Impact of Glycolipids Hydrophobic Chain Length, Headgroup Size on

Subscriber access provided by University | of Minnesota Libraries

Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Impact of Glycolipids Hydrophobic Chain Length, Headgroup Size on Self-Assembly and Hydrophobic Guest Release Kanaparedu P C Sekhar, Harikrishna Adicherla, and Rati Ranjan Nayak Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01401 • Publication Date (Web): 07 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Impact of Glycolipids Hydrophobic Chain Length, Headgroup Size on Self-Assembly and Hydrophobic Guest Release Kanaparedu P. C. Sekhar†,§, Harikrishna Adicherla ‡ and Rati Ranjan Nayak*,†,§ †

Centre for Lipid Science and Technology, CSIR-Indian Institute of Chemical Technology, Hyderabad-500 007, India

‡

CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500 007, India §

Academy of Scientific and Innovative Research (AcSIR), New Delhi-110 001, India

ABSTRACT: Encapsulation of hydrophobic guest molecule inside the micelle and their stimulisensitive release is useful strategy for target-specific drug delivery. Herein, nine bio-based glycolipids were derived from plant sources. The influence of headgroup on stability and aggregation pattern in water with different alkyl chain lengths was investigated to deduce structure-property relationship. External factors, such as temperature, pH, NaCl, and urea concentrations were employed to explore stimuli-response on glycolipid nano-assemblies. Further, the solvatochromic dyes, like pyrene, N-phenyl-1-naphthylamine, and curcumin were utilized to examine hydrophobe loading capacities of these glycolipids assemblies. Fluorescence study was applied to investigate the enzyme-sensitive hydrophobe release. Interestingly, the pH

ACS Paragon Plus Environment

1

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 36

sensitive hydrophobic guests showed pH responsive release from dynamic micelles. Finally, the synthesized glycolipids revealed their nano-assemblies as smart carriers for hydrophobic cargo.

1. INTRODUCTION Specifically designed bio-derived glycolipids have attracted considerable interest for biological applications.1−3 In general, self-assembly of amphiphilic molecules into distinct nano-assemblies (micelle, cylindrical micelle, vesicle, toroid, tube, and helical fiber) is a commonplace phenomenon. Structural modifications in the architecture of amphiphile alter structure of selfassemblies into various forms,4−7 thus affects the biological applications.8−9 Other than the molecular structure, external stimuli can also alter self-assemble pattern by varying the nature of dispersion medium.10 Light, pH, temperature, and redox-sensitive amphiphiles exhibit stimuli response on the self-assemblies.11−14 Some time, logically designed amphiphiles exhibits dual stimuli response.15 Further, the stimuli-sensitive assemblies enabled them as smart nano-carriers for potential drug delivery systems.16 Encapsulation of hydrophobic drug cargo inside nano-assemblies of amphiphilic molecules through non-covalent interactions improves the solubility, stability, and bioavailability of drug molecules.17 External stimulus such as temperature, pH, and light releases encapsulated drug molecules from stimuli-responsive nano-carriers.11,18,19 Biological stimuli like enzymes and proteins also release drug molecules by enzyme and protein induced disassembly of nano-carrier respectively.20,21 Enzyme-responsive nano-carriers are used to cure diseases related with overexpression of specific enzymes inside the infected tissue.22 Further, enzyme responsive prodrug based nano-carriers are utilized to improve the drug activity.23 Loading ability is high for pro-drug based nano-carriers while comparing with nano-carriers encapsulated the hydrophobic drug with non-covalent interactions.20 However, the pro-drug molecules with covalent bonds are


2

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

releasing drugs with slow rates than the non-covalently encapsulated micellar nano-carriers. Moreover, non-covalently encapsulated nano-carriers can encapsulate multiple hydrophobic drug cargos in water.24 In vitro drug release kinetics of nano-carriers is completely dependent on the rate of enzymatic disassembly. Further, these disassembly rates are greatly affected by monomer molecular geometry of the nano-carrier. Increase in hydrophobicity of monomer lower the disassembly rates of nano-carriers subsequently decreases the drug release rates.25−27 Thus, modular design of glycolipids with headgroup and chain length variation is useful to understand the structure-activity relation for effective in vitro drug delivery. In this study, we incorporated hydrophilic aspartic acid and glucamine in-between hydrophilic and hydrophobic moiety of linear glycolipids (I) to enhance its water solubility. Further, the influences of molecular geometry, specifically the hydrophobic chain length as well as the size of the headgroup on aggregation behavior, stimuli response, hydrophobe loading, and release studies of glycolipid were discussed.

2. EXPERIMENTAL SECTION 2.1. Materials. Cetylpyridinium chloride (CPC), pyrene, 1,6-diphenyl-1,3,5-hexatriene (DPH), N-phenyl-1-naphthylamine (NPN), curcumin, uranyl acetate, sodium hydroxide, hydrochloric acid, sodium chloride (NaCl), urea, lipase B candida antarctica (CAL-B), pepsin from porcine gastric mucosa (PEP), and trypsin from porcine pancreas (TRYP) were obtained from SigmaAldrich. Ultrapure water obtained from Milli-Q system (Siemens, Labostar 7) whose resistivity was 18.2 MΩ cm was used for sample preparation. N-(2,3,4,5,6-pentahydroxyhexyl)-Nmethylalkanamide, (1a− −c, Figure 1i) were synthesized by following the previous literature.28 The glycolipids 2a−c and 3a−c (Figure 1i) were synthesized according to Scheme S1. To get both the products in almost equal mole ratio 1.5 equivalents of N-methylglucamine was used. The


3

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 36

reactive α methyl ester is ammonalised first and essential minor peaks present in NMR data of glycolipids 2a−c and 3a− −c are due to the presence of rotamers. We note that the presence of characteristic β methyl ester signals of glycolipids 2a−c in their NMR spectra (δ 3.58 ppm (s, 3H, −OCH3) in 1H NMR and δ 48.61–5 ppm (−OCH3) in

13

C NMR spectra) confirmed the

asymmetrically branched (Ya) glycolipids structure. Hydrophilic-lipophilic balance (HLB) values of the synthesized glycolipids were calculated by using Griffin equation29 ‫= ܤܮܪ‬

௉

(1)

ହ

Where P is relative molecular mass percentage of hydrophilic group. 2.2. Fluorescence Measurements. Steady-state fluorescence studies were carried on a Varian (Cary Eclipse) fluorescence spectrometer. Emission spectra of 0.6 µM pyrene solutions were recorded in the range 347–600 nm at an excitation wavelength of 337 nm using excitation bandpass 5, emission band-pass 2.5 nm. Steady-state fluorescence anisotropy (r) of 1 µM DPH in surfactant micelle was measured on a fluorolog-3 spectrofluorometer (Horiba Jobin Yvon, UK) equipped with filter polarization accessory that uses L-format configuration. The sample was excited at 350 nm and the emission was followed at 450 nm, using both an excitation and emission slits with a band-pass of 5 nm. Temperature of the sample holder was controlled by Julabo FP 50 circulating water bath. 2.3. Dynamic Light Scattering. In order to determine the size distribution of micelles, scattered dynamic light intensities were measured using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK) with 90 degrees angle of detection. Sample was prepared in ultrapure water and filtered directly into the scattering DTS0012 through a Whatman syringe filter (sterile and endotoxin free, 0.45µm). Before measurement, scattering cell was equilibrated for 5 minutes in the DLS optical system at the mentioned temperatures. The change in refractive index (1.33-


4

Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1.35) and viscosity (0.89 cP (25 ºC), 1-0.47 cP (20-60 ºC), 0.89-1.71 cP (0-5 M NaCl), and 0.891.76 cP (0-10 M urea)) were included for sample analysis. 2.4. Transmission Electron Microscopy. For HR-TEM measurement, a drop of an aqueous solution of amphiphile was placed on a 200 mesh carbon-coated copper grid for two minutes. Excess solution (liquid) wicked off with a filter paper, and then negatively stained with freshly prepared 1.0% aqueous uranyl acetate solution for 45 seconds. The dried specimen was investigated with an electron microscope (JEOL-JEM 2100, Japan) operating at an accelerating voltage of 120 kV. For TEM experiments, the concentrations of glycolipids used are 1a (200 mM), 1b (50 mM), 1c (2 mM), 2a (10 mM), 2b (0.5 mM), 2c (0.5 mM), 3a (10 mM), 3b (0.5 mM) and 3c (0.5 mM). 2.5. Circular Dichroism Spectroscopy. Circular dichroism spectra were recorded using a JASCO J-180 (Japan) spectropolarimeter. Measurement was done in wavelength range of 300−185 nm with scan speed, bandwidth and response time of the device was set at 100 nm/min, 1 nm, and 2 s respectively. 2.6. Differential Scanning Calorimeter. Thermotropic behavior of aqueous surfactant sample (40 µL) in standard closed aluminium pan was determined by using DSC 6000 (Perkin Elmer) calorimeter by heating samples at 1 °C/min at a constant nitrogen flow of 50 mL/min. A cyclic process was carried out to get both heating and cooling profiles of glycolipid 1c and 2c. In order to get the time-dependent isothermal behavior of glycolipid 1c; an isothermal process was carried out immediately at 25 °C for 2 hours after completion of heating-cooling cycle. 2.7. UV-Vis Absorbance Measurements. Absorption studies were performed on Lambda 35 Perkin-Elmer UV/visible spectrophotometer. A kinetic turbidity measurement was carried out for


5

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 36

a pre-dissolved transparent 2 mM aqueous solution of glycolipid 1c (after attaining 25 °C) by measuring time-dependent absorbance at 400 nm, where the glycolipid has zero absorbance. 2.8. Encapsulation Efficiency. Hydrophobic guest (pyrene/NPN/curcumin) was dispersed into glycolipid solution (50 mL) at a concentration of 500 µM hydrophobe per 1 mM of glycolipid solution. The resultant mixture was stirred for 24 h and centrifuged at 5000 rpm for 30 minutes at 25 °C. The supernatant was filtered through a 0.45 µm Whatman syringe filter to remove nonincorporated guest molecules. The aliquot was diluted with methanol (90%) to release encapsulated guest.30 The absorbance maxima of corresponding hydrophobe in 90% methanol solution were recorded and encapsulation efficiency (µM of guest/mM of glycolipid) was determined by comparing with calibration curves. 2.9. Enzymatic Hydrophobe Release. To 10 mL of curcumin loaded aqueous glycolipid solution, enzyme (TRYP or PEP (at pH 1.5)) at a concentration of 10 mg per 1 mM of glycolipid was added at 37 °C. For the immobilized enzyme (CAL-B), in 10 sample vials, 1 mL of curcumin loaded glycolipid solution was taken per vial and 1 mg of enzyme per 1 mM of glycolipid was added at 37 °C. At certain time intervals 1 mL of solution was withdrawn, centrifuged at 5000 rpm, and filtered through a 0.45 µm filter. Emission spectra of curcumin solutions after filtration were recorded on a Varian (Cary Eclipse) fluorescence spectrometer in the range 450–800 nm at an excitation wavelength of 440 nm. The release percentage was calculated relative to initial fluorescence intensity of curcumin at 530 nm in absence of enzyme. 2.10. Acid/base Stimuli-responsive Release. For acid/base titration experiments, pH of the hydrophobe loaded aqueous glycolipid solution (3 mL) was adjusted by addition of HCl/NaOH solution. The emission spectrum was taken on a Varian (Cary Eclipse) fluorescence spectrometer. Pyrene, NPN, and curcumin containing aqueous glycolipid solutions were excited


6

Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

at 337, 340 and 440 nm, and the emission spectra were recorded between 347–600, 350–600 and 450–600 nm respectively. The release percentages of pyrene, NPN, and curcumin from the corresponding hydrophobe loaded glycolipid assemblies were calculated relative to initial fluorescence intensity at 375, 419, and 530 nm before addition of HCl/NaOH respectively.

Figure 1. (i) Molecular structure of I (1a−c), Ya (2a−c), and Ys (3a−c) glycolipids. (ii) Plot of pyrene micropolarity (I1/I3) versus glycolipid concentrations in water (a Studied at 55 °C). (iii) DSC thermograms of glycolipids (a Cooling curves).

3. RESULTS AND DISCUSSION This study is mainly focused, evaluation of structure-property relationship on glycolipid selfassembly in water by using various headgroup size and alkyl chain length (Figure 1i). The glycolipids are classified into three categories. First, the glucamine unit was directly coupled with fatty chain to form liner glycolipids (I). Second, the aspartic acid unit was incorporated


7

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 36

between the glucamine and fatty chain. Insertion of aspartic acid unit produced the asymmetrically branched glycolipids (Ya).Third, ammonalisis of β methyl ester of Ya glycolipids with another glucamine yields symmetrically branched glycolipids (Ys). Herein HLB values of linear (I) glycolipids and asymmetrically branched (Ya) glycolipids are in similar range (Table 1). Hydrophobic chain length of Ya glycolipids and symmetrically branched (Ys) glycolipids are same. This glycolipid Matrix was used to predict the results in two ways. First, effect of alkyl chain length with fixed headgroup geometry, later, the influence of headgroup size with a fixed hydrophobic chain on self-assembly behavior. Further studies were performed on glycolipid selfassemblies as nano-carriers. The experimental results were explained in three parts: (i) aggregation behavior of the glycolipids, (ii) stimuli-responsive self-assembly, and (iii) hydrophobic guest encapsulation and release. 3.1. Aggregation Behavior. Fluorescence emission studies of pyrene in aqueous glycolipid solution allow one to determine the micropolarity (I1/I3) and critical micelle concentration (CMC). The plot of pyrene I1/I3 ratio as a function of glycolipid concentration is shown in Figure 1ii. From Figure 1ii, I1/I3 shows a typical sigmoid of the Boltzmann type decrease as the surfactant concentration increases. The CMC values in Table 1 were obtained by Boltzmann fitting.31 While comparing the CMC values obtained for our synthesized glycolipids, in each series, with an increase in HLB values the CMC values are decreasing at 25 °C (omitting glycolipid 2c, whose Krafft point is 54.5 °C, Figure 1iii). On the other hand, for lauroyl glycolipids (glycolipids 1c, 2a, and 3a), irrespective of headgroup geometry, CMC values are in same order. This indicates hydrophobic chain length hydrophobic tail played an important role in micellisation process. Further, I1/I3 ratio of pyrene above the CMC is an index of micropolarity of micellar core. The observed micropolarity values presented in Table 1 are quite low as


8

Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

compared to the spherical micellar core. But similar values were reported for spherical micellar core of sugar based surfactants such as n-Octyl-β-D-glucopyranoside (1.01) and n-octyl-β-Dthioglucopyranoside (1.06).32,33 From Table 1, in each glycolipid series, micropolarity values decreases slightly with increase in alky chain lengths. In contrast to CMC values, micropolarity of symmetrically branched glycolipid (glycolipid 3a) has slightly higher value among the lauroyl glycolipids (n = 10). This may be due the penetration of water molecule inside micelle core of branched headgroups. Many authors have successfully used quenching of pyrene fluorescence by CPC to determine aggregation number (Nagg) of micelles using the following equation34 ln ቀ 0 ቁ = I

I

Nagg ሾQሿ

൫C-CMC൯

(1)

where I0 and I are fluorescence intensities of probe in absence and presence of quencher respectively. The steady-state fluorescence quenching of pyrene probe in micellar solution against quencher concentration is plotted in Figure S1. It should be noted that the Nagg obtained for glycolipids are showing typical order of micelle (Nagg < 100, Table 1). In each glycolipid series, the Nagg was decreased with increase in alkyl chain length. Moreover, steady-state fluorescence anisotropy (r) measurement can provide useful insights on nature of packing (microfluidity or rigidity) of hydrophobic tail group inside the micellar core. DPH is a well-known membrane fluidity probe and anisotropy value was calculated employing the following equation35 r=

IVV -GIVH IVV +2GIVH

(2)

where IVV and IVH are fluorescence intensities polarized parallel and perpendicular to excitation light, and G (IHV/ IHH) is instrumental grating factor. The anisotropy values for synthesized surfactants are presented in Table 1. It was found that the r value for lauroyl


9

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 36

glycolipids is increasing with headgroup branching. Similarly with increase in alkyl chain length, the r value increases for all the glycolipids studied at 25 °C, suggesting more rigid microenvironment. Table 1. Self-Assembly Properties of Glycolipids.

a

size (d, nm)

CMC (mM)

micropolarity (I1/I3)

Nagg

1a

47.152

1.166

94

1b

2.379

1.151

1c

0.395

2a

glycolipid

anisotropy (r)

HLB DLS

TEM

0.012

3.53

3.38

13.82

89

0.022

4.95

4.76

12.71

1.150

--

0.025

22.44

20.19

11.77

0.625

1.113

61

0.047

5.08

4.57

13.87

2b

0.011

1.064

43

0.095

6.41

5.77

12.48

2ca

0.026

1.053

--

0.088

26.68

23.41

10.86

3a

0.282

1.214

74

0.105

5.35

4.82

15.36

3b

0.024

1.177

33

0.131

6.25

5.62

14.17

3c

0.014

1.125

23

0.175

7.58

6.82

12.70

Studied at 55 ºC.

Size distribution and mean hydrodynamic diameter of micelle were measured by using DLS experiment. The monomodal size distribution was obtained for synthesized glycolipids at experimental condition, which indicates the formation of only one type of aggregate. As size of the aggregates found to be less than 10 nm (except 1c and 2c), suggesting formation of typical micelles. Interestingly, DLS size obtained for hydrophobic glycolipid of I glycolipid series (i.e. 1c) is 22.44 nm, which is quite high with respect to typical micelles. Further, size distribution does not undergo any changes even at an elevated temperature of 50 °C (Figure S2). The hydrodynamic size of glycolipid 1c resembles towards vesicles, tubules, and ribbons. Similarly,


10

Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

hydrophobic glycolipid of Ya glycolipid series (i.e. 2c) also showing particle size higher than the micelle range (26.68 nm). Further to understand aggregation pattern of synthesized glycolipids, HR-TEM study was carried out.

Figure 2. Negatively stained HR-TEM images of I (1a−c), Ya (2a−c), and Ys (3a−c) glycolipids. HR-TEM images obtained for the glycolipids were depicted in Figure 2 and diameter of nanostructures was presented in Table 1. It appears that the average size of aggregate obtained in both DLS measurements as well as TEM studies were almost in similar range. This analysis confirmed the formation of spherical micelles for glycolipids having HLB value greater than 12. Whereas, the hydrophobic glycolipids showed fiber like structure for 1c and bigger spherical aggregate for 2c with a core diameter of 20.19 and 23.41 nm respectively. Similar kind of observations were noticed, in case of polyglycerol dendrons with increase in headgroup size the fiber like structure changed to stable spherical micelles.5 This observations are relate to the solubility of the non-ionic surfactant in water. With increase in head group branching increases the head group polarity, thus by enhancing the water solubility. The lesser soluble molecule with


11

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 36

single head forms the fiber structure. The bigger spherical aggregates obtained for glycolipid 2c are ill-aggregate formed by association of glycolipids. Increase in hydrophobicity of glycolipid 2c favors self association of molecules towards the bigger aggregate formation. In order to confirm the conformational changes of glycolipids upon self-assembly, we have measured CD spectra of glycolipids at 25 °C (except glycolipid 2c). The glycolipids forming micellar aggregates (glycolipid 1a, 1b, 2a, 2b, 3a, 3b, and 3c) did not show any concentration dependence CD bands (Figure S3). At concentrations above CMC, the CD spectra of glycolipid 1c exhibit three bands. Among them, two negative peaks (minima) at 209 and 222 nm originate from π→ π* and n → π* transitions of amide groups in peptide bond respectively. Positive peak (maximum) near 195 nm originated from π→ π* of tertiary amides. The existence of a characteristic CD band as well as its concentration dependence in water indicates the formation of chiral aggregates for glycolipid 1c. Glycolipid 1a and Ys Glycolipids dissolved instantaneously in water at 25 °C due to their high hydrophilicity, whereas glycolipid 1b, 2a, and 2b dissolve slowly in water at 25 °C. Higher temperature is required to dissolve glycolipid 1c and 2c in water. We note that the aqueous solutions of glycolipids (except glycolipid 1c and 2c) are transparent and retain their aggregation behavior even after standing for months together. The aqueous solution of glycolipids 1c and 2c are clear at elevated temperature. Upon cooling, an aqueous solution of glycolipid 2c immediately develops turbidity, whereas glycolipid 1c develops turbidity after few minutes. Below CMC concentration of glycolipid 1c, time taken for turbidity is more than a day. To understand thermal behavior of synthesized surfactants in aqueous solution DSC experiment was carried out. As expected, aqueous micellar solutions of glycolipids (except glycolipid 1c and 2c) do not show any thermal response (Figure 1iii) and confirm that the


12

Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

micelles of these surfactants are thermally stable and stubborn. The glycolipid 2c showed temperature dependent solubility with Krafft point of 54.5 °C. In case of glycolipid 1c, turbid solution was heated from 20 to 60 °C and cooled from 60 to 20 °C (Figure 1iii) in a cycle. A sharp endothermic peak was observed at 46.8 °C during the heating cycle, which was missing while cooling. The endothermic peak may be due to solubilization temperature for glycolipid 1c and cooling profile indicates that the process is not instantly reversible.

Figure 3. Time-dependent aggregation behavior of glycolipid 1c: (i) visual turbidity observation in vial (ii) UV-kinetics study at 400 nm (iii) isothermal behavior (iv) HR-TEM images. Further, to understand the time-dependent aggregation behavior of glycolipid 1c, a kinetic spectrophotometric study was carried out for a pre-dissolved transparent 2 mM aqueous solution (after attaining 25 °C) by measuring the time-dependent absorbance at 400 nm, where the surfactant has zero absorbance. As clearly observed from Figure 3ii, the turbidity starts after 45 minutes and reaches a maximum at 110 minutes and the absorbance suddenly drop after that. The


13

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 36

appearance of turbidity after 45 minutes indicates a slow association of aggregates, which increase with an increase in time. In fact, a similar result was noticed in visual observation (Figure 3i), the solution was transparent up to 40 minutes and after that turbidity starts and finally phase separation occurs at 120 minutes. Any change in phase behavior of a solution will associate with a certain amount of internal energy which can be measured by change in enthalpy of the system. The change in aggregation behavior of glycolipid 1c was monitored through DSC experiment by performing isothermal process at 25 °C (Figure 3iii). A broad exothermic peak was observed between 40 to 80 minutes which further supports our turbidity study. The change in phase behavior, i.e. solution state to precipitation for glycolipid 1c was observed around 40 minutes. From time-dependent TEM images in Figure 3iv, it is observed that the ropes obtained by the self-assemblies of glycolipid 1c are elongating in the axial direction with a constant diameter and there is no sight of other self-assemblies in the solution. From our self-assembly study it may be concluded that the increase in hydrophobic chain length of the glycolipids reduced the CMC (except glycolipid 2c) and Nagg. The major problem associated with the long chain glycolipids as nano-carriers is their solubility and stability in biological media. However, increase in branched headgroup size enhanced the solubility with nominal difference in self-assembly behavior for long-chain glycolipids. In the following section, we focused our studies towards the external stimuli response of glycolipids nanoassembles on the way to drug carriers. 3.2. Stimuli Response. Physical, chemical, and biological stimuli-responsive nano-carriers received greater consideration in the area of nanomedicine.16 In this section external stimuli response on glycolipids whose self-assembled nanostructures are stable over a long period of time at room temperature were examined (glycolipid 1a (400 mM), 1b (100 mM), 2a (10 mM),


14

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

2b (1 mM), 3a (10 mM), 3b (1 mM) and 3c (1 mM)). Initially, the concentration dependence on self-assembly pattern was explored. The size of the glycolipids nano-assemblies are in same range even at higher concentrations below solubility limit (2-100 times of CMC, Figure S4). These glycolipids micelles in water are stable at the observed concentration range, they do not possess any secondary aggregates. To examine stimuli response, the effect of temperature, pH, NaCl concentration, and urea concentration on micropolarity, anisotropy and particle size of glycolipids assemblies were studied. Stimuli response on the micropolarity and anisotropy of glycolipids nano-assemblies explored through fluorescence technique by using pyrene and DPH probes respectively. The temperature effect on micropolarity and anisotropy were shown in Figure 4 (i and v). As observed, both parameters (micropolarity and anisotropy) are slightly decreased with an upsurge in temperature. Further, to understand temperature effect on micellar size of glycolipids, DLS experiments were performed (Figure S5). With an increase in temperature, a minute decrease in micellar size was noticed. The nominal effect of temperature was further supported by DSC experiments, in which the significant enthalpy changes were not observed for these glycolipids (Figure 1iii, glycolipid 1a, 1b, 2a, 2b, and 3a-c). The effect of pH on micropolarity and anisotropy were shown in Figure 4 (ii and vi). The micropolarity values of glycolipids are almost independent of pH, though slight differences in values were obtained at higher and lower pHs for I glycolipids. Similar observations were noticed in case of anisotropy study. The pH-dependent DLS measurements of glycolipids exhibited a nominal increase in micelle size upon acidification/alkalization (Figure S6). Probably, headgroups of glycolipids are not responding to pH changes, whereas the minute changes in self-assembly parameters are attributed to electrolyte effect on water structure. Further, to explore the effect of electrolyte, NaCl was added to aqueous glycolipid solutions.


15

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 36

Below 5 M NaCl concentration, micropolarity and anisotropy values are independent of salt concentration. At higher salt concentration (5 M) the micropolarity values are decreasing, whereas anisotropy values are increasing (Figure 4, iii and vii). Additionally, size of aggregate was increased at 5 M NaCl concentration (Figure S7). For sugar-based surfactants, salt disrupted the hydrophilic hydration of nonpolar tail and solvation of the –OH group containing hydrophilic head, causing easier self-organization of amphiphile. Further, the aggregation number increases with NaCl addition. Hence in turn it increases the size of the aggregate.36,37

Figure 4. Stimuli-responses on the micropolarity (i-iv), and anisotropy (v-viii) of glycolipids assemblies. In general addition of urea enhances salvation of glycolipid headgroup, consequently penetration of water into the micellar core.10 Herein, up to 1 M concentration, effect of urea on micropolarity is insignificant for all the glycolipids (Figure 4iv). Above 1 M urea concentration, micropolarity values are increasing due to water penetration. This effect is more for I glycolipids. In case of anisotropy, values are slightly increasing at lower urea concentration and then approaching the anisotropy value around 0.1. Further DLS measurements showed stable micellar aggregate up to 4 M urea concentration (Figure S8). This indicates up to 4 M urea concentration glycolipids assemblies are stable. Above 4 M urea concentration, urea molecules


16

Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

disrupted the micelle core and solvated the DPH molecules. From above all stimuli response studies it may be inferred that the glycolipids assemblies are resistant to temperature, pH changes, and additives (Urea and NaCl) at physiological conditions. Later, influence of glycolipid geometry on encapsulation of hydrophobic guest and release studies by biological stimuli such as enzyme was explored as discussed in following section. Table 2. Encapsulation capacity and size of Hydrophobic Guest-Loaded Glycolipid Assemblies.

Conc. encapsulation capacity (µM/mM) glycolipid (mM) pyrene curcumin NPN

size (d, nm) pyrene

curcumin

NPN

1a

400

0.96

1.75

7.60

3.54

3.61

3.70

1b

100

3.47

5.28

24.73

4.98

5.07

5.21

2a

10

13.42

21.29

48.71

5.35

6.16

6.53

2b

1

23.94

42.76

144.80

7.15

8.17

11.44

3a

10

9.50

17.89

41.05

5.49

5.93

6.13

3b

1

20.04

37.70

122.62

6.77

7.91

11.06

3c

1

56.01

128.43

244.78

8.86

11.32

12.40

3.3. Hydrophobe Encapsulation and Release. Hydrophobe loaded nano-particles gained immense importance in the area of nano-medicines and agrochemicals.11,38,39 In this section, fluorescent hydrophobic guest molecules were incorporated inside host glycolipid micelles to understand structure-loading relationship. The selected aromatic guest molecules are pyrene, acid responsive NPN, and alkali sensitive curcumin. The guest molecules are incorporated by direct solid dispersion into glycolipid assemblies in water. The characterization of obtained hydrophobe-loaded nano-particles was listed in Table 2. For linear comparison, encapsulation capacities (µM/mM) are represented as µM of guest-loaded for mM of glycolipids in water.


17

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 36

From Table 2, for all guest molecules the encapsulation capacities of glycolipids are increased with increase in alkyl chain length. As expected, sizes of hydrophobe-loaded micelles are increased with increase in loading capacity of glycolipids (Table 2). At a specific alkyl chain length (n = 10 or 14, Figure 1i) encapsulation capacities of Ya glycolipids are higher than Ys glycolipids. Increase in hydrophobicity of the host molecule can improve miscibility with the lipophilic guests and increase the driving force for encapsulation.40 At a particular hydrophobic chain length (n), hydrophobicity of the Ya glycolipids is more than Ys glycolipids, thus enhances the encapsulation capacity. It is interesting to note that 3c shows excellent loading capacity among all synthesized glycolipids. Further highly branched headgroup is always useful for the enhance water solubility of encapsulated glycolipid and long-time stability in aqueous phase to find a practical application in biological system. Usually, nano-carriers can encapsulate multiple hydrophobic guest molecules by non-covalent interaction and releases the guest molecules faster than covalently encapsulated drug molecules.20 After encapsulation of fluorescent guest molecules, release study of guest molecules through the biological stimuli such as enzymes were carried out. Moreover, enzymes are well known biological stimuli and show the time-dependent micellar disassembly, in consequence non-covalently encapsulated drug molecules get released.20,22−26,41−46 Herein, glycolipids under investigation having one or more amide bonds in their molecular structure. Breaking of the amide bond by enzymes (amidases) leads to micellar disassembly and finally the drug release.23,26,41,43,44 HPLC and fluorescence techniques are used to examine the time-dependent drug release profile. Among them, fluorescence method is easy to handle and less time taking method than the HPLC method. However, the results obtained from both method had an excellent correlation.20,25,44 In fluorescence study, after micelle disassembly the water-insoluble


18

Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

solvatochromic dye encapsulated inside the nano-carrier release into water medium and starts precipitating. As a consequence, the fluorescence intensity gradually decreases with micelle disassembly. The fluorescence intensities in the absence of enzyme and after addition of enzyme (at specific time intervals) are used to quantify the percentage of drug release by micelle disassembly.20,25,26,42,44,47 As NPN is acid sensitive, hence judging the release will be extremely difficult in case of PEP responsive hydrophobe release studies at pH 1.5. Similarly in case of pyrene, release could not be quantified as water soluble pyrene has significant fluorescence. Hence the solvatochromic dye curcumin was used for present hydrophobe release studies. Upon addition of enzyme, we observed gradual decrease in color of curcumin loaded aqueous glycolipid solution with time by precipitation of curcumin at bottom. Precipitated poorly water soluble curcumin from the glycolipid assembly was removed by centrifugation followed by filtration, and the percentage release of curcumin was calculated relative to initial fluorescence intensity at 530 nm in absence of specific enzyme.

Figure 5. Enzyme-responsive hydrophobic guest release kinetics of glycolipids: (i) TRYP, (ii) PEP, and (iii) CAL-B. The systematic study on enzyme-sensitive hydrophobe release profile from glycolipid assemblies with the help of amide breaking enzymes (TRYP, PEP, and CAL-B) in water was shown in Figure 5. These enzymes were successfully released the guest molecules by micellar


19

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 36

disassembly of glycolipids. Among all enzymes, PEP showed fast release profile. In general, monomeric glycolipids and self-assembled glycolipids are always in dynamic equilibrium with each other. The monomeric glycolipids present in solution are responsible for enzymatic hydrolysis. Sometimes the increase in hydrophobicity of nano-carriers enables them as enzyme resistant.25,26 For each glycolipid series, the hydrophobe release percentage is decreasing with increasing hydrophobicity (alkyl chain length) and this behavior is independent of enzyme. It should be noted that headgroup of studied glycolipids do not follow any trend on release dynamics. Despite the monomeric concentration, disassembly rates also depend on specific enzyme-glycolipid interactions. For I, Ya, and Ys glycolipid series, number of amide bonds present in the head groups are 1, 2, and 3 respectively. Overall, the simultaneous complex properties such as free monomer concentration, number of cleavable bonds per molecule, and enzyme-glycolipid interactions are collectively responsible for enzyme-sensitive micellar disassembly of the glycolipids and consequent hydrophobe release. Initially, enzyme release of pyrene and NPN solvatochromic dyes encapsulated inside the glycolipids assemblies were studied. For pyrene chemosensors, micelle disassembly caused decrease in excimer emission intensity along with the increase in monomer emission intensity. Cao et al. have shown the pyrene excimer fluorescence intensity is also helpful to quantify the amount of pyrene present inside micelle core.48 Surprisingly, in case of NPN loaded glycolipid assemblies, after attaining pH 1.5 for PEP responsive NPN release studies, the fluorescence intensity is immediately reduced to ~ 50% for glycolipids 1a, 2a, and 3a. In the absence of enzyme (PEP), a similar reduction in fluorescence intensity of NPN was observed at pH 1.5. Moreover, the decrease in fluorescence was happening immediately and it was independent of time. After 24 hours of incubation, fluorescence intensity is matching with intensity obtained


20

Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

prior to the incubation. This instantaneous release shifted our focus towards the pH-sensitive hydrophobe from glycolipid micelles.

Figure 6. Reversible acid/base stimuli-responsive release of acid sensitive NPN (i-viii) and base sensitive curcumin (ix-xvi) guest molecules encapsulated inside the glycolipid assemblies (inset: Repeatability study up to 5 cycles). PY-pyrene, CCM-curcumin, Ac-Acidification, AlAlkalization, and Nu-Neutralization. In general, hydrophobic guest molecules encapsulated in palisade zone of micelles (anionic, cationic, or nonionic) interact with the surrounding aqueous medium. These interactions are responsible for favorable solvation dynamics of hydrophobe in micellar media.49 In the present study, a series of pH-dependent experiments were executed for the acid sensitive NPN and base


21

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 36

sensitive curcumin. For NPN loaded glycolipid assemblies, pH-induced release experiments were performed from pH 7 to 1 by acidification (Figure 6i-vii). Similar release studies were also carried out for acid insensitive pyrene probe in same experimental condition to understand the effect of acid on hydrophobe release (Figure 6i-vii). Further, reloading ability of glycolipid was also monitored through the neutralization study (Figure 6i-vii). The acid/base titration on NPN loaded glycolipid assemblies showed reversible release behavior. The reversibility was examined for 5 cycles and presented in inset of Figure 6i-vii. The complete NPN release was not achieved at pH 1 and maximum release percentage in each glycolipid series is decreasing with an increase in alkyl chain length (Figure 6viii). At acidic pH, NPN molecules at palisade layer of the glycolipid micelles are protonated due to solvation dynamics of hydrophobe in micellar media. Consequently, the increase in hydrophilicity of NPN molecule caused hydrophobe release from micelles. In brief, pH-sensitive hydrophobe release from glycolipid assemblies was observed.

Figure 7. (i) DLS measurements of unloaded and hydrophobe-loaded micelles of glycolipid 3c. Acid/base stimuli-response on size of (ii) NPN and (iii) curcumin (CCM) loaded micelles of glycolipid 3c.


22

Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

In case of curcumin loaded nano-assemblies similar type of pH-induced release experiments were performed from pH 7 to 13 by alkalization with base (Figure 6ix-xvi). The release profile of curcumin showed a sharp release after pH 10 (Figure 6xvi). It should be noted that the third pKa of curcumin is ~ 10 which may be responsible for observed trend in release. At pH 13 encapsulated curcumin was completely released (~ 100%) from glycolipid micelle core. The release was further confirmed by pH-dependent DLS measurements for glycolipid 3c. The aggregate size of glycolipid 3c before and after loading of NPN/ curcumin is shown in Figure 7i. For NPN loaded assembly of glycolipid 3c at pH 1 (31.9% NPN release), the size distribution curve is slightly shifted to the lower side (Figure 7i). At pH 13 (97.3 % curcumin release), size distribution curve of curcumin loaded glycolipid 3c assembly is almost shifted towards the empty micelle range (Figure 7i). Similar to the previous acid/base stimuli-response release studies, pH-sensitive reversibility in size of the aggregate of NPN/ curcumin loaded micelles of glycolipid 3c were observed up to 5 cycles (Figure 7ii/iii). Our findings disclosed the release of pH-sensitive hydrophobic cargo from hydrophobe encapsulated dynamic micelles was achieved by changing pH of system. 3.4. Structural Impact on Glycolipid as Nano-carriers. Specifically designed and synthesized bio-sourced glycolipids can act as biologically active molecules.2,3 Moreover, structurally rigid polymeric glycolipid assemblies showed less biological activity than dynamic micelles of monomeric glycolipid.8 The other aggregates (except micelles) obtained by structural modification of glycolipids also had applications in various fields.4 For example, metal nanowire fabrications by using hollow cylindrical nano-tubes of glycolipid.50 To show the specific activity in water, the aggregates formed by glycolipids should be stable under requisite conditions. In I glycolipid series, CMC is decreasing with an increase in alkyl chain length. A


23

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 36

further rise in alkyl chain length caused the instability in water at 25 ºC and showing temperature- and time-dependent aggregation behavior (glycolipid 1c). Replacing the single headgroup of the glycolipid with multiple headgroups may improve the solubility by retaining their aggregation pattern.9 Further incorporation of hydrophilic aspartic acid successfully improved the solubility of glycolipid (glycolipid 2a). Addition of another glucamine unit to second carbonyl of aspartic acid further enhanced the solubility up to n = 20 (glycolipid 3c). Branching in headgroup retained the formation of nano-micellar aggregates with insignificant change in self-assembly behavior (Figure 8i).

Figure 8. Influence of glycolipids hydrophobic chain length on (i) self-assembly behavior, (ii) stimuli-response, (iii) hydrophobe loading capacity, (iv) enzyme-response, and (v) pHresponsive release of pH-sensitive hydrophobes. To design a stimuli-responsive glycolipid, glycolipid should contain a specific unit that can respond for corresponding external changes. For example, the photosensitive diacetylene or uracil units present in an amphiphile enables them as photosensitive nano-carriers.11,15 The


24

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

temperature (clouding) and NaCl concentration unstable polypropylene glycol and polyethylene oxide present in surfactant headgroup facilitates thermal and NaCl sensitivity in water.15,18 The acid/base responsive groups present in molecular structure of glycolipid shows the pH-driven transformation of glycolipid assemblies (micelle/bilayer/fiber/vesicle).12,19,51,52 In the present study, the water-soluble/stable glycolipids (at 25 ºC) under investigation (glycolipid 1a, 1b, 2a, 2b, 3a, 3b, and 3c) do not have the specific stimuli-sensitive units in their molecular structures. Unlike the ethylene oxide based amphiphiles, linear glycolipid of I glycolipid series with n = 7 exhibited the NaCl concentration and temperature insensitive self-assembly pattern.37 Similarly in the present study, the room temperature soluble glycolipids showed resistance towards NaCl concentration and temperature. Additionally, these glycolipids exhibited resistance towards pH changes due to lack of acid/base responsive groups. Further, the glycolipid assemblies are stable up to 4 M urea concentration. Overall, the synthesized glycolipid nano-assemblies are stable at physiological conditions like temperature, pH, NaCl and urea concentrations (Figure 8ii). The fluorescence micropolarity values of glycolipid solutions inferred the feasibility of these amphiphiles to encapsulate hydrophobic molecules with host-guest mechanism.24,53 In general, covalent encapsulation capacity of guest molecules is higher than the non-covalent encapsulation. However, the release rates are slow for covalently encapsulated hydrophobic cargo.20 In present study, glycolipid assemblies have the feasibility to encapsulate multiple hydrophobic guests. Interestingly, the rise in headgroup size via branching improved solubility without altering the self-assembly behavior (micelle formation) and increase in hydrophilic chain enhanced the loading abilities to a greater extent (Figure 8iii). The presence of enzyme cleavable groups in glycolipid molecular structure enabled them as “enzyme-responsive nano-carriers”.22 Normally, micellar disassembly may happen either by protein binding or amide bond


25

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 36

breaking.21,42,44,54 The synthesized glycolipids are having one or more amide bond in their molecular structure. Therefore the amidases can cleave hydrophilic and hydrophobic groups of monomeric glycolipid molecules for in vitro release.42,44 All the synthesized glycolipid showed release dynamics after addition of enzyme. Generally, the enzymatic micellar disassembly rates change with a change in molecular structure of amphiphile.25,26,44 For example, the increase in hydrophobicity or oxidative dimerization of amphiphilic PEG-dendrons transformed them from enzyme-responsive to irresponsive micellar aggregates.25,44 The decrease in disassembly rate with increase in hydrophobicity among each glycolipid series was observed in current study (Figure 8iv). Interestingly, pH-responsive guest molecules showed reversible pH-responsive encapsulation/release without disturbing the glycolipid self-assemblies (Figure 8v). Finally, these glycolipids improve the solubility and bioavailability of hydrophobic drugs, which may find application in targeted drug delivery system.

4. CONCLUSION In summary, stimuli-responsive self-assembly nature of glycolipid amphiphiles with various headgroup geometry and tail lengths was investigated to establish the structure-property relationship of glycolipid as nano-carriers. Initially, incorporation of hydrophilic part (aspartic acid and glucamine) successfully enhanced the water solubility of glycolipids at higher tail lengths (n = 20). Moreover, branched headgroup geometry retained spherical micelle of size < 10 nm even for higher chain length glycolipid (n = 20). Further, all the glycolipids nano-assemblies exhibits excellent stability towards external stimuli in aqueous medium such as temperature (up to 60 °C), pH (1 to 13), NaCl (up to 1 M), and urea (up to 4 M). Hydrophobic guest encapsulation capacities are significantly enhanced with an increase in hydrophobic chain length of the glycolipid. Amidases such as PEP, TRYP, and CAL-B were successfully dissembled


26

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

hydrophobic guest-loaded glycolipid assembly and released the hydrophobic cargo. However, influence of headgroup geometry on hydrophobic guest release is dependent on the enzyme. For pH-sensitive guest molecules, the glycolipids assemblies exhibited pH-responsive reversible hydrophobic cargo release. Overall, these outcomes are useful to understand the role of glycolipid molecular geometry for efficient drug encapsulation and release.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ACKNOWLEDGMENTS KPCS thanks, UGC, New Delhi for providing fellowship.

ASSOCIATED CONTENT Supporting Information Synthesis, fluorescence quenching, CD spectra, and size distribution of the glycolipids are available.

REFERENCES (1) Valotteau, C.; Calers, C.; Casale, S.; Berton, J.; Stevens, C. V.; Babonneau, F.; Pradier, C. M.; Humblot, V.; Baccile, N. Biocidal Properties of a Glycosylated Surface: Sophorolipids on Au(111). ACS Appl. Mater. Interfaces 2015, 7, 18086–18095. (2) Prasad, Y. S.; Miryala, S.; Lalitha, K.; Ranjitha, K.; Barbhaiwala, S.; Sridharan, V.; Maheswari, C. U.; Srinandan, C. S.; Nagarajan, S. Disassembly of Bacterial Biofilms by the


27

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 36

Self-Assembled Glycolipids Derived from Renewable Resources. ACS Appl. Mater. Interfaces 2017, 9, 40047–40058. (3) Verma, Y. K.; Reddy, B. S.; Pawar, M. S.; Bhunia, D.; Kumar, H. M. S. Design, Synthesis, and Immunological Evaluation of Benzyloxyalkyl-Substituted 1,2,3-Triazolyl α-GalCer Analogues. ACS Med. Chem. Lett. 2016, 7, 172–176. (4) Jung, J. H.; John, G.; Yoshida, K.; Shimizu, T. Self-Assembling Structures of Long-Chain Phenyl Glucoside Influenced by the Introduction of Double Bonds. J. Am. Chem. Soc. 2002, 124, 10674–10675. (5) Trappmann, B.; Ludwig, K.; Radowski, M. R.; Shukla, A.; Mohr, A.; Rehage, H.; Böttcher, C.; Haag, R. A New Family of Nonionic Dendritic Amphiphiles Displaying Unexpected Packing Parameters in Micellar Assemblies. J. Am. Chem. Soc. 2010, 132, 11119–11124. (6) Milkereit, G.; Garamus, V. M.; Gerber, S.; Willumeit, R. Self-Assembly Properties of Alkyloxyethyl β-Glycosides with Different Types of Carbohydrate Headgroups. Langmuir 2007, 23, 11488–11495. (7) Percec, V.; Leowanawat, P.; Sun, H. J.; Kulikov, O.; Nusbaum, C. D.; Tran, T. M.; Bertin, A.; Wilson, D. A.; Peterca, M.; Zhang, S.; et al. Modular Synthesis of Amphiphilic Janus Glycodendrimers and Their Self-Assembly into Glycodendrimersomes and Other Complex Architectures with Bioactivity to Biomedically Relevant Lectins. J. Am. Chem. Soc. 2013, 135, 9055–9077. (8) Schaeffer, E.; Dehuyser, L.; Sigwalt, D.; Flacher, V.; Bernacchi, S.; Chaloin, O.; Remy, J. S.; Mueller, C. G.; Baati, R.; Wagner, A. Dynamic Micelles of Mannoside Glycolipids are more


28

Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Efficient than Polymers for Inhibiting HIV-1 trans -Infection. Bioconjug. Chem. 2013, 24, 1813– 1823. (9) Bhattacharya, S.; Samanta, S. K. Surfactants Possessing Multiple Polar Heads. A Perspective on their Unique Aggregation Behavior and Applications. J. Phys. Chem. Lett. 2011, 2, 914–920. (10) Hierrezuelo, J. M.; Molina-Bolívar, J. A.; Ruiz, C. C. On the Urea Action Mechanism: A Comparative Study on the Self-Assembly of Two Sugar-Based Surfactants. J. Phys. Chem. B 2009, 113, 7178–7187. (11) Gravel, E.; Ogier, J.; Arnauld, T.; MacKiewicz, N.; Ducongé, F.; Doris, E. Drug Delivery and Imaging with Polydiacetylene Micelles. Chem. - A Eur. J. 2012, 18, 400–408. (12) Baccile, N.; Selmane, M.; Le Griel, P.; Prévost, S.; Perez, J.; Stevens, C. V.; Delbeke, E.; Zibek, S.; Guenther, M.; Soetaert, W.; et al. pH-Driven Self-Assembly of Acidic Microbial Glycolipids. Langmuir 2016, 32, 6343–6359. (13) Zahid, N. I.; Abou-Zied, O. K.; Hashim, R.; Heidelberg, T. Fluorescence Probing of the Temperature-Induced Phase Transition in a Glycolipid Self-Assembly: Hexagonal ↔ Micellar and Cubic ↔ Lamellar. Langmuir 2012, 28, 4989–4995. (14) Alkan, A.; Wald, S.; Louage, B.; De Geest, B. G.; Landfester, K.; Wurm, F. R. Amphiphilic Ferrocene-Containing PEG Block Copolymers as Micellar Nanocarriers and Smart Surfactants. Langmuir 2017, 33, 272–279.


29

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

(15) Gebeyehu, B. T.; Huang, S. Y.; Lee, A. W.; Chen, J. K.; Lai, J. Y.; Lee, D. J.; Cheng, C. C. Dual Stimuli-Responsive Nucleobase-Functionalized Polymeric Systems as Efficient Tools for Manipulating Micellar Self-Assembly Behavior. Macromolecules 2018, 51, 1189–1197. (16) Mura, S.; Nicolas, J.; Couvreur, P. Stimuli-Responsive Nanocarriers for Drug Delivery. Nature Materials. 2013, 12, 991–1003. (17) Granata, G.; Paterniti, I.; Geraci, C.; Cunsolo, F.; Esposito, E.; Cordaro, M.; Blanco, A. R.; Cuzzocrea, S.; Consoli, G. M. L. Potential Eye Drop Based on a Calix[4]arene Nanoassembly for Curcumin Delivery: Enhanced Drug Solubility, Stability, and AntiInflammatory Effect. Mol. Pharm. 2017, 14, 1610–1622. (18) Karimi, M.; Zangabad, P. S.; Ghasemi, A.; Amiri, M.; Bahrami, M.; Malekzad, H.; Asl, H. G.; Mahdieh, Z.; Bozorgomid, M.; Ghasemi, A.; et al. Temperature-Responsive Smart Nanocarriers for Delivery of Therapeutic Agents: Applications and Recent Advances. ACS Appl. Mater. Interfaces 2016, 8, 21107–21133. (19) Breitenbach, B. B.; Schmid, I.; Wich, P. R. Amphiphilic Polysaccharide Block Copolymers for pH-Responsive Micellar Nanoparticles. Biomacromolecules 2017, 18, 2839– 2848. (20) Rosenbaum, I.; Harnoy, A. J.; Tirosh, E.; Buzhor, M.; Segal, M.; Frid, L.; Shaharabani, R.; Avinery, R.; Beck, R.; Amir, R. J. Encapsulation and Covalent Binding of Molecular Payload in Enzymatically Activated Micellar Nanocarriers. J. Am. Chem. Soc. 2015, 137, 2276–2284.


30

Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(21) Molla, M. R.; Prasad, P.; Thayumanavan, S. Protein-Induced Supramolecular Disassembly of Amphiphilic Polypeptide Nanoassemblies. J. Am. Chem. Soc. 2015, 137, 7286– 7289. (22) Zelzer, M.; Todd, S. J.; Hirst, A. R.; McDonald, T. O.; Ulijn, R. V. Enzyme Responsive Materials: Design Strategies and Future Developments. Biomater. Sci. 2013, 1, 11–39. (23) Li, N.; Cai, H.; Jiang, L.; Hu, J.; Bains, A.; Hu, J.; Gong, Q.; Luo, K.; Gu, Z. EnzymeSensitive and Amphiphilic PEGylated Dendrimer-Paclitaxel Prodrug-Based Nanoparticles for Enhanced Stability and Anticancer Efficacy. ACS Appl. Mater. Interfaces 2017, 9, 6865–6877. (24) Aluri, R.; Jayakannan, M. Development of L-Tyrosine-Based Enzyme-Responsive Amphiphilic Poly(ester-urethane) Nanocarriers for Multiple Drug Delivery to Cancer Cells. Biomacromolecules 2017, 18, 189–200. (25) Segal, M.; Avinery, R.; Buzhor, M.; Shaharabani, R.; Harnoy, A. J.; Tirosh, E.; Beck, R.; Amir, R. J. Molecular Precision and Enzymatic Degradation: From Readily to Undegradable Polymeric Micelles by Minor Structural Changes. J. Am. Chem. Soc. 2017, 139, 803–810. (26) Harnoy, A. J.; Buzhor, M.; Tirosh, E.; Shaharabani, R.; Beck, R.; Amir, R. J. Modular Synthetic Approach for Adjusting the Disassembly Rates of Enzyme-Responsive Polymeric Micelles. Biomacromolecules 2017, 18, 1218–1228. (27) Feiner-Gracia, N.; Buzhor, M.; Fuentes, E.; Pujals, S.; Amir, R. J.; Albertazzi, L. Micellar Stability in Biological Media Dictates Internalization in Living Cells. J. Am. Chem. Soc. 2017, 139, 16677–16687.


31

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

(28) Jie, Y.; Sumita, B. M.; Yi, H.; Brian, A. S.; Naimul, K.; Afshin, F.; Richard, B. R.; Paul, R. K.; George, W. G. Substituted Saccharide Compounds and Dental Compositions. WO2012036838 A2, Mar 22, 2012. (29) William, C. G. Calculation of HLB Values of Non-Ionic Surfactants. J. Soc. Cosmet. Chem. 1954, 5, 249−256. (30) Tavano, L.; Mauro, L.; Naimo, G. D.; Bruno, L.; Picci, N.; Andò, S.; Muzzalupo, R. Further Evolution of Multifunctional Niosomes Based on Pluronic Surfactant: Dual Active Targeting and Drug Combination Properties. Langmuir 2016, 32, 8926–8933. (31) Maiti, M.; Roy, A.; Roy, S. Effect of pH and Oxygen Atom of the Hydrophobic Chain on the Self-Assembly Property and Morphology of the Pyridyl Boronic Acid Based Amphiphiles. Langmuir 2013, 29, 13329–13338. (32) Pastor, O.; Junquera, E.; Aicart, E. Hydration and Micellization Processes of n-Octyl β-DGlucopyranoside in Aqueous Solution. A Thermodynamic and Fluorimetric Study in the Absence and Presence of Salts. Langmuir 1998, 14, 2950–2957. (33) Hierrezuelo, J. M.; Aguiar, J.; Ruiz, C. C. Micellar Properties of a Mixed Surfactant System Constituted by N-Octyl-β-D-Thioglucopyranoside and Sodium Dodecyl Sulphate. Colloids Surfaces A Physicochem. Eng. Asp. 2005, 264, 29–36. (34) Turro, N. J.; Yekta, A. Luminescent Probes for Detergent Solutions. A Simple Procedure for Determination of the Mean Aggregation Number of Micelles. J. Am. Chem. Soc. 1978, 100, 5951–5952. (35) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York, 2006.


32

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(36) Phillies, G. D. J.; Yambert, J. E. Solvent and Solute Effects on Hydration and Aggregation Numbers of Triton X-100 Micelles. Langmuir 1996, 12, 3431–3436. (37) Pan, A.; Mati, S. S.; Naskar, B.; Bhattacharya, S. C.; Moulik, S. P. Self-Aggregation of MEGA-9 (N-Nonanoyl-N-Methyl-D-Glucamine) in Aqueous Medium: Physicochemistry of Interfacial and Solution Behaviors with Special Reference to Formation Energetics and Micelle Microenvironment. J. Phys. Chem. B 2013, 117, 7578–7592. (38) Chen, G.; Roy, I.; Yang, C.; Prasad, P. N. Nanochemistry and Nanomedicine for Nanoparticle-Based Diagnostics and Therapy. Chem. Rev. 2016, 116, 2826–2885. (39) Nuruzzaman, M.; Rahman, M. M.; Liu, Y.; Naidu, R. Nanoencapsulation, Nano-guard for Pesticides: A New Window for Safe Application. J. Agric. Food Chem. 2016, 64, 1447–1483. (40) Bickerton, S.; Jiwpanich, S.; Thayumanavan, S. Interconnected Roles of Scaffold Hydrophobicity, Drug Loading, and Encapsulation Stability in Polymeric Nanocarriers. Mol. Pharm. 2012, 9, 3569–3578 (41) Cheng, Y.; Huang, F.; Min, X.; Gao, P.; Zhang, T.; Li, X.; Liu, B.; Hong, Y.; Lou, X.; Xia, F. Protease-Responsive Prodrug with Aggregation-Induced Emission Probe for Controlled Drug Delivery and Drug Release Tracking in Living Cells. Anal. Chem. 2016, 88, 8913–8919. (42) Harnoy, A. J.; Rosenbaum, I.; Tirosh, E.; Ebenstein, Y.; Shaharabani, R.; Beck, R.; Amir, R. J. Enzyme-Responsive Amphiphilic PEG-Dendron Hybrids and Their Assembly into Smart Micellar Nanocarriers. J. Am. Chem. Soc. 2014, 136, 7531–7534.


33

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 36

(43) Samarajeewa, S.; Shrestha, R.; Li, Y.; Wooley, K. L. Degradability of Poly(Lactic Acid)Containing Nanoparticles: Enzymatic Access through a Cross-Linked Shell Barrier. J. Am. Chem. Soc. 2012, 134, 1235–1242. (44) Rosenbaum, I.; Avinery, R.; Harnoy, A. J.; Slor, G.; Tirosh, E.; Hananel, U.; Beck, R.; Amir, R. J. Reversible Dimerization of Polymeric Amphiphiles Acts as a Molecular Switch of Enzymatic Degradability. Biomacromolecules 2017, 18, 3457–3468. (45) Azagarsamy, M. A.; Sokkalingam, P.; Thayumanavan, S. Enzyme-Triggered Disassembly of Dendrimer-Based Amphiphilic Nanocontainers. J. Am. Chem. Soc. 2009, 131, 14184–14185. (46) Wang, F.; Klaikherd, A.; Thayumanavan, S. Temperature Sensitivity Trends and MultiStimuli Sensitive Behavior in Amphiphilic Oligomers. J. Am. Chem. Soc. 2011, 133, 13496– 13503. (47) González, D. C.; Savariar, E. N.; Thayumanavan, S. Fluorescence Patterns from Supramolecular

Polymer

Assembly

and

Disassembly

for

Sensing

Metallo-

and

Nonmetalloproteins. J. Am. Chem. Soc. 2009, 131, 7708–7716. (48) Cao, X.; Casier, R.; Little, H.; Duhamel, J. Characterization of the Distribution of Pyrene Molecules in Confined Geometries with the Model Free Analysis. J. Phys. Chem. B 2017, 121, 11325–11332. (49) Adhikary, R.; Carlson, P. J.; Kee, T. W.; Petrich, J. W. Excited-State Intramolecular Hydrogen Atom Transfer of Curcumin in Surfactant Micelles. J. Phys. Chem. B 2010, 114, 2997–3004.


34

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(50) Yang, B.; Kamiya, S.; Shimizu, Y.; Koshizaki, N.; Shimizu, T. Glycolipid Nanotube Hollow

Cylinders

as

Substrates:

Fabrication

of

One-Dimensional

Metallic-Organic

Nanocomposites and Metal Nanowires. Chem. Mater. 2004, 16, 2826–2831. (51) Baccile, N.; Babonneau, F.; Jestin, J.; Pehau-Arnaudet, G.; Van Bogaert, I. Unusual, pHInduced, Self-Assembly of Sophorolipid Biosurfactants. ACS Nano 2012, 6, 4763–4776. (52) Baccile, N.; Cuvier, A. S.; Prévost, S.; Stevens, C. V.; Delbeke, E.; Berton, J.; Soetaert, W.; Van Bogaert, I. N. A.; Roelants, S. Self-Assembly Mechanism of pH-Responsive Glycolipids: Micelles, Fibers, Vesicles, and Bilayers. Langmuir 2016, 32, 10881–10894. (53) Tyagi, R.; Malhotra, S.; Thünemann, A. F.; Sedighi, A.; Weber, M.; Schäfer, A.; Haag, R. Investigations of Host-Guest Interactions with Shape-Persistent Nonionic Dendritic Micelles. J. Phys. Chem. C 2013, 117, 12307–12317. (54) Azagarsamy, M. A.; Yesilyurt, V.; Thayumanavan, S. Disassembly of Dendritic Micellar Containers due to Protein Binding. J. Am. Chem. Soc. 2010, 132, 4550–4551.


35

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 36

TOC Graphic


36

Impact of Glycolipids Hydrophobic Chain Length, Headgroup Size on

Recommend Documents