution of 1 resulted in the formation of a highly viscous solution. With further increase in the DMSO content up to 4:1 v/v DMSO−water, 1 formed a gel having minimum gelation concentration (MGC) = 8.4 mg mL−1 (Table 1) that Table 1. MGCs of 1 and 2 (in mg mL−1) in Different Solventsa MGC

a

MGC

solvents

1

2

solvent

1

2

DMSO DMF THF acetonitrile DMSO−water (4:1 v/v) DMF−water (2:3 v/v) THF−water acetonitrile−water

S S S Ins 8.4 8.2 S Ins

S S S Ins 6.5 6.1 S Ins

n-hexane chloroform ethanol benzene toluene o-xylene chlorobenzene nitrobenzene

S S S 7.8 7.2 7.2 6.6 5.8

S 7.9 S 6.1 5.0 4.8 5.4 5.0

S = soluble, Ins = insoluble.

was “stable-to-inversion” upon turning down the vial. It also formed stable gel in DMF (dimethylformamide)-water (2:3 v/v) mixture with a MGC of 8.2 mg mL−1. Amphiphile-1 showed C

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Figure 2. Photographs of vesicular solution in water and gels in different solvents for amphiphile-1 and -2.

These elongated vesicles gradually stuck with each other to form ribbon like hierarchical nanostructures in 1:1 v/v DMSO−water (Figure 3c). With further increase in DMSO content (4:1 v/v DMSO−water), the ribbon like structures possibly integrated to form the intertwined fibrillar network having diameter of 20−30 nm and length of several micrometers. This fibril network restricted the mobility of solvents leading to the formation of gel (Figure 3d). Amphiphile-2 showed analogous solvent dependent self-aggregation like 1. Amphiphile-2 formed vesicles in water having diameter of 70−140 nm with 2−3 nm wall thickness (Figure 3e and inset). In 1:4 v/v DMSO−water, smaller vesicles gradually fused with each other to form bigger aggregates (Figure 3f). Further increase in DMSO content (DMSO−water 1:2 and 1:1 v/v) resulted in the elongation of the fused vesicles along a particular array (Figure 3g,h). Complete transition from vesicles to fibril network with twisted morphology took place in 4:1 v/v DMSO−water mixture (Figure 3i) that led to the formation of gel. The fibers were 20−30 nm in diameter and length of several micrometers. The magnified image showed the helical nature of the fibers, which are termed as nanoribbons (inset of Figure 3i). The coexistence of vesicles and fiber in varying compositions of DMSO−water mixtures delineates the pathway of vesicle-to-gel transition upon changing the solvent from water to 4:1 v/v DMSO−water. In pure DMSO, 1 and 2 did not show any well-defined structure as they were completely soluble in that solvent (Figure S1a,b, Supporting Information). Solvent triggered transition in self-aggregated structure of 2 from vesicle-to-fiber was also observed in the respective field emission scanning electron microscopic (FESEM) images in water and DMSO−water mixture (Figure 3j−l). The formation of different topologies in solvent dependent self-aggregation was further investigated from the fluorescence microscopic images of calcein (fluorescent probe) entrapped self-assemblies of 2. In concurrence with the morphological changes observed in HRTEM, here also the changes from green

efficient organogelation ability in aromatic solvents like benzene, toluene, nitrobenzene, etc (Figure 2) but failed to gelatinize aliphatic solvents such as chloroform, n-hexane, and ethanol (Table 1). To investigate the influence of HLB on structure dependent gelation, we have slightly tuned the hydrophobicity of the linker moiety by introducing C-6 long chain (hexan1,6-diamine) in amphiphile-2 (Figure 1) in place of 2,2′(ethylenedioxy)bis(ethylamine). Amphiphile-2 formed an opaque solution in water. Upon addition of DMSO to the aqueous solution of 2, high viscous solution was formed which gradually transformed to translucent gel in DMSO−water (4:1 v/v) at MGC = 6.5 mg mL−1. Analogous behavior was observed when DMF was added to the aqueous solution of 2 and it formed gel in 2:3 v/v DMF−water mixture with a lower MGC (6.1 mg mL−1, Table 1, Figure 2) compared to that of 1. Amphiphile-2 formed organogels in aromatic organic solvents as well as in chloroform. In most solvents, amphiphile-2 exhibited better gelation efficiency compared to that of 1 (Table 1). Presumably, the optimal HLB required for efficient gelation was attained by 2 comprising of more hydrophobic linker. Microscopic Investigations of the Self-Assemblies. This solvent dependent transformation in the physical state of the self-assemblies intrigued us to investigate the change in the nature of self-aggregation (if any) during the transition from transluscent/opaque solution in water to gels in DMSO−water (4:1 v/v) with increasing DMSO content. To this end, we have taken the high resolution transmission electron microscopy (HRTEM) images of the self-assemblies of both amphiphiles in varying composition of water and DMSO. HRTEM image of aqueous solution of 1 showed spherical vesicles having average diameter of 80−130 nm with a thin wall and hollow core (Figure 3a). The thickness of the vesicular wall was around 2−4 nm which may be correlated to the bilayered organization of the amphiphilic molecules (inset of Figure 3a).29,30 In 1:4 v/v DMSO−water, the vesicular aggregates started to get elongated and aligned in a specific direction (Figure 3b). D

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Figure 3. HRTEM images of (a) vesicle-1 in water (in set magnified image of bilayered vesicle) and self-assemblies of 1 in DMSO−water mixture (v/v = (b) 1:4, (c) 1:1, (d) 4:1); HRTEM images of (e) vesicle-2 in water (in set magnified image of the bilayered vesicle) and self-assemblies of 2 in DMSO−water (v/v = (f) 1:4, (g) 1:2, (h) 1:1, (i) 4:1); FESEM images of 2 taken (j, in set magnified image of vesicle) in water, in DMSO−water (v/v = (k) 1:2, (l) 4:1).

emitting spherical aggregates to green emitting fiber was evinced. The green emitting vesicular self-assemblies of 2 in water gradually fused with each other forming hierarchical aggregates with increasing DMSO in DMSO−water mixtures. Ultimately, it transformed to green emitting fiber in 4:1 v/v DMSO−water (Figure S2, Supporting Information). At this instant, we were curious to find out whether the concentration of amphiphiles or the incubation time in varying solvents have any influence in the vesicle-to-fiber transformation. Accordingly, we incubated the aqueous solution of 1 and 2 for 30 days and observed the sample under HRTEM. However, no notable change in the vesicular self-assembly was noticed (Figure 4a,c). Also upon increasing the concentration of 1 and 2 in water, the vesicular aggregates got stuck with each other to form bigger aggregates but morphological changes in the aggregates structure was not observed (Figure 4b,d). Thus, the observed structural change from vesicle-to-fiber is primarily influenced by the variation in the solvent compositions. Intertwined fibrillar cross-linked network was also evident in the HRTEM images of organogel-1 and -2 in benzene (Figure S3, Supporting Information). Hence, the solvent dependent vesicle-to-gel transition mainly progressed through three stages of selfassembly that includes fusion of vesicles, elongation of fused vesicles to nanoribbons and twisting of nanoribbons to generate fibrillar network. Generally, the dielectric constant (ε) provides a rough measurement of a solvent’s polarity. High polar solvent have high dielectric constant (such as water has ε = 79.5 at 25 °C). The variation of dielectric constant (or polarity) of the binary solvent mixtures with increasing DMSO or DMF content might have played a

Figure 4. HRTEM image of (a) aqueous solution of 1 (0.9 mg mL−1) after incubation of 30 days and (b) at concentration of 8.4 mg mL−1 (∼45 times higher than CAC of 1); HRTEM images (c) aqueous solution of 2 (0.65 mg mL−1) after incubation of 30 days and (d) at concentration of 6.5 mg mL−1 (∼50 times higher than CAC of 2).

crucial role in vesicle-to-fiber transformation. The self-association between amphiphilic molecules gradually shifted toward gelation upon moving from highly polar solvent like water (εwater = 79.5, Table S1, Supporting Information) to comparatively low polarity binary solvent mixture, (DMSO/DMF−water).31,32 E

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Figure 5. (a) DLS and (b) complex viscosity measurement of 2 in water and DMSO−water.

Participation of Intermolecular Noncovalent Forces in Self-Assembly. Various intermolecular noncovalent interactions operating between the amphiphilic molecules during selfaggregation were investigated by FTIR, temperature and solvent dependent 1H NMR spectroscopy.12,30,31 FTIR spectra of 2 taken in KBr pellet (non-self-assembled state) showed sharp transmittance signals at 1523, 1652, and ∼3327−3372 cm−1 due to amide δN−H (amide II, bending), νCO (amide I, stretching), and coexistence of νN−H (amide A) with νO−H peaks, respectively (Figure 6). Interestingly, the transmittance signals were shifted to

The dielectric properties of mixed solvents were reported to be εDMSO−water for DMSO−water (4:1 v/v) is 64.0 ± 0.25 and εDMF−water for DMF−water (2:3 v/v) is 72.1 ± 0.25 (Table S1, Supporting Information).31,32 Both amphiphile-1 and 2 were found to be soluble in pure DMSO or DMF (εDMSO = 47.0 ± 0.6, εDMF = 39.8). Decrease in the dielectric constant with increase in DMSO content (Table S1) possibly resulted in the morphological transition of aggregates from fusion, elongation and twisting of vesicles. An intermediate dielectric constant (ε = 64.0−73.0) between water and DMSO/DMF might have facilitated the gelation of 1 and 2. In addition different solvophobic and solvophilic forces particularly an optimal HLB also played a pivotal role in gelation. Characterization of the Self-Assemblies. The critical aggregation concentration (CAC) at which the amphiphiles started to form self-aggregation was recorded to be 181.6 and 130.5 μgmL−1 for 1 and 2, respectively in water (measured by surface tension method, Figure S4, Supporting Information).29 For further investigations, we have chosen amphiphile 2 as representative due to its better gelation efficiency. According to dynamic light scattering (DLS) study, the mean hydrodynamic diameter of the vesicles formed by 2 in water was found to be around 80−150 nm (Figure 5a), which is in well concurrence with microscopic evidence (Figure 3). Notably, in DMSO− water (1:4 v/v), an additional peak was found around 300 nm along with the peak at 80 nm. The existence of both peaks was also observed in 1:1 v/v DMSO−water mixture (Figure 5a). The peak raised at ∼300 nm could be due to the formation of self-aggregated nanoribbon (Figure 3h). The coexistence of vesicles and nanoribbons in varying DMSO−water mixture further supports vesicle-to-fiber transition toward gelation. DLS measurement for 2 could not be carried out in 4:1 v/v DMSO− water mixture as it formed gel. The extent of fiber formation with increasing DMSO content might be assessed from the increase in the peak intensity at 300 nm (Figure 5a). Rheology measurement provides information about the viscoelastic nature of materials.33,34 A strain controlled (strain fixed at 1%) frequency sweep experiment was carried out with 2 (6.5 mg mL−1) in water and in DMSO−water mixtures to measure complex viscosity (η*) as a function of angular frequency (10−3 to 10 rad s−1). At low frequency range, η* value sharply increased with increasing DMSO content (Figure 5b). This observation suggests possible transition from low viscous solution (vesicles) to high viscous self-aggregates like nanoribbons and consequently to fibers. The remarkably high η* value for 2 in 4:1 v/v DMSO−water indicates the formation of self-aggregated gel.33,34 The solvent dependent vesicle-to-fiber transition as found from microscopic evidence is in good agreement with DLS and rheological study.

Figure 6. FTIR spectra of 2 in different solvents.

1534, 1637 and ∼3335−3488 cm−1 (broad band), respectively in D2O. Similar shifts were noticed for organogel 2 in chloroform where the transmittance peaks appeared at 1543, 1639, and 3278−3492 cm−1, respectively.35,36 These shifts in stretching and bending frequencies at self-assembled state both in D2O (vesicles) and in chloroform (gel) indicate the strong intermolecular hydrogen bonding between carbonyl (CO) and amide N−H moieties during the process of self-assembly. Next, we carried out solvent dependent 1H NMR of 2 at self-assembled state in D2O, DMSO-d6−D2O (4:1 v/v), and benzene-d6 at room temperature and compared with the 1 H NMR of 2 in methanol-d4 (non-self-assembled state). In methanol-d4, cholesteryl and C-6 long chain protons of 2 showed characteristic NMR signals at δ = 0.8−2.0 ppm while the amide proton signals appeared at δ = 7.42−7.55 ppm (Figure 7). Interestingly, in D2O, DMSO-d6−D2O (4:1 v/v), and benzened6, these 1H NMR signals of 2 got notably suppressed possibly F

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Mode of Self-Aggregation and Morphological Transformation. On the course of solvent dependent transition of the aggregated structures, we were keen to find the change in aggregation pattern of the amphiphile. In this context, circular dichroism (CD) spectra were recorded for 2 in different solvent compositions (Figure 8a). In DMSO−water (4:1 v/v), 2

Figure 8. (a) CD spectra of 2 in water and DMSO−water; (b) small angle and (c) high angle XRD pattern of vesicle-2 in water and gel-2 in DMSO−water; (d) UV−vis spectra of ANS in the presence of 2 in water and DMSO−water.

showed characteristic CD signals with a negative peak at 219 nm and a positive peak at 204 nm which can be correlated with β-sheet-like supramolecular aggregation as usually found in peptide based aggregates. In the present case, intermolecular hydrogen bonding between terminal glucose and hydrophobic interaction between steroidal moieties gives rise to sheet like aggregation pattern in modified solvent polarity (DMSO− water 4:1 v/v). Also this hydrophobic and hydrogen bonding interaction render helicity in the nanoribbon’s topology which further led to the formation of gel fibrillar network.12 However, amphiphile-2 in water (vesicles) produced different type of CD signal with a single negative Cotton effect at 211 nm. This dissimilar CD spectral pattern of vesicles and nanofibers in varying solvent compositions indicates the solvent dependent different morphology of self-aggregates. The X-ray diffraction (XRD) study (Figure 8b,c) further supported the formation of sheetlike structures during the gel formation.12 Gel-2 in DMSO−water (4:1 v/v) showed an XRD peak at 2θ = 18.23° (d = 4.86 Å) corresponding to the spacing between intermolecular steroidal backbones. Another peak at 2θ = 3.99° (d = 22.07 Å) indicates the spacing between intersheet of the self-organized amphiphiles. Several other peaks generated in the range 4−10° due to the periodic ordered stacking of sheets.12 In contrast, vesicle-2 showed peaks at 2θ = 21.47° and 4.25° (d = 4.13 and 20.76 Å, respectively), that were correlated to the spacing between intermolecular steroidal moieties and bilayer, respectively. The characteristic diffraction peaks delineate that vesicle-to-fiber transition proceeded through bilayered stacking (in vesicle) to sheet like molecular aggregation (in nanoribbon). Generally molecular aggregations can be categorized to two types (i) H-type (parallel plane-to-plane stacking)

Figure 7. Solvent dependent 1H NMR spectra of 2 (2.5 mg mL−1).

due to the participation of hydrophobic interaction between the steroidal moieties, C-6 alkyl chain and intermolecular hydrogen bonding (through CO···H−N linkage) during self-aggregation (Figure 7).35,36 In the case of temperature dependent 1H NMR study, the temperature of gel-2 taken in DMSO-d6−D2O (4:1 v/v) and benzene-d6 gradually increased up to 95 and 80 °C, respectively. With increase in temperature the 1H NMR peaks owing to cholesteryl, C-6 alkyl chain and amide protons became prominent with increased intensity (Figure S5, Supporting Information). Self-aggregation between the amphiphiles through noncovalent interactions is expected to get dissociated at elevated temperature. Consequently, transition from aggregated state to molecular state took place where the characteristic 1H NMR signals were observed (Figure S5, Supporting Information). This observation further confirmed the participation of noncovalent forces in selfaggregated gelation. G

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Langmuir and (ii) J-type (head-to-tail fashion arrangement).37−39 In UV−vis spectra, H-aggregation shows blue-shifted absorption while J-aggregation exhibits red-shifted absorption maxima compared to their nonself-assembled monomers in the solution.37−39 Since the presently used amphiphiles are devoid of any chromophore moiety, we used fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS) to understand the aggregation pattern.37−39 Hydrophobic probe ANS is known to locate itself at the hydrophobic region of the supramolecular aggregate.37−39 UV−vis spectra of ANS (1 × 10−6 M) in the presence of 2 (0.65 mg mL−1) were recorded in different solvents (methanol, water and DMSO−water mixtures). In methanol (molecularly dissolved 2, i.e., in non-self-assembled state), ANS showed absorption maxima at 372 nm (Figure 8d). Notably, in water, a blue-shifted UV signal of ANS was observed at 358 nm (Figure 8d). It indicates the probable occurrence of H-aggregation during the formation of vesicles by 2. However, in DMSO−water (2 formed gels), the UV absorption maxima gradually red-shifted (365 to 385 nm) with increasing DMSO content. Variation in the solvent compositions as well as its polarity possibly led to the change in the aggregation pattern from H-type in water to J-type in DMSO−water through reorientation of amphiphiles. Consequently, the solvent dependent reorganization of H- to J-type molecular packing might have assisted in vesicle-to-fiber transition. To this end, steady state fluorescence anisotropy (r) of 1,6-diphenyl-1,3,5-hexatriene (DPH) entrapped within the selfaggregates of 2 was examined.29,40,41 Hydrophobic DPH probe is known to intercalate between the hydrophobic interior of the aggregates due to its rod-like shape. Higher is the steady state anisotropy (r) value, higher will be the restricted movement of DPH.29,30 We observed that with increasing concentration of 2, the (r) value in water increased from 0.15 to 0.33 and reached a steady value 0.33 at 1.9 mg mL−1 and above. The hydrophobic DPH got confined within the hydrophobic bilayer of vesicle and the restricted movement of DPH showed high (r) value (Table S2, Supporting Information).40,41 With increase in the DMSO content in aqueous solution of 2 (1.9 mg mL−1), the (r) value gradually dropped to 0.21 (Table S3, Supporting Information) possibly due to reorientation of amphiphiles to J-type arrangement during structural transition from vesicle to fiber. Proposed Pathway of Structural Transition of SelfAggregates. On the basis of the above-mentioned findings, a probable model (Scheme 1) is proposed to elucidate the pathway through which the amphiphiles formed nanoassemblies with different structural characteristics. It is evident that that the formation of vesicle by compound-1 and 2 in water crucially depends on the tendency of cholesterol unit to keep away from the contacts with water and its solvophobic interaction between each other. The model delineates that the hydrophobic steroidal part (cholesteryl) of the amphiphiles possibly facilitates the formation of vesicle through bilayer stacking (H-type) in water while the hydrophilic glucose terminals are exposed toward the aqueous domain (directed toward the surrounding aqueous medium and inner aqueous core, Scheme 1). Also the hydrogen bonding between water molecules and the exposed polar headgroup (glucose) played a vital role to stabilize vesicular system in water. In presence of DMSO, the vesicles fused with each other and elongated to form twisted nanoribbon like topology with increasing DMSO amount. The compact molecular arrangement as found in the vesicular system was slightly disfigured in DMSO−water

Scheme 1. Proposed Model for Vesicle-to-Nanoribbon Transformation from Bilayered H-Aggregated Molecular Packing to J-Aggregated Sheetlike Molecular Arrangement

(as evident from fluorescence anisotropy). Consequently, the bilayered H-aggregated molecules gradually reorient themselves in J-type pattern in the reduced solvent polarity. Furthermore, sheet like molecular packing originating from intermolecular hydrogen bonding as well as hydrophobic interaction between the amphiphiles (Scheme 1) resulted in the formation of nanoribbon like fibrillar network

4. CONCLUSION In summary, cholesterol based glucose appended amphiphiles were developed which showed solvent dependent diverse selfaggregation behavior. It formed vesicles in water and gels in different organic solvents. Interestingly, in DMSO−water mixture, progressive morphological transformation was observed from vesicle-to-fiber depending on the solvent compositions. This transformation took place due to the changes in the aggregation pattern of the amphiphiles in varied solvent polarities. The present study describes a facile approach to develop solvent triggered multifaceted self-aggregated structure from single molecular backbone. Also it establishes the route of forming hierarchical self-assembly through direct visualization of the intermediate topologies.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b02165. Dielectric constant for varying solvent compositions, fluorescence anisotropy, synthetic scheme, HRTEM images, fluorescence microscopic images, critical aggregation concentration (CAC) plots, and temperature dependent 1H NMR spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. H

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ACKNOWLEDGMENTS P.K.D. is thankful to Council of Scientific and Industrial Research (CSIR), India (ADD, CSC0302) for financial assistance. D.M., S.D., and P.C. acknowledge CSIR, India for Research Fellowships.

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