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Supplementary Data
Fabrication of nanostructures through self-assembly of non-ionic amphiphiles for biomedical applications
Suchita Prasad,a Katharina Achazi,b Christoph Bӧttcher,c
Rainer Haag,b,* Sunil K. Sharmaa,*
aDepartment of Chemistry, University of Delhi, Delhi 110007, IndiabInstitut für Chemie und Biochemie, Freie Universität Berlin, Takustraße 3,
14195 Berlin, GermanycForschungszentrum für Elektronenmikroskopie, Institut für Chemie und Biochemie,
Freie Universität Berlin, Fabeckstraße 36a, 14195 Berlin, Germany
Table of Contents
1. Experimental Section1.1. Materials1.2. Methods and Instrumentation1.3. Synthetic Procedures2. FiguresFigure S1: 1H & 13C NMR spectra of 2,2-di(prop-2-yn-1-yl)propane-1,3-diyl bis(4-(decyloxy)benzoate) (9)Figure S2: 1H & 13C NMR spectra of 2,2-di(prop-2-yn-1-yl)propane-1,3-diyl bis(4-(octadecyloxy)benzoate)
(10) Figure S3: 1H & 13C NMR spectra of methoxypolyethylene glycol carboxymethyl ether (13) Figure S4: 1H & 13C NMR spectra of methoxypolyethylene glycol carboxymethyl ether (14)Figure S5: 1H & 13C NMR spectra of (methoxypolyethylene glycol carboxymethyl ether)ethylester (15)Figure S6: 1H & 13C NMR spectra of (methoxypolyethylene glycol carboxymethyl ether)ethylester (16)Figure S7: 1H & 13C NMR spectra of 2-azidopropane-1,3-diyl bis(methoxypoly(oxyethylene)oate) (18) Figure S8: 1H & 13C NMR spectra of 2-azidopropane-1,3-diyl bis(methoxypoly(oxyethylene)oate) (19) Figure S9: 1H & 13C NMR spectra of amphiphile 20 Figure S10: 1H & 13C NMR spectra of amphiphile 21 Figure S11: 1H & 13C NMR spectra of amphiphile 22 Figure S12: DEPT-135 NMR spectrum of amphiphile 21 Figure S13: 2D HETCOR and COSY NMR spectra of amphiphile 22 Figure S14: Gel permeation chromatogram of amphiphiles 20-22 Figures S15, S16 and S17: Critical aggregation concentration (CAC) of amphiphiles 20 and 21 in aqueous
solution by surface tension and fluorescence measurements at 25 oCFigure S18: Size distribution profile (by intensity, volume and number) of 20-22 before encapsulation Figure S19: Cryo-TEM micrographs of amphiphiles 20 and 21
Figure S20: Size distribution profile (by volume) of 20-22 after encapsulation Figure S21: Variation of the fluorescence intensity of Nile red with varying amounts of amphiphile 20 and Nile
red in water Figure S22: UV absorbance spectra of Nile red encapsulated samples in methanolFigure S23: UV absorbance spectra of nimodipine with varying amounts of nimodipine in aqueous solution of
amphiphile 20 at 5 mg mL-1 concentration Figure S24: UV absorbance spectra of nimodipine encapsulated samples in ethanol Figure S25: Calibration curve of curcumin in methanol Figure S26: UV absorbance spectra of curcumin encapsulated samples in methanol using (a) 1 mg and (b) 2.5
mg of curcuminFigure S27: HPLC chromatogram of dexamethasone encapsulated samples using 1 mg of dexamethasoneFigure S28: HPLC chromatogram of dexamethasone encapsulated samples using 2.5 mg of dexamethasone Figure S29: Confocal laser scanning fluorescence microscopy images from A549 cells after 5 and 24 h
incubation with Nile red encapsulated by amphiphiles 20 and 22 and non-treated control cells3. References
1. Experimental Section
1.1. Materials
All the chemicals and solvents used were obtained from Spectrochem Pvt. Ltd., India and Sigma-
Aldrich Chemicals, USA. Immobilized Candida antarctica lipase (Novozym 435) was procured
from Julich Chiral Solutions GmbH (Jülich, Germany). All the dyes/drugs used for encapsulation
studies were purchased from Fluka Chemie GmbH, (Buchs, Switzerland) and Sigma-Aldrich
Chemicals, USA with maximum purity. The solvents used in the reactions were dried and
distilled prior to use. Pre-coated TLC plate (Merck silica gel 60F254) was used to monitor the
progress of the reactions with visualization of the spots on TLC using cerric solution. Silica gel
(100-200 mesh) was used for column chromatography. Benzoylated dialysis tubing (molecular
weight cut-off (MWCO) 2000 Da), procured from Sigma-Aldrich was used for the purification
of amphiphiles. Millipore water used for preparing samples for physico-chemical
characterization and transport analysis, was obtained from Merck Millipore Milli-Q Integral
System. Cremophor® ELP (purified grade of Cremophor® EL (polyethoxylated castor oil)) was
obtained from BASF, Ludwigshafen, Germany.
1.2. Methods and Instrumentation
Infrared spectra (IR) of the samples were recorded using a Perkin-Elmer FT-IR model 9
spectrometer. The 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on
JEOL 400 MHz, Bruker DRX 400, and Bruker AMX 500 MHz spectrometers with referencing
done using the residual solvent peak. The chemical shift values are on a δ scale and the coupling
constant values (J) are in Hertz. High-resolution mass spectrometry (HRMS) data were recorded
on Q-TOF LCMS-Agilent Technology-6530 and HPLC/MS - Agilent 6210 (Agilent
Technologies).
1.2.1. Gel permeation chromatography (GPC)
An Agilent GPC system equipped with Agilent 1100 pump, refractive index detector, and PLgel
columns, was used to determine the molecular weight w, n and z of amphiphiles using M M M
tetrahydrofuran (THF) as an eluent at a flow rate of 1.0 mL min-1 and molecular weight
dissolved in dimethylformamide (DMF) (50 mL) were stirred at room temperature for 30 min
followed by the addition of 1-bromoalkane (1.1 equiv.). The reaction mixture was stirred at 40 oC and on completion of the reaction (6 h), it was poured over ice. The solid so obtained was
filtered, washed with water and dried. Further, a solution of ethyl 4-(n-alkyloxy)benzoate (5/6) (1
equiv.) in ethanol (50 mL), refluxed along with aqueous potassium hydroxide (KOH) solution (4
equiv., 3 mL) for 4 h followed by acidification with hydrochloric acid (HCl) solution (1N) led to
the formation of the hydrolyzed product, 4-(n-alkyloxy)benzoic acid (7/8) in almost quantitative
yield.
1.3.1.4. Synthesis of 2,2-di(prop-2-yn-1-yl)propane-1,3-diyl bis(4-(n-alkyloxy)benzoate) (9/10).
To a stirred and clear solution of 2,2-di(prop-2-yn-1-yl)propane-1,3-diol (3) (0.5 g, 1 equiv.) and
4-(n-alkyloxy)benzoic acid (7/8) (2.5 equiv.) in anhydrous dichloromethane (DCM):DMF (4:1,
100 mL) was added N-(3-dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC) (3
equiv.) followed by 4-dimethylaminopyridine (DMAP) (1.2 equiv.) at 0 oC. The reaction mixture
was stirred at 30 oC for 48 h with subsequent removal of the solvent under reduced pressure. The
resulting product was extracted with chloroform (3 x 100 mL) and dried over anhydrous sodium
sulphate. Removal of the solvent and subsequent purification by column chromatography using
silica gel (Ethyl acetate:Pet. ether :: 1:49) afforded 2,2-di(prop-2-yn-1-yl)propane-1,3-diyl bis(4-
Figure S14. Gel permeation chromatogram of amphiphiles (A) 20 (B) 21 (C) 22.
A
B
C
Figure S15. Critical aggregation concentration (CAC) of amphiphiles 20 and 21 in aqueous solution by surface tension measurements at 25 oC.
Figure S16. Critical aggregation concentration (CAC) of amphiphiles 20 and 21 in aqueous solution by pyrene fluorescence measurements at 25 oC.
Figure S17. Critical aggregation concentration (CAC) of amphiphiles 20 and 21 in aqueous solution by Nile red fluorescence measurements at 25 oC.
20 21
20 21
20 21
Figure S18. Size distribution profile (by intensity, volume and number) of 20-22 before encapsulation.
Figure S19. Cryo-TEM micrographs of amphiphiles (a) 20 and (b) 21 showing spherical micellar particles in the < 5 nm range. Scale bar is 100 nm.
a
b
Figure S20. Size distribution profile (by volume) of 20-22 after encapsulation.
Figure S21. Variation of the fluorescence intensity of Nile red with varying amounts of amphiphile 20 and Nile red in water.
Figure S22. UV absorbance spectra of Nile red encapsulated samples in methanol.
Figure S23. UV absorbance spectra of nimodipine with varying amounts of nimodipine in aqueous solution of amphiphile 20 at 5 mg mL-1 concentration.
Figure S24. UV absorbance spectra of nimodipine encapsulated samples in ethanol.
0 2 4 6 8 10 12 14 160
0.2
0.4
0.6
0.8
1
Concentration (µM)
Abs
orba
nce
Figure S25. Calibration curve of curcumin in methanol.
Figure S26. UV absorbance spectra of curcumin encapsulated samples in methanol using (a) 1 mg and (b) 2.5 mg of curcumin.
a b
Figure S27. HPLC chromatogram of (a) blank dexamethasone (1 mg) (b) dexamethasone (1 mg) encapsulated in 5 mg mL-1 of 20 (c) dexamethasone (1 mg) encapsulated in 5 mg mL-1 of 21 (d) dexamethasone (1 mg) encapsulated in 5 mg mL-1 of 22.
Figure S28. HPLC chromatogram of (a) blank dexamethasone (2.5 mg) (b) dexamethasone (2.5 mg) encapsulated in 5 mg mL-1 of 20 (c) dexamethasone (2.5 mg) encapsulated in 5 mg mL-1 of 21 (d) dexamethasone (2.5 mg) encapsulated in 5 mg mL-1 of 22.
a b
c d
a b
c d
Figure S29. Confocal laser scanning fluorescence microscopy images from A549 cells after 5 and 24 h incubation with Nile red encapsulated by amphiphiles 20 and 22 and non-treated control cells. Nile red is shown in red color and labeled early endosomes and lysosomes in green. The bright field channel is shown in grey scale.
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1951.3. A. C. Rodrigo, A. Barnard, J. Cooper and D. K. Smith, Angew. Chem. Int. Ed., 2011, 50, 4675-
4679.4. E. Fleige, B. Ziem, M. Grabolle, R. Haag and U. Resch-Genger, Macromolecules, 2012, 45,
9452-9459.5. V. Kumar, B. Gupta, G. Kumar, M. K. Pandey, E. Aiazian, V. S. Parmar, J. Kumar and A. C.
Watterson, J. Macromol. Sci., Pure Appl. Chem., 2010, 47, 1154-1160.6. S. Das, A. Bedi, G. R. Krishna, C. M. Reddy and S. S. Zade, Org. Biomol. Chem., 2011, 9,
6963-6972.7. L. Q. Xu, F. Yao and G. D. Fu, Macromolecules, 2009, 42, 6385-6392.
8. X. Y. Hu, K. Jia, Y. Cao, Y. Li, S. Qin, F. Zhou, C. Lin, D. Zhang and L. Wang, Chem. Eur. J., 2015, 21, 1208-1220.
9. S. Gupta, B. Schade, S. Kumar, C. Böttcher, S. K. Sharma and R. Haag, Small, 2013, 9, 894-904.