Supramolecular gels from sugar-linked triazole amphiphiles for drug entrapment and release for topical application

A simple molecular framework obtained by cross-linking a hydrophobic chain with S,S- and R,R-tetritol by the copper-catalysed azide–alkyne cycloaddition reaction is found to serve as an excellent bioisostere for self-assembly. The hexadecyl-linked triazolyl tetritol composite spontaneously self-assembles in n-hepane and methanol to form hierarchical organogels. Microscopic analyses and X-ray diffraction studies demonstrate eventual formation of nanotubes through lamellar assembly of the amphiphiles. A rheological investigation shows solvent-dictated mechanical properties that obey power law behavior similar to other low molecular weight gelators (LMOGs). The gel network was then utilized for the entrapment of drugs e.g. ibuprofen and 5-fluorouracil, with tunable mechanical behaviour under applied stress. The differential release profiles of the drugs over a period of a few hours as a result of the relative spatio-temporal location in the supramolecular network can be utilized for topical formulations.

R, R -and S, S -diethyl tartrate, molecular iodine, sodium borohydride, propargyl bromide, sodium hydride, copper sulphate and sodium ascorbate were purchased from Sigma-Aldrich Chemicals Ltd. 5-Fluoro uracil and racemic ibuprofen were obtained from Sigma. Solvents used for the chemical synthesis acquired from commercial sources were of analytical grade and were used without further purification unless otherwise stated. Solvents for reactions were dried by standard procedures and stored over activated molecular sieves (3Å). Reactions were monitored by TLC, which was performed with 0.2 mm Merck pre-coated silica gel 60 F254 Aluminium sheets.
Compounds were detected by charring i.e. dipping the TLC plates in an ethanolic solution of phosphomolybdic acid (5% v/v) and heating. NMR spectra were recorded on Bruker Advance DPX (400 MHz) spectrometer. Chemical shift are reported in parts per million (ppm) units relative to tetramethylsilane (TMS) as internal standard. Coupling constant (J) are reported in Hertz (Hz).

Synthesis & Characterizations 2.1. Procedure for synthesis of Diethyl-2,3-O-isopropylidene-R, R-tartrate (7a) 1
R, R -Diethyl tartrate (10 g, 48.5 mmol) was added to anhydrous acetone (500 mL) in 1 lit round bottom flask then, iodine (1.29 g, 11.8 mmol) was added to it. The reaction mixture was stirred at room temperature for 3 h. Completion of the reaction was monitored by TLC in 2:1 eluent, after which, aqueous solution of sodium thiosulphate was added to the reaction mixture till it become colorless. The reaction mixture was then concentrated to 1/3rd of its original volume and was diluted with CH2Cl2. The organic layer was washed successively with NaHCO3 solution and water. It was then dried over anhydrous

General procedure for synthesis of various triazole derivatives
Di-O-propargyl derivatives (9a/9b) (1 mmol) and alkyl azide (11) (2 mmol) were taken in stainless steel cup containing 10 stainless steel balls (10 mm diameter). Further CuSO4.7H2O (0.4 mmol) and sodium ascorbate (0.8 mmol) were added to it. The reaction mixture was allowed to grind in S5 PM-100 ball mill at 450 rpm for 6-7 h. The reaction was continuously monitored by TLC using 3:5 eluent. After the completion of the reaction the reaction mixture was dissolved in CH2Cl2 and MeOH. The solvent was evaporated and the crude reaction mixture was purified by column chromatography.

Gelation Study
Gelation was performed by dissolving 0.7 mg (R, R -derivatives) and 1.5 mg (S, S -derivatives) in 1 ml of HPLC grade solvents viz. n-hexane, n-heptane, n-octane, methanol, ethanol dichloromethane, toluene, DMSO, water and 1:1 mixtures of dichloromethane/n-hexane, nhexane/water and methanol/water in a 2 ml glass vials with heating. After complete dissolution, solutions were allowed to remain undisturbed at room temperature for 10 min to obtain gels.

Microscopic study
To get clear TEM images of nanostructures and to avoid occlusion of copper grids by densely packed gel fibres the gel sample was diluted to 0.02% w/v. Then, 5 µL aliquot of the suspension of n-hexane/n-heptane was drop casted on a clean copper grid, the solvent was evaporated completely and the sample was examined under TEM. In case of solvents which does not result organogels such as dicholoromethane and toluene, the samples were not diluted, instead 0.7 %w/v concentration was used and directly placed on copper grid, dried and observed under TECNAI G 2 F-20 high resolution transmission electron microscope (HR-TEM) from FEI.

Circular dichroism spectra
Circular dichroism spectra were recorded using a quartz cell of 1 mm pathlength and data pitch 0.1 nm with a scanning speed of 50 nm/min. For the CD studies of the gels, a weak gel with a concentration of 1.5 mM was first prepared and CD spectra were recorded at 25 o C. Each spectrum is the average of two consecutive scans. The spectra show mirror images of the cotton band for the diluted gels of 15a, 15b in methanol, suggesting supramolecular chiral nature of the assembly. S12

XRD Analysis
The organogels in n-heptane were heated to sol, and 100 L of the sols were individually transferred carefully on a pre-cleaned glass slide and left to air dry for 8 h to form the selfsupported cast films on which measurements were performed using a Model-D8 Advance X-ray diffractometer. The X-ray beam generated with a Cu anode at the wavelength of K1 beam at 1.5418 Å was directed toward the film edge, and scanning was done upto a 2 value of 22°. Data were analysed and interpreted in terms of higher order reflections.
The low intensity peaks at higher angles indicate higher order of packing in the arrangement of gel networks.

In-vitro drug release study HPLC analysis:
For Ibuprofen: The samples for calibration curve were prepared by making 10 ml of 1mg/ml stock solution of ibuprofen. 6 dilutions of 100 ppm, 200 ppm, 400 ppm, 600 ppm, 800 ppm and 1000 ppm were prepared and calibration curve as obtained. The samples at different time points were taken and given directly, without dilution, for HPLC analysis. The HPLC was carried out on LC-10AT VP Shimadzu, using Phenomenex ® , Luna C18(2) HPLC column (with particle size 5 μ and pore size100 Å). Acetonitrile (A) and phosphoric acid (B) (pH 2.50) in the ratio of A:B = 55:45 were used as mobile phase and the flow rate was kept at 2 ml/min. UV-visible detector SPD-10A VP Shimadzu, was used with the detection wavelength of 230 nm at room temperature. The retention time of ibuprofen was found out to be 7.56 min. The samples for calibration curve were prepared by making 10 ml of 1mg/ml stock solution of 5fluorouracil. 6 dilutions of 100 ppm, 200 ppm, 400 ppm, 600 ppm, 800 ppm and 1000 ppm were prepared and calibration curve as obtained. The samples at different time points were taken and given directly, without dilution, for HPLC analysis. The HPLC was carried out on LC-10AT VP Shimadzu, using Phenomenex ® , Luna C18(2) HPLC column (with particle size 5 μ and pore size100 Å). Acetonitrile (A) and phosphoric acid (B) (pH 2.50) in the ratio of A:B = 55:45 were used as mobile phase and the flow rate was kept at 1 ml/min. UV-visible detector SPD-10A VP Shimadzu, was used with the detection wavelength of 260 nm at room temperature. The retention time of 5-fluorouracil was found out to be 3.24 min. From the drug release profile of ibuprofen and 5-fluorouracil (Fig S6), we observed that the maximum cumulative release of 5-fluorouracil i.e. up to 95% occurred within the first 8 h of the study when water was used as the dissolution media. In the case of ibuprofen the release was relatively slower, and after 36 h only 62% of the drug was found released.