Hydrogen-Bonding-Induced Chain Folding and Vesicular Assembly of

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Hydrogen-Bonding Induced Chain Folding and Vesicular Assembly of an Amphiphilic Polyurethane Tathagata Mondal, Krishna Dan, Jolly Deb, Siddhartha S Jana, and Suhrit Ghosh Langmuir, Just Accepted Manuscript • DOI: 10.1021/la401008y • Publication Date (Web): 12 May 2013 Downloaded from http://pubs.acs.org on May 20, 2013

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Hydrogen-Bonding Induced Chain Folding and Vesicular Assembly of an Amphiphilic Polyurethane †

†

‡

‡

†

Tathagata Mondal, Krishna Dan, Jolly Deb, Siddhartha S. Jana and Suhrit Ghosh * †

Polymer Science Unit, Indian Association for the Cultivation of Science, Kolkata- 700032,

India; ‡ Biological Chemistry Department, Indian Association for the Cultivation of Science, Kolkata- 700032, India KEYWORDS. Amphiphilic Polyurethane; Hydrogen-Bonding; Chain-Folding; Self-Assembly; Zwitterionic-Polymersome. ABSTRACT. In this article we have reported synthesis and vesicular-assembly of a novel amphiphilic polyurethane with hydrophobic backbone and hydrophilic pendant carboxylic acid groups which were periodically grafted to the backbone via a tertiary amine group. In aqueous medium the polymer chain adopted a folded conformation which was stabilized by intra-chain Hbonding among the urethane groups. Such a model was supported by concentration and solventdependent FT-IR, powder XRD and urea-mediated “denaturation” experiments. Folded polymer chains further formed vesicular assembly which was probed by dynamic light scattering, TEM, AFM, SEM and fluorescence microscopic studies and dye encapsulation experiments. pHdependent DLS and fluorescence microscopic studies revealed stable polymersome in entire tested pH window of 3.5 to 11.0. Zeta potential measurements showed negatively charged

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surface in basic pH while charge-neutral surface in neutral and acidic pH. MTT-assay with CHO cell line indicated good cell viability. INTRODUCTION Self-assembly of amphiphilic polymers

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has been studied extensively with diverse

macromolecular architectures including block copolymer, random copolymer, homopolymer, dendrimer, hyper-branched polymers, graft copolymer and so forth. Key motivation is to understand structural effects on nature of self-assembly (micelle, vesicle, hydrogel etc) and their properties such as stability, size, bio-compatibility, guest encapsulation ability and critical aggregation concentration. These properties are highly relevant in context of applicability of a particular material in biomedical domain

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such as delivery of therapeutics, tissue engineering

and imaging. Remarkable advancement in various controlled radical polymerization techniques has helped in enriching this field by bringing new functional monomers into the fore which are capable of influencing the self-assembly by precisely defined directional supramolecular interactions such as H-bonding, pi-stacking, metal-ligand interactions and others.10-19 A review in this field will reflect that such supramolecular engineering with macromolecular scaffolds mostly revolves around polymers those have been synthesized by living/controlled chain polymerizations. This is possibly due to the coincidental overlap between the growth of these two independent research areas during last three decades. On the other hand many of the polymers in commercial use (polyester, polyamide, polyurethane, polyimide, various semiconducting polymers) are made by step growth polymerization routes which offer more options in terms of structural variation in the polymer backbone compared to chain polymerization. Thus a revisit to those systems, in light of the knowledge gathered in the area of supramolecular interactions and self-assembly of block copolymers, may offer newer opportunities to develop future materials for


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biological applications. In this context we are particularly interested about polyurethane

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because (i) their synthesis is straightforward and well-documented in literature (ii) selfcomplementary H-bonding functionality urethane is inherently embedded in the polymer backbone (iii) they possess excellent biocompatibility and have been used as biomaterials since long.21-22 Many water soluble/dispersible polyurethanes have already been reported and their aggregation in aqueous medium is well-documented.23-29 But surprisingly in most cases amphiphilicity is introduced by incorporation of hydrophilic segments in the backbone itself while only few reports are known on molecular engineering in the pendant.30-34 Even those few do not elaborate on self-assembly aspects. We envisaged grafting hydrophilic groups onto the polymer backbone will be advantageous because it would not hinder chain folding-induced self-assembly stabilized by intra-chain H-bonding among the urethane groups. Based on these logistics we have synthesized an amphiphilic polyurethane (P1, Scheme 1) with hydrophobic backbone and pendant hydrophilic carboxylic acid group which is periodically attached to the backbone by a tertiary nitrogen atom. Co-inclusion of carboxylic acid and tertiary amine in the hydrophilic segment is by design to realize pH-response schizophrenic behavior.35 In this article we report spontaneous pH-responsive anionic/ zwitterionic vesicular assembly 36-39 of P1 by intra-chain Hbonding induced chain-folding and preliminary cell-culture results.


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HO

OH

N

DABCO, THF, 60 oC H3COOC

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*

O

N

O

1 ROOC

+ OCN

H N

O

NCO

R = CH3, P1E R = H, P1

O N H

* n

NaOH. H2O/MeOH, rt, H+

2

Scheme 1: Synthesis (top) and intra-chain H-bonding induced vesicular assembly (bottom) of P1

RESULTS AND DISCUSSION Synthesis and characterization: Commercially available di-ethanol amine was treated with methyl acrylate to produce diol 1 in almost quantitative yield which was condensed with hexamethylene di-isocyanate (2) in presence of catalytic amount of DABCO to produce the prepolymer P1E (Scheme 1) in 75 % yield.40 It was structurally characterized by 1H NMR (Figure 1) and FT-IR (Figure S1). Molecular weight (Mn = 10000 gm/mol, PDI = 2.5) was estimated by GPC analysis (Figure S2). It was then hydrolyzed under basic condition to produce P1 which unlike P1E was not easily soluble in any solvent except H2O and DMSO. Ester hydrolysis could be confirmed by 1H NMR which showed complete disappearance of the –COOCH3 peak at δ = 3.57 ppm (Figure 1). However other peaks remained almost invariant suggesting polymer backbone was stable under basic hydrolysis condition.


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Figure 1: 1H NMR spectra of P1E (bottom, black) and P1 (top, blue) in DMSO-d6 (* indicates peaks from solvent); inset shows urethane peak corresponding to urethane proton

Self-assembly studies: To check self-assembly, P1 was directly dissolved in aqueous NaOH solution (repeat unit: NaOH = 1: 2) which was then subjected to extensive dialysis for removal of excess base prior to physical studies. Transmission electron microcopy images (Figure 2a, S3) of this solution (pH = 8.0 ) showed presence of spherical aggregate with thin wall (wall thickness ~ 3 nm) and hollow interior with diameter in the range of 200-250 nm suggesting vesicular assembly. Morphology was confirmed by atomic force microscopic studies (Figure 2b) which revealed similar spherical aggregates with width and height in the range of 280 and 38 nm (a-b, Figure 2c), respectively. Significantly less height compared to the width is attributed to flattened spheres which is expected for soft vesicular assembly and is consistent with literature reports.41-42 Scanning electron microscopic (SEM) images also showed (Figure S4) collapsed near spherical morphology supporting vesicular assembly. Microscopic results were corroborated well with DLS (Figure 2d) measurements which revealed average hydrodynamic diameter of the aggregates


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to be 290 ± 50 nm. Further concentration dependent DLS measurements were performed which showed (Figure 2e) almost similar particle size in a wide concentration window of 2.0-0.015 mM suggesting similar vesicular-assembly even at very dilute condition.

Figure 2: (a) TEM and (b) AFM images of aqueous P1 solution (C = 0.5 mM, pH = 8.0); (c) height profile along a-b in figure 2b; (d) DLS data (C = 0.5 mM, pH = 8.0) showing size distribution (number average) of aggregates formed by P1; (e) Concentration dependent DLS profile of aqueous P1. Dye encapsulation: Vesicular morphology can be verified by presence of confined water pool inside the assembly which can be tested by the ability of the aggregate to encapsulate hydrophilic guest molecules.

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Such guest encapsulation ability of P1 was tested using a hydrophilic

fluorescent dye Calcein. Calcein was treated with aqueous P1 (C = 0.5 mM, pH = 8.0) and the resulting solution was extensively dialyzed to completely remove any non-encapsulated free dye. The dialyzed solution showed characteristics emission peak of Calcein with λmax = 512 nm (Figure 3a) indicating dye encapsulation. Please note any non-encapsulated dye should have been removed during dialysis process. Further, emission intensity of P1 encapsulated Calcein was compared to that of the absorption matched solution of the free dye in water. Much reduced


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emission was notice for vesicle encapsulated dye which can be attributed to self-quenching in the confined water-pool inside the vesicle.43-44 Dye encapsulated solution was further examined under fluorescence microscope which showed green emitting spherical particles (Figure 3b) confirming effective Calcein encapsulation inside polymersome. To determine the critical aggregation concentration (CAC), emission spectra of Calcein encapsulated polymer solution was monitored as a function of dilution (Figure S5). Emission intensity showed linear relationship with polymer concentration till ~10-5 M of P1 suggesting no disassembly at least till this point. In case of disassembly, one would expect release of vesicle encapsulated dye to bulk water. That should have caused increase in emission intensity due to absence of self-quenching effect in bulk water at that dilution. Thus it can be concluded CAC of P1 is below 10-5 M. Further encapsulation of pyrene, a hydrophobic dye, was also tested while intense emission peaks (Figure 3c) were noticed in the range of 370-425 nm with vibrational fine structure along with a broad excimer emission band around 450-500 nm. But the ratio of the first (I1) and third (I3) vibrational peaks was intriguingly found to be 1.46 suggesting the probe was located in rather hydrophilic environment.45 It is proposed that tightly packed H-bonding stabilized chain-folded bilayer (Scheme 1) did not allow guest insertion to avoid any possible disruption of the H-boding network and thus pyrene was probably located in outer surface of the bilayer and more exposed to aqueous environment.


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Figure 3: (a) Absorbance matched photoluminescence spectra (λex = 450 nm) of Calcein encapsulated in P1 vesicles (P1 = 0.5 mM and pH = 8.0) and free water; (b) Fluorescence microscopic images (λex = 450 nm) of Calcein encapsulated P1 vesicle; (c) Emission spectra of pyrene (λex= 337 nm) encapsulated in aqueous P1 vesicle. Proposed model of self-assembly: Vesicular assembly of P1 is depicted in Scheme 1 which assumes bilayer formation by folding of the hydrophobic polymer backbone driven by intra-chain H-bonding among the self-complementary urethane groups. To support this model FT-IR spectra of P1 in MeOH and H2O were compared (Figure 4a). It can be clearly seen that the peak corresponding to carbonyl stretching of urethane group which appeared at 1688 cm-1 in MeOH was shifted by ~ 44 cm-1 in H2O and appeared at 1644 cm-1 clearly demonstrating that they remained H-bonded in water. Concentration-dependent FT-IR studies were performed in aqueous medium which showed (inset-Figure 4a) no change in the position of the peak corresponding to


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the carbonyl stretching in entire tested concentration window (4.0-0.015 mM) indicating intrachain H-bonding. However, several of this chain folded polymers assemble together to form a vesicular particle of diameter ~ 300 nm. H-bonding mediated self-assembled proteins can be denatured in presence of urea due to its ability to interrupt with H-bonding network.46 Similar examples are also known for few synthetic systems.47-48 To test such possibilities in proposed Hbonding mediated vesicular assembly of present systems Calcein encapsulated P1 solution was treated with urea while with gradual urea addition emission intensity increased and eventually reached saturation point (Figure 4b).

Figure 4: (a) FT-IR spectra (selected region) of P1 in H2O (C= 1 wt %, pH = 8.0) and MeOH solution; inset-concentration dependent spectra in water. (b) Effect of urea addition on the PL spectra (λ

ex

= 450 nm) of Calcein encapsulated in P1 vesicle; inset- variation of emission

intensity at 512 nm with urea addition. (c) Effect of urea addition on the emission spectra of aqueous solution of Calcein (C = 1.5 x 10-6 M). (d) Size distribution (number average) of aggregates from by P1 (C = 0.5 mM, pH = 8.0) before (blue) and after (green) urea (50 mg) addition in a 2 ml polymer solution.


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It can be recalled (Figure 3a) that when Calcein was residing inside the vesicle its emission intensity was reduced due to self-quenching. Now the fact that in presence of urea the selfquenching effect is lifted indicates dye is released to bulk water due to urea-mediated disassembly of vesicles. In an independent experiment urea was added to an aqueous solution of Calcein in absence of any polymer while no change in the emission intensity was notice (Figure 4c) eliminating the possibility of any inherent effect of urea on Calcein emission due to some non-specific interaction or some other unknown effect. Further DLS measurements revealed in presence of urea the peak around 300 nm due to vesicular assembly completely disappeared confirming disassembly. In turn a new peak appeared around ~ 50 nm (Figure 4d) which may represent some undefined aggregate the nature of which is not clear to us at the moment. Additional evidence supporting the proposed chain-folding based model for vesicular assembly (Scheme 1) could be gathered by powder XRD measurements. X-ray diffraction pattern of a dried film generated from aqueous solution of P1 (Figure 5a) revealed a sharp peak at low angle (2θ = 2.84°) from which d spacing was estimated to be 31.048 Å which nearly matched with the theoretically calculated length (30. 6 Å) of the chain-folded bilayer (Figure 5b). This result further supports intra-chain H-bonding mediated folding and self-assembly of P1 that is depicted in Scheme 1.


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Figure 5: (a) XRD pattern of the dried film generated out of aqueous P1 solution (C =10 mg/ ml, pH = 8.0). (b) Energy minimized structure of chain folded layer obtained by molecular modelling done in Chem 3D-ultra 8.0 using MM2 for energy minimization. Effect of pH on aggregation and cell viability studies: We also examined effect of pH on selfassembly of P1 anticipating state of protonation of the carboxylic acid and nitrogen atom to have significant role on self-assembly. DLS measurements were carried out at pH 3.5, 7.2 and 11.5 while it was found (Figure 6a) that size of the aggregates remained almost invariant (~ 250-340 nm) in the entire tested pH range (3.5- 11.5) suggesting stable vesicular assembly.

Figure 6: (a) Size distribution in DLS and (b) zeta potential of the P1 solution (Concentration = 0.5 mM) at various pH.


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Further Calcein encapsulated vesicular solutions at three different pH s (3.5, 7.2 and 9) were examined under fluorescence microscope which showed presence of almost identical green emitting particles (Figure S6) in all three samples indicating dye encapsulated vesicles. However to our surprise we found (Figure 6b) the zeta potential value was almost close to zero (-5.8 mV and –1.4 mV at pH 7.2 and 3.5, respectively) in neutral and acidic pH indicating almost chargeneutralized states which is rather unexpected considering the presence of a tertiary amine functional group in the polymer chain. This can be attributed to highly stable zwitter-ionic state4950

generated from protonation of the tertiary nitrogen atom by the pendant carboxylic acid group.

On the other hand zeta potential of -12.0 mV at pH 11.5 suggested mild negatively charged surface at basic pH. The zwitterionic nature of polyurethane assembly prompted us to examine cell viability in presence of P1 (Figure S7). Chinese hamster ovarian (CHO) cell line was cultured in high glucose Dulbecco's minimal essential medium (DMEM) supplemented with 10% FBS and 1% L-glutamine-penicillin-streptomycin. Cells were maintained in a humidified incubator at 37˚C and 5% CO2. Almost 100% cells were found to be viable even at a polymer concentration of 100 µm. Kinetic stability: Polymersome are known to be stable aggregates. To test this property in the present systems, fluorescence emission intensity of Calcein encapsulated P1 was monitored over a period of 5 days at pH 7.2 while no significant change was noticed (Figure 7). This indicates no dye leakage and high kinetic stability of present vesicular assembly.


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Figure 7: Emission spectra of Calcein (C = 3.3 x 10-6 M) encapsulated P1 (C = 1mM) at pH 7.2 as a function of time. Absorption normalized emission spectra of the dye in water (without polymer) is also shown for comparison.

CONCLUSIONS In this article we have demonstrated utility of a structurally simple but functionally diverse monomer to synthesize novel amphiphilic polyurethane consisting of a hydrophobic backbone segmented by zwitterionic hydrophilic moieties. It showed spontaneous charge-neutralized vesicular assembly in aqueous medium driven by folding of the hydrophobic polymer backbone by intra-chain H-bonding which in broader context can be correlated to controlled hydrophobic collapse of classical polymers known as ionenes analogues

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or even specific types of folded oligomers

or their recently introduced non-ionic 54-59

and polymers.60 Considering easy

synthesis, possibilities of versatile pendant functionalization, facile self-assembly and excellent cell viability we are highly optimistic about developing future responsive biomaterials based on this classical polymeric scaffold.

EXPERIMENTAL

Materials and methods: Solvents and reagents were purchased from commercial sources and purified by reported methods1. 1H NMR spectra were taken from Bruker DPX-500 MHz


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spectrometer and calibrated using TMS as the internal standard. FT-IR spectra were recorded in a Perkin Elmer Spectrum 100FT-IR spectrometer. TEM studies were done in a JEOL-2010EX machine operating at an accelerating voltage of 200KV. SEM images were recorded in a JEOL6700F microscope. Dynamic Light Scattering (DLS) measurements were carried out in Malvern instrument. Fluorescence emission spectral studies were carried out in a FluoroMax-3 spectrophotometer, from Horiba Jobin Yvon. Fluorescence microscopic images were obtained by an Olympus (1x2-KSP, 6M 24413) machine, Japan. Molecular weight of the polymers was calculated with respect to polystyrene standards in Water’s GPC machine (515 HPLC Pump, Waters 2414 RI detector, HSPgelTM Column) equipped with a RI detector and using DMF as eluent. Synthesis of compound 1: A solution of methyl acrylate (1.8 gm, 0.0209 moles) in 10 ml methanol was added drop-wise to an ice cold solution di-ethanolamine (2.0 gm, 0.019 moles) in methanol (5.0 ml) and the reaction mixture was stirred at rt for 24 h. Then solvent was evaporated and the product was purified by column chromatography using silica gel (60-120 mesh) as a stationary phase and dichloromethane/methanol (95:5) as eluent to obtain the pure product as a light yellow liquid in 92 % yield. 1H NMR (500 MHz, CDCl3, TMS): δ (ppm) = 2.49 (t, 2H), 2.62 (t, 4H), 2.83 (t, 2H), 3.55 (t, 4H), 3.68 (s, 3H). FT-IR (wavenumber / cm-1): 3358(-OH), 2957(CH alkyl), 1730 (C=O). Synthesis of P1E: Compound 1 (200 mg, 0.001 moles), hexamethylene di-isocyanate (2 in Scheme 1) (176 mg, 0.001 moles) and 0.5 ml freshly dried THF were placed in a reaction vial under continuous flow of argon and to this a solution of DABCO (4.0 mg, 4x10-5 moles) in 1.0 ml THF (dry and degassed) was added and the reaction mixture was stirred for 5 h at 60 ° C. Heating was stopped and the reaction mixture was allowed to cool at rt and to this another 1 ml THF was added to get a viscous solution from which the polymer was precipitated out in excess


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diethyl ether. It was centrifuged, washed with diethyl ether and dried under vacuum to isolate the polymer as colorless sticky mass in 75% yield. Mn = 10,000 g/mol (PDI = 2.5) from GPC; 1H NMR (500 MHz, DMSO-D6, TMS): δ (ppm) = 1.21 (4H, broad peak ), 1.35 (4H, broad peak ), 2.40 (2H, broad peak), 2.64 (4H, broad peak ), 2.77 (2H, broad peak ), 2.92 (4H, broad peak ), 3.56 (3H, s ), 3.92 (4H, broad peak ), 7.06(2H, broad peak). FT-IR (wavenumber /cm-1): 3333 (N-H), 2940 (CH alkyl), 1699 (C=O of urethane), 1545 (N-H, N-C=O), 1257 (C-N). Synthesis of P1: A solution of P1E (260 mg, 7.2x10-4 mol) in 1.5 ml methanol was added with an aqueous NaOH solution (144 mg in 1.5 ml H2O) and the resulting mixture was stirred at rt for 16 h. Then 1 mole equivalent (with respect to NaOH) tri-fluoro-acetic acid was added drop-wise to neutralize the base and then MeOH was evaporated and rest of the solution was freeze dried to get the hydrolyzed polymer P1 as white sticky mass in 92 % yield. 1H NMR (500 MHz, DMSOD6, TMS): δ (ppm) = 1.21 (4H, broad peak), 1.36 (4H, broad peak), 2.24 (2H, broad peak), 2.63 (4H, broad peak), 2.72 (2H, broad peak), 2.93 (4H, broad peak), 3.94 (4H, broad peak), 7.06 (2H, broad peak). FT-IR (wavenumber /cm-1): 3440 (-OH), 3338 (N-H), 2920 (CH alkyl), 1690 (C=O), 1543 (N-H, N-C=O), 1262 (C-N). The polymer was found to be extremely hygroscopic in nature. TEM studies: Measured amount of polymer (P1) was dissolved in 2 mM NaOH solution to make the concentration of polymer 1 mM with respect to repeat unit molecular weight. Then the solution was subjected to dialysis for 24 h against water using dialysis bag of MWCO = 3000 Da. After that the polymer solution was diluted to 0.5 mM by water and drop casted in a TEM grid which was air dried for 24 h before images were captured. Very similar solution preparation method was used for other studies unless other methods are mentioned specifically. Dynamic Light Scattering (DLS) and zeta potential studies: Aqueous solution of P1 which was prepared for TEM studies (described in the previous section) was added appropriate amount


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of aqueous NaOH or HCl solution to adjust the pH and from those solutions DLS and zeta potential measurements were done at rt. In all measurements final polymer concentration (with respect to the repeat unit molecular weight) was maintained at 0.5 mM. For concentration dependent experiments stock solution was prepared at highest concentration and it was subsequently diluted quantitatively to get the series of samples with desired concentrations. Calcein encapsulation study and determination of critical aggregation concentration: A stock solution of Calcein (0.03 ml, 10-3 M) and P1 (0.1 ml, 30 mM) in MeOH were mixed and to this 2.870 ml aqueous NaOH (2 mM) was added drop-wise and the resulting mixture was subjected to dialysis against water (MWCO = 3000 Da) for 24 h. Then the UV/vis spectra of the solution were recorded which showed a peak with λmax = 490 nm due to Calcein. From the absorbance and known extinction coefficient data of the dye in free water concentration of encapsulated Calcein was estimated to be 1.5x10-6 M. Emission spectra were recorded from this solution and compared with absorption matched emission spectra of free dye in water. For determination of CAC, Calcein encapsulated P1 solution (0.3 mM) was transferred to a cuvette and emission spectra were recorded as a function of dilution. A measured amount of dye encapsulated polymer solution was taken out from the cuvette and dilution was made by adding equal volume of water and spectra was recorded after stirring the solution for 2 min. This procedure of successive dilution was continued till 10-5 M polymer concentration below which the emission intensity of Calcein was extremely weak suggesting it reached the detection limit of the instrument. Pyrene encapsulation study: A stock solution of pyrene (0.02 ml, 10-3 M) and P1 (0.200 ml, 102

M) in MeOH were mixed and solvent was removed to get a thin film which was then added

with 2 mM aqueous NaOH solution (2 ml) so that eventually polymer and pyrene concentrations


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were 1mM and 10-5 M, respectively. The solution was sonicated for 5 min before spectral measurements. Fluorescence microscopic study: Fluorescence microscopic studies of Calcein encapsulated polymer solution were done with polymer (P1) concentration 1 mM and encapsulated Calcein concentration 1.5 x 10-6 M. The solution was sandwiched between two glass slides before images were taken. FT-IR study: A solution of the polymer (P1) (1 wt %) was prepared in methanol and aqueous NaOH (pH = 0.8) and FT-IR spectra were recorded by placing these solutions between two CaF2 windows (path length = 0.2 mm) and spectral measurements were carried out with scan range = 4000-600 cm-1, resolution = 0.5 cm-1, number of scans = 30, T = 25 °C. Urea addition experiment by PL and DLS: For PL studies weighted amount of solid urea was added to a Calcein (C = 1.5 x 10-6 M) encapsulated polymer solution (1.0 mM) and the solution was stirred to make the entire solid dissolved before spectral measurements were done. For DLS studies a 0.5 mM P1 solution was added with measured amount of urea at once and measurements were done before and after urea addition. Calcein encapsulation in different pH solution: A Calcein encapsulated aqueous solution of P1 was added with appropriate amount of NaOH or HCl solution to adjust the desired pH and then fluorescence microscopic images were taken individually. Powder X-Ray diffraction studies: Aqueous solution of P1 (10 mg/ml, pH =8.0) was dropcasted repeatedly on a glass slide to make a thick film and it was then air dried for 12 h. Data was recorded with this sample from 1° to 30 ° with sampling interval of 0.02 Å per state. AFM and SEM studies: 0.5 mM aqueous P1 solution was drop casted on a glass slide and the sample was allowed to stand at ambient temperature for 24 h before taking the AFM images. Similar sample preparation method was also followed for SEM studies.


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Cell viability studies using MTT assay: Briefly, 104 cells/well were seeded in a 96-well plate and left to attach overnight. Polymer was prepared as a stock (500 µM) in sterile water, prior to addition to the cells. The culture medium was replaced with medium containing the polymer at various concentrations (1 - 100 µM) and incubated for another 24 h. The medium was then replaced with 100 µl of 1.0 mg/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) per well, incubated for 3 h at 37˚C and subjected to addition of DMSO (150 µl/well). Incubation was carried out for 10 min at 37˚C, and the absorbance was recorded at 570 nm using a plate reader (Various scan, ThermoFisher). The Yellow MTT, a tetrazole, gets oxidized by the cells with intact mitochondria and produce purple formazan crystals which gets dissolved in DMSO. Hence, only live cells can convert tetrazole to formazen giving a characteristic purple color.

ASSOCIATED CONTENT Supporting Information. Synthetic scheme, materials and methods and additional spectral data. This material is available free of charge via the Internet at http://pubs.acs.org.” AUTHOR INFORMATION Corresponding Author ** Dr. Suhrit Ghosh Associate Professor, Polymer Science Unit Indian Association for the Cultivation of Science 2A & 2B Raja S. C. Mullick Road, Kolkata, India- 700032 Email: [email protected]


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Author Contributions All authors have given approval to the final version of the manuscript.

Funding Sources Department of Biotechnology (Project No: BT/01/CEIB/11/V/13), India. ACKNOWLEDGMENT TM and KD thanks CSIR and JD thanks DBT, India for a research fellowship. REFERENCES 1. Moffitt, M.; Khougaz, K.; Eisenberg, A. Micellization of Ionic Block Copolymers. Acc. Chem. Res. 1996, 29, 95-102. 2. McCormick, C. L.; Lowe, A. B.

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For Table of Contents Use Only Hydrogen-Bonding Induced Chain Folding and Vesicular Assembly of an Amphiphilic Polyurethane Tathagata Mondal, Krishna Dan, Jolly Deb, Siddhartha S. Jana and Suhrit Ghosh*


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Hydrogen-Bonding-Induced Chain Folding and Vesicular Assembly of

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