Supramolecular Hydrogel Derived from A C3-symmetric Boronic acid

Supramolecular Hydrogel Derived from A C3-symmetric Boronic acid Derivative for Stimuli Responsive Release of Insulin and Doxorubicin. Koushik Sarkar ...
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Supramolecular Hydrogel Derived from A C3-symmetric Boronic acid Derivative for Stimuli Responsive Release of Insulin and Doxorubicin Koushik Sarkar, and Parthasarathi Dastidar Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03326 • Publication Date (Web): 11 Dec 2017 Downloaded from http://pubs.acs.org on December 14, 2017

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Supramolecular Hydrogel Derived from A C3-symmetric Boronic acid Derivative for Stimuli Responsive Release of Insulin and Doxorubicin Koushik Sarkar, and Parthasarathi Dastidar* Department of Organic Chemistry, Indian Association for the Cultivation of Science, 2A & 2B Raja S. C. Mullick Road, Kolkata-700032, India KEYWORDS: Boronic acid, Hydrogel, Honeycomb network, Drug release, Cell imaging ABSTRACT: A C3-symmetric triazine based triboronic acid (HG1) was designed and synthesized. HG1 was found to give hydrogel in DMSO-water (1:9). The hydrogel was rheo-reversible and thermoreversible over a few cycles. Single crystal X-ray diffraction (SXRD) studies on the crystals of HG1 established the presence of honeycomb network in which solvent molecules (DMSO and water) were occluded. SXRD data corroborated well with the hypothesis based on which HG1 was designed. Stimuli responsive release (in vitro) of insulin and doxorubicin from the hydrogel was also achieved.

INTRODUCTION Gelation is a phenomenon wherein a large amount of liquid (solvents) is entrapped and immobilized within a 3D fibrous network arising due to covalent crosslinking of monomers (polymer gel)1,2 or anisotropic self-assembly of small molecules sustained by various supramolecular interactions (supramolecular gel).3-9 The resulting solid-like soft-materials popularly known as gels are becoming increasingly important in material science because of their various potential applications.10-17 Supramolecular hydrogel18 is a special type of supramolecular gel wherein, instead of organic solvent as in organogel, the gelling solvent is pure water or aqueous solvent.19-22 Unlike organogel wherein strong and directional hydrogen bonding is the main driving force for self-assembly of gelator molecules, such hydrogen bonding becomes weak in aqueous media in hydrogel; consequently hydrophobic interactions that lack the strength and directionality become important in aqueous media. Thus a perfect balance between hydrophobic and hydrophilic interactions must be achieved in order to obtain hydrogel. Water being benign and intrinsically biogenic, supramolecular hydrogel has enormous potential in biomedical applications;23,24 it is becoming an attractive alternative of polymeric counterpart mainly because of the following properties that are unattainable by polymer hydrogel – i) rapid response to external stimuli, ii) inherent thermoreversibility and iii) rapid excretion of low molecular weight gelator molecule from the system. However, designing a hydrogelator a priori is nontrivial because the molecular level mechanism of gelation phenomenon (including hydrogelation) is unclear. Nevertheless, there are a few groups involved in designing hydrogelators following systematic molecular approach; for example, van Esch and co-workers reported a rational design of a hydrogelator based on 1,3,5-triamide cis,cis-cyclohexane core;25 Bing Xu and co-workers exploited both hydrophobic (aromatic…aromatic interactions) and hydrogen bonding to achieve the required self-assembly in water resulting in hydrogels.26

We20,21 and others27 reported crystal structures of hydrogelators derived from low molecular weight gelators (LMWGs) that showed lattice inclusion phenomenon.28 Intrigued by the striking analogies that exist between supramolecular gels and lattice occluded crystalline solids (i.e. in both the cases, a large amount of solvents are entrapped within the corresponding networks that are formed by the supramolecular self-assembly of the molecules), we29-31 and others32 made deliberate attempts to develop lattice inclusion compounds suitable for gelation. Lattice inclusion materials may be designed by synthesizing a molecule which either cannot pack in all directions and therefore needs solvent molecules as guest for crystallization or it has sticky functionality that recognizes each other via supramolecular interactions (for example hydrogen bonding) resulting in porous architecture that requires solvent molecules as guests to stabilize the crystal structure. The design strategy for generating such porous architecture lies on the judicious selection of small building units (small molecules) known as molecular tectons33 that can recognize themselves via supramolecular interactions such as H-bonding in a more specific and directional manner. Such supramolecular system, under suitable conditions, may result in gel.

Scheme 1

Like carboxylic acid (-COOH), boronic acid (-B(OH)2) also forms hydrogen bonded dimers; in majority cases, the hydroxyl groups display syn-anti conformation with respect to

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Scheme 2: An overview of the results described in the present work.

the location of the hydrogen atom (type I) whereas dimers displaying anti-anti and syn-syn conformation (type II) are also reported34,35 (Scheme 1). Thus boronic acid functionality can act as sticky site to self-assemble the molecules to form a network structure. Cambridge Structural Database (CSD) analyses reveal that boronic acid functionality has hitherto been marginally exploited to generate designed network structure (Supporting Information). For example, Wuest et al. installed boronic acid functionality on a tetrahedral C and Si and successfully generated 5-fold interpenetrated diamondoid network sustained by boronic acid hydrogen bonding.36 It is understandable that if boronic acid functionality is installed in strategic positions like 1, 3 and 5 position of a C3 symmetric molecule, it can drive the molecules to self-assemble to form a honeycomb network structure having large pores to occlude solvent molecules, which under suitable conditions may result in gel. In this article, we disclosed a new hydrogelator derived from a C3-symmetric triboronic acid derivative of triazine (HG1) having boronic acid functionality installed in the strategic 1,3,5 positions; the hydrogel so derived displayed the ability to entrap and pH responsive release of an anti-cancer drug (doxorubicin or DOX), and also showed glucose responsive release of hydrogel-entrapped insulin. The single crystal structure of HG1 displayed hitherto unknown honeycomb network involving boronic acid dimeric hydrogen bonding interactions and corroborated well with the hypothesis based on which the hydrogelator (HG1) was designed. The gelator molecule being photoluminescent was also exploited to per-

form cell imaging (Scheme 2). Boronic acid based supramolecular hydrogelators were scarcely reported.14, 37-39

EXPERIMENTAL SECTION Materials and Physical measurements: All the chemicals were commercially available and used without further purification. Solvents were laboratory reagent (LR) grade and were used without any further purification. Mouse macrophage RAW 264.7 was purchased from the American Type Culture Collection (ATCC). FT-IR spectra were obtained from a FT-IR instrument (FTIR-8300, Shimadzu). The elemental compositions of the purified compounds were confirmed by elemental analysis (Perkin Elmer Precisely, Series-II, CHNO/S Analyser-2400). TGA analyses were performed on a SDT Q Series 600 Universal VA.2E TA instrument. X-ray powder diffraction patterns were recorded on a Bruker AXS D8 Advance Powder (CuKα1 radiation, λ=1.5406 Å) X-ray diffractometer. TEM images were recorded using a JEOL instrument with 300 mesh copper TEM grid. Scanning electron microscopy (SEM) was performed with a JEOL JMS6700F microscope. Rheology studies were performed using an SDT Q series AR 2000 advanced rheometer. UV−Vis spectroscopic measurements were carried out on a VARIAN CARY 50 Bio UV-Visible Spectrophotometer. NMR spectra (both 1H and 13C) were recorded using 500 or 400 MHz spectrometer (Bruker Ultrasheild Plus-500/400). Emission spectra were recorded with a Horiba Jobin Yvon Fluoromax-4 spectrofluorometer. MTT assay were conducted using a multiplate ELISA reader (Varioskan Flash Elisa Reader, Thermo Fisher). Fluorescence microscopy was done in a Nikon Ti-U eclipse invert-

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Langmuir ed microscope. CD data were collected in a JASCO CD spectrometer (model-J815). Viscosity was collected using a Brookfield viscometer. ImageJ software was used to determine the size of the fibers in TEM and SEM. Synthesis of the precursor ((1,3,5-triazine-2,4,6triyl)tris(benzene-4,1-diyl))triboronic acid (HG1): HG1 was synthesized by acid catalyzed trimerisation reaction with 4-cyanophenylboronic acid. 1.47 g (10 mmol) of 4cyanophenylboronic acid was taken in a 100 ml round bottomed flask and 10 ml of trifluoromethanesulfonic acid was added under ice bath. The reaction mixture was rotated 1 hour in ice bath and at room temperature for overnight. The mixture was worked up by adding drop wise in ice cold water with continuous stirring. The precipitate was collected by filtration and washed with water thoroughly. Yield: 380 mg. elemental analysis of the crystals of HG1: calcd (%) for C23H32B3N3O11S: C 46.74, H 5.46, N 7.11; found: C 46.31, H 5.24, N 7.29; ESI-MS on crude: m/z: 479.339 [M+K]+; FT-IR (KBr): 3724-2717, 1945, 1824, 1613, 1575, 1518, 1466-1205, 1173, 1122, 1014, 841, 809, 720 cm-1. 1H NMR (500 MHz, DMSO-d6, 25°C): δ= 8.712-8.696 (d, J= 8 Hz, 6H), 8.350 (s, 6H), 8.055-8.039 (d, J= 8 Hz, 6H). 13C NMR (500 MHz, DMSO-d6, 250 C): 171.33, 139.60, 136.76, 134.62, 127.66. Synthesis of the precursor 4,4',4''-(1,3,5-triazine-2,4,6triyl)trianiline (NG1): NG1 was synthesized by acid catalyzed trimerisation reaction with 4-cyanoaniline. 1.16 g (10 mmol) of 4-cyanoaniline was taken in a 100 ml round bottomed flask and 10 ml of trifluoromethanesulfonic acid was added under ice bath. The reaction mixture was rotated 1 hour in ice bath and at room temperature for overnight. The mixture was worked up by adding drop wise in ice cold water with continuous stirring. The precipitate was collected by filtration and washed with water thoroughly. Yield: 330 mg. elemental analysis of the crystals of NG1: calcd (%) for C25H26N6O2: C 67.86, H 5.92, N 18.99; found: C 67.51, H 5.64, N 19.29; ESIMS: m/z: 355.6 [M+H]+; FT-IR (KBr): 3282, 2966, 2871, 1629, 1509, 1452, 1373, 1360, 1199, , 892, 705 cm-1. 1H NMR (400 MHz, DMSO-d6, 25°C): δ= 8.36-8.34 (d, J= 8 Hz, 6H), 6.70-6.68 (d, J= 8 Hz, 6H), 5.92 (s, 6H). 13C NMR (400MHz, DMSO-d6, 250 C): 170.80, 154.33, 131.69, 124.01, 114.31. Gelation experiment 10 mg of the HG1 was taken in a vial and 40 µL of DMSO was added and heated. After that 360 µL of water was added to the hot solution and kept undisturbed. After ∼ 2-3 minutes gel was formed. Tgel was measured by the dropping ball method at various gelator concentrations. In this experiment, a glass ball weighing 205.50 mg was placed on a 0.4 mL of hydrogel (at their respective minimum gelator concentration, MGC) taken in a test tube (15x100 mm). The test tube was then immersed in an oil bath placed on a magnetic stirrer to ensure uniform heating. The temperature was noted when the ball touched the bottom of the test tube. TEM sample preparation The sample for TEM was prepared by smearing the hydrogel (2.5 wt %) on a carbon-coated Cu (300 mesh) TEM grid. The grid was dried under vacuum at room temperature for one day and used for recording TEM images using an accelerating voltage of 100 kV without staining. SEM sample preparation SEM sample was prepared by smearing the hydrogel (2.5 wt %) on a SEM stub. The grid was dried under vacuum at room temperature for one day and used for recording SEM images.

Rheological studies: Rheology studies were carried out with an Anton Paar Modular Compact Rheometer MCR 102 on at 25 °C in parallel-plate geometry (25 mm diameter, 1 mm gap). Single crystal X-ray diffraction: Single crystal X-ray data for HG1 was collected using CuKα (λ = 1.54184 Ǻ) radiation on a XtaLAB Synergy, Dualflex, Pilatus 200K four-circle diffractometer equipped with CCD plate Pilatus 200K detector. Data collection, data reduction, structure solution and refinement were carried out using the software package of CrysAlisPro 1.171.39.9b (Rigaku Oxford Diffraction, 2015). The crystal was non-merohedrally twinned (59:41). Single crystal X-ray data for NG1 was collected using MoKα (λ = 0.7107 Ǻ) radiation on a SMART APEX- II diffractometer equipped with CCD area detector. Data collection, data reduction, structure solution and refinement were carried out using the software package of SMART APEX-II. The structure was solved by direct methods and refined in a routine manner. The solvent dioxane molecule was found to be disordered over three positions (SOF- 0.35414, 0.30393 and 0.34195). In all the cases, non-hydrogen atoms were treated anisotropically except for the disordered atoms. Whenever possible, the hydrogen atoms were located on a difference Fourier map and refined. In other cases, the hydrogen atoms were geometrically fixed at their idealized positions. Crystallographic data for the structural analysis of compounds reported herein have been deposited at the Cambridge Crystallographic Data Centre, CCDC Nos. 1541748-1541749. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: +44 1233 336 033; e-mail: [email protected] or http://www.ccdc.cam.ac.uk/deposit). Powder X-ray diffraction: PXRD data were collected using Bruker AXS D8 Advance Powder (CuKα1 radiation, λ = 1.5406 Å) Diffractometer equipped with super speed LYNXEYE detector. The sample was prepared by making a thin film of finely powdered sample (~30 mg) over a glass slide. The experiment was carried out with a scan speed of 0.3 sec/step (step size = 0.02˚) for the scan range of 5-35˚ 2θ. Insulin release study Loading of insulin on the gel matrix: 14 µL of human insulin (1.388 mgmL-1 or 40 IUmL-1) stock solution was diluted by 346 µL of PBS. In a separate vial 13.2 mg of HG1 has been taken and 40 µl of biological grade DMSO has been added and slightly heated and cooled to room temperature to form a colloidal solution. 360 µL insulin solution in PBS, prepared earlier, has been added to the gelator and kept for few minutes until the gel formation takes place. Release of insulin from the gel matrix: Glucose solutions of different concentrations (5, 10, and 20 mM) were prepared in PBS solution of pH 7.4. These glucose solutions (1 mL) were placed on top of the insulin loaded gel and incubated for several hours. For control experiment, PBS solution (without glucose) was layered on the top of the gel bed. The release of insulin has been monitored by taking the supernatant solution each time, and subjected to UV-Vis analysis at λ=276 nm. Doxorubicin adsorption within gel matrix: In a typical experiment, 2 mL solution of 0.1 mg/mL doxorubicin hydrochloride (DOX) has been placed on the top of the gel bed (2.5 wt%) and kept in dark for one week. After one week, the supernatant has been decanted and the amount of DOX adsorbed in the gel bed has been estimated by UV-Vis spectroscopy.

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DOX release study: The release of DOX has been scanned in two different buffer solution e.g. PBS (pH = 7.4) and acetate buffer (pH = 5.0). 2 ml of each PBS and acetate buffer was placed separately on the top of DOX loaded gel samples and incubated at 37 °C for several hours. In each time (3, 6, 9, 12, 24, 48 hours) 200 µL of supernatant has been taken out from the vials and equal amount of respective buffer solutions have been added. The release has been studied from UV-Vis analysis. These release profile has been investigated in triplicate. Biocompatibility studies MTT assay: RAW 264.7 macrophage cells were purchased from American Type Culture Collection (ATCC) and maintained following their guidelines. The cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin and kept in a humidified incubator at 37° C and 5% CO2.The cytotoxicity of HG1 was evaluated in RAW 264.7 cells by using a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay. In a 96-well plates, the cells were seeded keeping density approximately 1×104 cells per well. After 24 h of seeding, the cells were treated with various concentrations (0.25, 0.50, 0.75 and 1.0 mM) of the HG1 or DMEM alone for 72 h in a humidified incubator at 37° C and 5% CO2. The culture medium was then replaced with 100 mg of MTT per well and kept at 37° C and 5% CO2 for 4 h. The formazan produced by mitochondrial reductase from live cells was dissolved by adding DMSO (100 µL per well) and incubated for 30 min at 37° C. The absorbance of formazan was recorded at 570 nm by using a multiplate ELISA reader (Varioskan Flash Elisa Reader, Thermo Fisher). The percentages of survival of cells in HG1 precursor treated samples were calculated by considering the DMEM-treated sample to be 100%. Cell imaging: For cell imaging, RAW 264.7 cells were cultured by using DMEM supplemented with 10% FBS and 1% penicillin– streptomycin on ethanol etched cover slips kept in a 35 mm tissue culture dishes. The dishes were then kept in a humidified incubator at 37° C overnight. Then the cells were washed with PBS and incubated in serum-free media (SFM) for half an hour. DMSO solution of HG1 at IC50 concentration was made by mixing it in serum-containing medium keeping Serum-containing medium: DMSO = 99:1 (v/v). The mixture was shaken thoroughly. The solution was poured cautiously into the cells and incubated for 30 minutes. After incubation, media was discarded and the cells were fixed by using 4% paraformaldehyde for 10 minutes at room temperature. Then the cells were washed with PBS and mounted on glass slides for microscopy.

RESULTS AND DISCUSSION The hydrogelator HG1 was synthesized by acid catalyzed trimerization of 4-cyanophenyl boronic acid at ambient condition in more than 90 % yield (Supporting Information). HG1 was then subjected to gelation studies. Fourteen different solvents (polar, nonpolar and aqueous) were scanned for gelation (Table S1). Interestingly, it could gel only aqueous solvent (1:9 v/v DMSO/water) resulting in hydrogel. The minimum gelator concentration (MGC) was found to be 2.5 wt % with a gel to sol dissociation temperature (Tgel) 57 °C. The hydrogel

was found to be stable at rt. for several weeks and thermoreversible over quite a few heat-cool cycles. Gradual increase in Tgel with the increase in gelator concentration supported the supramolecular nature of the gel network (Figure S6b).40

Figure 1: a) Visco-elastic response in frequency sweep, b) thixotropy and c) rheoreversibility of the hydrogel of HG1.

To study the morphology of the gel network, we then performed high-resolution transmission electron microscopy (HR-TEM) and scanning electron microscopy (SEM) on the hydrogel. Entangled tape like morphology was observed in both the experiments (width 220-525 nm in HR-TEM and 320-650 nm in SEM) (Figure S5). Dynamic rheology (frequency sweep) of the hydrogel (2.5 wt %) revealed that the storage modulus G′ was largely frequency invariant and higher than the loss modulus G″ – a typical characteristics of visco-elastic material like gel. The hydrogel was found to be rheoreversible – an important criterion for biomedical applications such as in drug delivery via topical or subcutaneous routes. In a typical rheoreversibility test, we applied a constant strain (50 %) higher than that of the critical strain (12.8 %) and allowed the sample (2.5 wt %) to relax for 200 s followed by lowering the strain to 0.5 %. At higher strain the sample showed a viscous response (G″ > G′) indicating disruption of the gel network and at lower strain it displayed an elastic response (G′ > G″) confirming gel like behavior; these data clearly established the rheoreversible property of the hydrogel. Such thixotropic behavior of the hydrogel was further confirmed by converting mechanically (vigorously shaken by hand) the hydrogel to a sol which readily reverted back to hydrogel within 15 mins upon resting and such gelsol-gel conversion was possible over a few cycles (Figure 1).

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Langmuir To probe structure-property correlation based on which HG1 was designed (vide supra), attempts were made to grow single crystal of HG1. After several trials, we could crystallize HG1 by slow cooling of a hot solution (DMSO-water) of the hydrogelator. Single crystal X-ray diffraction (SXRD) revealed that the crystal was non-merohedrally twinned (59:41) and belonged to the noncentrosymmetric orthorhombic space group Pna21. The asymmetric unit comprised of fourteen molecules of HG1 and quite a few scattered electron densities which could not be modeled. Analyses revealed that HG1 selfassembled to form a hitherto not reported 2D honeycomb network sustained by boronic acid dimer hydrogen bonding interactions resulting in a large pore (~27 x 30 Å); each pore was further interpenetrated by seven 2D honeycomb network displaying a 8-fold interpenetrated hydrogen bonded network (Figure 2). The scattered electron densities were attributed to disordered solvent molecules (1 DMSO and 4 H2O per asymmetric unit) located within the interstitial space of the network as suggested by the SQUEEZE calculations.41 TGA data on freshly grown crystals of HG1 indicated ~25.8 % weight loss in the temperature range of ~31-132 °C which corroborated well with the SQUEEZE calculation (calc. weight loss for 1 DMSO and 4 H2O ~25.4 %).

inclusion compound from a hot solution of NG1 in 1,4dioxane wherein one of the –NH2 group was found to be involved in N-H…O hydrogen bonding interaction with the O atom of 1,4-dioxane (Figure S10). To probe further the role of boronic acid hydrogen bonded dimer in hydrogelation, we carried out gelation experiments as a function of pH. HG1 was able to produce hydrogel at pH 7.4, 5 and 3 whereas it produced clear solution at pH 10. Boron being a strong Lewis acid, it is expected to be coordinated by hydroxyl ion at alkaline pH (10) thereby changing the coordination geometry of B (in boronic acid) from planar to tetrahedral consequently disrupting the boronic acid hydrogen bonded dimer resulting in a transition from gel to sol. Understandably, the hydroxyl group of boronic acid is not readily available for protonation at lower pH (e.g. 3 and 5) thereby keeping the gel undisturbed. The corresponding morphology in HR-TEM revealed intriguing results; highly entangled 1D tape type of morphology at pH 7.4 was observed indicating stability of the gel network. On the other hand, the tape type of fibers appeared to have been broken into small pieces at lower pH (5 and 3). The effect of lowering the pH on the morphology was more prominent at pH 3 compared to that at pH 5. This could be because of the partial disruption of gel network arising due to protonation of the ring N of triazine moiety of HG1. Such observation was corroborated well with the corresponding viscosity data; a plot of average viscosity (η) vs. pH showed that η gradually increased with the increase in pH up to pH 7.4 and then abruptly dropped to a minimum at pH 10 (Figure 3).

Figure 2: a) Honeycomb network, b) eight fold interpenetration of the honeycomb network in the crystal structure of HG1.

To provide further support to the existence of occluded solvent molecules, freshly grown crystals of HG1 were soaked in MeOD and 1H NMR of the filtrate was recorded; the appearance of a quintet peak at δ 2.05 supported the existence of DMSO (Figure S11-S12). Elemental analysis also supported the SQUEEZE calculations (see experimental section). The forgoing data clearly indicated that the hydrogelator HG1 indeed formed lattice occluded crystalline solid which was in agreement with the hypothesis based on which HG1 was designed (vide supra). However, it was not possible to decipher the exact supramolecular structure of the network in the xerogel; while the PXRD of the bulk solid and the xerogel displayed excellent match, the simulated pattern calculated from SXRD data showed several discrepancies with the other two patterns.6 This may be attributed to the loss of the occluded solvents from the bulk solid as well as the xerogel (Figure S13). The data presented thus far clearly indicated that boronic acid dimeric hydrogen bonding in HG1 played a crucial role in gelation. To support this further, we synthesized a tris-amine analogue of HG1 (devoid of boronic acid functionality, designated as NG1). Expectedly NG1 was unable to gel any solvent studied herein. Since –NH2 functionality cannot form dimeric hydrogen bonding akin to boronic acid moiety, NG1 was unable to form the similar 2D honeycomb network required for gelation. In fact, NG1 was found to crystallize as 1,4-dioxane

Figure 3: Variation of average viscosity and morphology with pH. To probe the role of boronic acid hydrogen bonding in crystalline network formation as well as hydrogelation, we exploited cis-diol-boronic acid chemistry.42 It is well known that cis diol reacts with boronic acid by coordinating the lone pair of electrons of the hydroxyl groups to B atom thereby eliminating one hydroxyl group from B resulting in expected disruption of the hydrogen bonding interactions. Thus, an aqueous solution containing glucose was carefully layered over a hydrogel bed of HG1 at rt. After 48 hrs., the gel bed started breaking and after one week, it turned out to be a colloidal mass indicating complete disruption of boronic acid hydrogen bonding resulting in the breakage of the gel. It may be noted that HG1 was photoluminescent displaying emission band at (λem ≈ 400 nm, λex ≈ 330 nm in DMSO) and the negative

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charge generated on B atom after cis-diol reaction in presence of glucose was expected to increase the PL intensity of HG1. Interestingly, HG1 in solution could easily sense glucose as revealed by PL studies (Figure S15-S16). These results prompted us to study glucose responsive release of insulin from the hydrogel matrix.37 HG1 was found to produce gel in DMSO-Phosphate buffered saline (1:9, pH = 7.4) with slightly higher MGC (3.3 wt %) as compared to that of in DMSO-water (2.5 wt %). The increase in MGC could be because of the increase in polarity of the medium due to the presence of salts in PBS. We first made hydrogel (3.3 wt % containing insulin) in DMSO-PBS in such a way that the final concentration of insulin remained 0.05 mg/ml. Remarkably insulin containing hydrogel thus prepared was found to be stable for more than a week and HR-TEM data revealed that the gel network retained the tape like morphology observed for the hydrogel alone (Figure S17b). We then assessed the glucose responsive insulin release from the hydrogel using varying concentration of glucose in PBS. It was observed that the release of insulin gradually increased with the increase in concentration of glucose (32% in PBS only, 39 % in 5 mM glucose, 58 % in 10 mM glucose and 75 % in 20 mM glucose). Thus, under simulated diabetic conditions43 (10 and 20 mM glucose in human blood serum), the insulin entrapped hydrogel could release a significant amount of insulin. From

The ratio of both these bands (Φ209/Φ223) provides a qualitative idea of the overall conformation of insulin.37, 44 The ratio for excreted insulin from the hydrogel under various glucose concentration and PBS alone was found to be within the range 1.25-1.21 which is nearly equal to that of native insulin (Table S3). Therefore, no significant conformational change took place for the released insulin from the hydrogel network compared to that of the standard one. The data presented thus far clearly indicate the crucial role of boronic acid hydrogen bonding in gelation. Therefore, the hydrogel matrix is may be exploited as a pH responsive drug delivery vehicle. In a model study, we loaded an anti-cancer drug doxorubicin in its hydrophilic form i.e. doxorubicin hydrochloride (DOX) within the hydrogel matrix and monitored its pH responsive release by UV-Vis spectroscopy. It may be noted here that DOX which is devoid of any cis-diol is not expected to react with boronic acid moiety. In a typical experiment, 0.1 mM aqueous solution of DOX was layered on the top of 400 µL gel bed (2.5 wt%) and kept at rt. for one week. The data revealed that the maximum loading of the drug was 76% (Figure S20). The DOX entrapped hydrogel bed was then subjected to be in contact with phosphate buffered saline (PBS, pH = 7.4) and acetate buffer (pH = 5.0) by layering the corresponding buffer solution on the top of the hydrogel bed in separate experiments.

Figure 5: a) Image of DOX adsorption on the HG1 gel matrix, b) release of DOX from HG1 gel matrix in a pH responsive manner.

Figure 4: a) Release of insulin from the gel matrix of HG1 in presence of varying concentration of glucose, b) CD spectra of standard and released insulin. circular dichroism (CD) spectra it was evident that no significant denaturation of insulin was taken place under the experimental conditions (Figure 4). Native insulin displays two negative bands at λ= 209 and 223 nm in the CD spectra due to the existence of α and β-helical structure of insulin, respectively.

Time dependent UV-Vis data of the supernatant solution revealed that the pH responsive release of DOX was much higher (~56 %) in the case of acetate buffer compared to that of in PBS (~25 %) (Figure 5). The result is quite intriguing as it is well known that in cancer cells, the pH is quite acidic45 and such DOX loaded hydrogel may be used in treating skin cancer (for example Kaposi sarcoma) via topical route.46 To demonstrate further the slow and sustained release of DOX from the hydrogel matrix, we carried out in vitro studies using RAW 264.7 cell line. In these experiments, we first loaded a known amount of DOX in the hydrogel matrix, prepared a xerogel (DOX@HG1) and carried out MTT assay on RAW 264.7 cells. The results were then compared with the MTT assay data obtained using only DOX. It was observed that the percentage survival of the cells was much better in the case of DOX@HG1 (∼62% at 1 µM) compared to that of free DOX (∼46% at 1 µM) indicating slow and sustained release of the drug from the gel matrix (Figure S21). Photoluminescence of HG1 (vide supra) prompted us to undertake cell imaging studies. MTT assay on RAW 264.7 cells revealed that the IC50 of HG1 was 1 mM after 72 hrs (Figure S22). Thus for cell imaging studies, we kept the concentration of HG1 to 1 mM. The cells treated with 1 mM HG1 could be nicely observed under fluorescence microscope with green emission (λem = 560 nm) when excited with blue light (λex = 480 nm) upon 30 mins

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Langmuir incubation at 37 °C (Figure 6). It may be mentioned here that such experiments with 4 hrs incubation did not produce any emission which may be because of the phagocytic behavior47 of RAW 264.7 cells (Figure S23). Manual Z stacking also ruled out the possibility of adherence of HG1 on the cell and confirmed the uptake of the compound within the cells (for Z stacking animation video, see supporting information). The possibility of auto fluorescence was also ruled out by a control experiment in which imaging was done in the absence of HG1 (Figure S24). To probe that the green emission was indeed due to internalization, we excited the solution of HG1 at 480 nm (blue) and the corresponding emission peak was observed at ~540 nm (green) (Figure S25). This observation clearly established that HG1 was indeed internalized. It may be noted that DMSO in low concentration (~1%) is routinely being used in various biological assays.48, 49

Figure 6: Fluorescence microscopic images of RAW 264.7 cells incubated with HG1; a) bright field, b) fluorescence and c) overlay.

CONCLUSION Thus, we presented a unique design of a boronic acid functionalized C3-symmetric molecule (HG1) that was capable of forming hydrogel. The hitherto unreported 8-fold interpenetrated honeycomb network sustained by hydrogen bonded boronic acid dimer left enough void space to occlude solvent molecules (DMSO/water) in the crystal lattice of HG1 – an observation that corroborated well with the strategy of designing the hydrogelator. The hydrogel displayed glucose responsive release of insulin and pH responsive release of doxorubicin in in vitro studies. HG1 was also found to be internalized in RAW 264.7 cell line as revealed by the cell imaging studies. Thus, the result presented herein is an elegant example of the design of a functional hydrogelator based on a rational approach.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental section, gelation studies, ORTEP plots, crystal data, TGA, PXRD patterns and biological studies.

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

ACKNOWLEDGMENT K.S thanks CSIR (Grant No. 09/080(0882)/2013-EMR-1) for research fellowship. P.D thanks DST (Grant No. EMR/2016/000894) for financial support. Single crystal X-ray data of HG1 has been collected in a Rigaku oxford diffractometer

and partly solved by Dr. Dyanne Cruickshank, UK. Single crystal X-ray diffraction facility at the Department of Organic Chemistry, IACS supported by CEIB program of DBT (BT/01/CEIB/11/V/13) was used for data collection of NG1. We thank Mr. Saswat Mohapatra, IICB Kolkata for helping in cell imaging.

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Boronic acid hydrogen bonding interactions have been exploited to design a supramolecular hydrogel displaying glucose responsive and pH responsive release of insulin and doxorubicin, respectively.

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