Stimuli-Responsive Directional Vesicular Assembly with Tunable

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Stimuli-responsive directional vesicular assembly with tunable surface functionality and impact on enzyme inhibition Amrita Sikder, Debes Ray, Vinod Kumar Aswal, and Suhrit Ghosh Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01652 • Publication Date (Web): 25 Jul 2017 Downloaded from http://pubs.acs.org on July 28, 2017

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Stimuli-responsive directional vesicular assembly with tunable surface functionality and impact on enzyme inhibition Amrita Sikder, a Debes Ray,b Vinod K Aswal b and Suhrit Ghosha* a

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

b

Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai, India-400085

ABSTRACT. The article describes self-assembly of a series of unsymmetrical bola-shape πamphiphiles (NDI-1, NDI-1a, NDI-2, NDI-3 and NDI-4) consisting of a hydrophobic naphthalene-diimide (NDI) chromophore attached with a non-ionic hydrophilic wedge and an anionic head group in the two opposite arms of the central NDI. By design only a single hydrazide group is linked either on the ionic or non-ionic arm of the NDI. NDI-1 and NDI-1a are regioisomers differing only in the location of the hydrazide group, placed in the non-ionic or ionic arm, respectively. While NDI-2, NDI-3 and NDI-4 are similar to NDI-1 in the placement of the hydrazide group, but differ in the nature of the ionic head groups. Except NDI-2, all of them exhibit spontaneous vesicle structures in water (pH 9.0) as established by electron microscopy, small-angle

neutron

scattering,

dynamic

light

scattering

ACS Paragon Plus Environment

and

spectroscopy

studies.

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Supramolecularly assembled oligo-oxyethylene chains of the hydrophobic wedge exhibited a lower critical solution temperature (LCST) at ~ 40 °C similar to covalent polymers. Consequently above LCST, the bola-amphiphile was converted to single head group surfactant resulting in collapse of the vesicular structure to nano-particles. In all examples, dominant Hbonding force among the hydrazide groups resulted in uni-directional orientation leading to the formation of unsymmetric membrane with the H-bonded chain located at the inner wall. Therefore the functional group display in these vesicles could be fully dictated by the location of the hydrazide group. Thus for NDI-1, NDI-3 or NDI-4, the hydrazide group, located at the nonionic arm, directed the non-ionic wedge to converge at the inner wall of the vesicle by displaying the anionic head groups towards outer surface. In contrast, NDI-1a formed a non-ionic vesicle as in this case anionic head groups were located at the inner-wall of the membrane. Furthermore, amongst NDI-1, NDI-3 and NDI-4, the charge density of the anionic surface and accordingly the radius of curvature and particle size could be tuned precisely as a function of the extent of charge delocalization in the phenoxide or carboxylate head group. These distinct self-assembly modes resulted in very different ability of these vesicles for electrostatic interaction driven biomolecular recognition which was studied by testing their ability to bind with a cationic protein Chymotripsin and inhibit its enzymatic activity. The enzyme inhibition ability followed the order NDI-1> NDI-3> NDI-4> NDI-2~NDI-1a which could be rationalized by their distinct functional group display and surface charge density factors.

INTRODUCTION Considering enormous importance of vesicle1 or polymersome other biomedical applications,

4-5

2-3

for targeted drug delivery and

it is of significant interest to gain control on the functional


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group display at the inner and outer surface of the membrane which may independently control the specific interaction mediated guest encapsulation or communication with exo-vesicular solution (relevant in targeted delivery or sensing), respectively. Unsymmetrical bolaamphiphiles 6-7

bearing two dissimilar head groups have been explored to make such unsymmetric monolayer

vesicle (MLV). However the success is limited to only a few examples8-10 as normally random or antiparallel orientations are preferred to minimize steric or electrostatic repulsion. To circumvent this issue, recently we have communicated10 a supramolecular strategy utilizing H-bonding as the driving force to generate unsymmetric MLV from unsymmetrical bola-shape πamphiphiles11-20 (NDI-1 and NDI-1a, Scheme 1). They consist of a naphthalene-diimide (NDI) πsystem22-27 as the hydrophobic segment which is connected to a non-ionic hydrophilic wedge and an anionic head group. It also contains a single hydrazide group which is located either at the non-ionic or ionic arm in NDI-1 and NDI-1a, respectively. Extended H-bonding among the hydrazide group not only acted as the dominant force to produce unidirectional stack but further could dictate the direction of bending. As discussed in our earlier report, 10 it is believed that the specific direction of bending originates from the preference of the hydrazide groups to be located at the inner wall of the membrane to avail stronger H-bonding as in this case the intermolecular distance between the adjacent hydrazides should be shorter compared to the situation if they would have been located at the outer wall of the membrane. Consequently, the functional groups attached to the hydrazide arm were converged at the inner surface while the other head groups were displayed at the outer surface of the vesicle and thus NDI-1 and NDI-1a formed anionic and nonionic vesicles, respectively.10 Among these two, NDI-1 is particularly interesting as in this case the ionic head groups were very effectively displayed at the outer surface which is important for multi-valent binding, targeting and biomolecular recognition. In an attempt to


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examine generalized applicability of this strategy and further explore structural diversity in this family of bola-shape π-amphiphiles, we have now introduced NDI-2, NDI-3 and NDI-4 which are similar to NDI-1 in terms of the location of the hydrazide group but differ in the structure of the ionic head group. In this article, we disclose comprehensive self-assembly studies of these πamphiphiles including structure-property correlation, impact of head group structure on the mesoscopic structure, surface charge density and their enzyme inhibition activity. Furthermore, we disclose lower critical solution temperature (LCST) exhibited by these supramolecular polymers similar to a few water soluble covalent polymers and implication in thermo-responsive vesicle to nanoparticle transition.

Scheme 1. Structures of the amphiphiles (top) and a schematic depiction (bottom) on their directional vesicular assembly


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RESULTS AND DISCUSSION Vesicular assembly: Structures of the amphiphiles studied in this article are shown in Scheme 1. NDI-1 and NDI-1a differ in the location of the hydrazide group which is connected to either the non-ionic or ionic arm. On the other hand, in all amphiphiles (NDI-2, NDI-3 and NDI-4), the hydrazide is located at the non-ionic arm similar to NDI-1, but they differ in the nature of the head groups. Instead of phenoxide as in NDI-1, NDI-2 and NDI-3 contain a carboxylate head group connected to the NDI core by an aliphatic or aromatic spacer, respectively. On the other hand, NDI-4 contains a phenoxide head group but it is linked at the meta-position with respect to the imide group. Self-assembly was studied by solvent dependent UV-Vis spectroscopy (Figure 1). In a good solvent THF, sharp absorption band between 300-400 nm affirms monomeric species in all cases, while in aqueous solution (pH-9.0) significant reduction of absorption band with concomitant reversal of peak intensity indicates aromatic interaction between the NDI chromophore in all examples except NDI-2.22 This possibly reflects the essential role of hydrophobic shielding of the hydrogen-bonding site for self-assembly in water which is achieved in all cases with the aromatic spacer but not in NDI-2 as it contains a flexible aliphatic spacer. Therefore NDI-2 was excluded from the list for further self-assembly studies.


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Figure 1. UV-Visible spectroscopy for NDI-1, NDI-1a, NDI-2, NDI-3 and NDI-4 in THF (solid line) and in water (dotted line). (c= 0.5 mM; l=0.1cm, pH = 9.0). Concentration dependent UV/Vis studies showed (Figure S1) no significant change in the nature of the absorption spectra up to 10-6 M suggesting very low critical aggregation constant in all cases which is attributed to synergy between H-bonding and aromatic interaction. While the later is evident from UV/Vis spectra, FT-IR spectra provided direct evidence of H-bonding. In THF a sharp peak at 1700 cm-1 corresponds to non-bonded C=O stretching of the hydrazide group which appeared (Figure S2) in significantly lower frequency in D2O ascertaining H-bonding among the hydrazide groups. Urea is a well known reagent for denaturing protein secondary structure

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or destroying self-assembled abiotic nanostructure26 by jeopardizing H-bonding.

However with addition of excess urea, none of the NDI-amphiphilic systems show any change in the UV-Vis spectra (Figure S3) reflecting the H-bonded chain among the hydrazides is inaccessible to interact with urea which is conceivable if they are located at the inner surface of the membrane so that urea may not cross the membrane consisting of rigidly placed NDI array. To look into the mesoscopic structures of these amphiphiles, small-angle neutron scattering (SANS) measurements were performed (Figure 2). The SANS data of all the NDIs were analyzed using a model of spherical shell for large unilamellar vesicles (LUV). In the low-Q region of the data, the scattering intensity decreases in almost a straight line as 1/Q2 and it indicates formation of vesicles in these systems. The absence of lower cut-offs in the data indicates that the size of the vesicles could be much larger than what can be determined from the present Qmin and therefore the size of vesicle was kept fixed higher than to a value 2π/Qmin, i.e., ~350 Å. These LUVs have been characterized by the membrane thickness as the measurement of the size of vesicles is limited by the Qmin of the SANS instrument. The fitted parameters are


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given in Table 1. Similar SANS profiles for NDI-1, NDI-3 and NDI-4 are reflected in the membrane thickness of the vesicles, which is in the order of the length of the hydrophobic block obtained from the energy minimized structures (Figure S4) and therefore supports mono-layer vesicular assembly by these unsymmetric bola-amphiphiles. To further establish the vesicular structure, their ability to sequester hydrophilic guest molecules was probed by encapsulation of Calcein dye which is known to exhibit self-quenching of fluorescence at higher concentration. Individual NDI amphiphile was treated with Calcein and the non-encapsulated dye was removed by dialysis.

Figure 2. Plot of scattering intensity as a function of scattering vector Q for NDI-1, NDI-3 and NDI-4 and the corresponding fit reveals vesicular structure for all the bolaamphiphiles. The plots are vertically shifted for clarity. Table 1. Size and membrane thickness of NDI-amphiphiles. The size is greater than that can be fitted. System

Size of Vesicle (Å)

Thickness of Hydrophobic Shell (Å)

NDI-1

> 350 Å

8.3 ± 0.6 Å

NDI-3

> 350 Å

7.5 ± 0.5 Å

NDI-4

> 350 Å

8.4 ± 0.6 Å


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Fluorescence spectra (Figure 3) of the Calcein treated NDI amphiphile(s) solutions exhibit characteristics emission band in the range of 500-600 nm indicating successful entrapment of the dye. Absorption spectra of the solutions (Figure S5) further confirm dye encapsulation and allow estimating the dye load efficiency as 13%, 16% and 17% for NDI-1, NDI-3 and NDI-4, respectively which corroborate with literature reports on vesicles of comparable size.29 Furthermore, emission intensity of encapsulated Calcein was found to be significantly low compared to those of free Calcein in water at the same dye concentration (absorption same) indicating self-quenching which can be attributed to the entrapment of the dye in confined lacuna of the vesicular assembly.30

Figure 3. Absorption normalized emission spectra of Calcein encapsulated NDI-1, NDI-3 and NDI-4 and corresponding emission spectra of free Calcein in water (pH-9; 25oC).


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Head group effect on surface property and enzyme inhibition: Dynamic light scattering (DLS) studies show a significant variation in the particle size of different vesicles. NDI-1 vesicle was found to be largest with average hydrodynamic diameter (Dh) = 140 nm (Figure 4a) while NDI-3 and NDI-4 exhibit much smaller size (Dh = 45 nm and 60 nm, respectively). Transmission electron microscopy images (Figure S6) reveal hollow spherical structures with average diameter of 180 nm, 50 nm and 80 nm for NDI-1, NDI-3 and NDI-4 vesicles, respectively, which roughly matches with the DLS data. The observed difference in particle size can be rationalized on the basis of different extent of charge delocalization in the aromatic ionic head groups.31 The negative charge of the phenoxide head group in NDI-1 can be delocalized by extended conjugation over the benzene ring due to the presence of the electron withdrawing imide group connected to the para-position of the phenoxide group. On the other hand for NDI-4, due the meta-connectivity, the delocalization is expected to be less effective.32 Likewise in NDI-3, there is no scope for the negative charge on the carboxylate group to be delocalized over the benzene ring. Therefore, it is proposed that due to localized charge in NDI-3 and NDI-4, there is a stronger charge-charge repulsion causing smaller radius of curvature of the membrane.

Figure 4. (a) DLS and (b) zeta potential of NDI-amphiphiles in water (pH =9; c=0.5 mM, RT)


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As a consequence smaller vesicles was formed for these two amphiphiles compared to that in NDI-1 in which due to effective delocalization, the repulsion among the ionic head groups is less severe. As all the bola-amphiphiles contain different types of negatively charged head groups we were keen to examine the effect of difference in their structure on the surface charge density of these vesicles. A rather high negative value of zeta potential (-45 mV) was observed for NDI-1 while for NDI-3 and NDI-4, the zeta potential was -34 mV and -20 mV, respectively (Figure 4b). This is attributed to charge repulsion induced lack of efficient packing of the head groups which eventually leads to the presence of less number of ionic head group per unit volume for NDI-3 and NDI-4. The impact of the observed difference in surface charge density on protein surface binding was examined using enzyme activity assay of α-Chymotripsin (Cht) in presence of these vesicles.33-37 It is known that the active site of Cht is surrounded by cationic residues and thus it can bind with anionic surface by complementary electrostatic interaction which retards its enzymatic activity. Enzymatic activity assay was performed using N-succinyl-l-phenylalanine-para-nitroanilide (SPNA) as the substrate. Pre-incubated Cht with NDI-1, NDI-3 or NDI-4 vesicles was added to the substrate solution and the enzymatic action was probed by monitoring the absorption spectra of the p-nitro-aniline (Figure S7) generated by enzymatic action on SPNA. NDI-1 vesicle was found to be highly potent surface-based inhibitor revealing ~80% inhibition while moderate (45 %) or low (20%) inhibition was observed by NDI-3 and NDI-4, respectively (Figure 5). Interestingly NDI-2 that did not show any self-assembly due to lack of the hydrophobicity (Figure 1), also showed no enzyme inhibition. This reveals strong impact of multi-valent effect that arise due to self-assembly for enzyme binding and inhibition of its activity. Interestingly for


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vesicle forming amphiphiles (NDI-1, NDI-3, NDI-4), the trend in their ability for enzyme inhibition nicely corroborate with the zeta potential values of the corresponding vesicles and therefore highlight strong impact of the head group structure on the surface binding properties. In contrast NDI-1a that also forms vesicle, but with non-ionic surface due to the location of the hydrazide group does not act as an efficient Cht inhibitor as it shows less than 20 % inhibition. Important to note that no significant difference in CD (Figure S8) or fluorescence spectra (Figure S9) of Cht were observed after treatment with NDI vesicles affirming unperturbed protein secondary structure upon binding with any of NDI vesicles and therefore confirms that the observed enzyme activity inhibition is due to blockage of the active site upon binding with vesicular surface.

Figure 5. Comparison of enzyme activity in presence of different NDI-amphiphiles. Concentration of enzyme =3.2µM, amphiphile =0.1mM. Activity assay was carried out at pH =9 (5Mm PBS buffer) and at T = 25oC. Thermo-responsive morphology transition and dye release: Thermal stability of the vesicles were investigated by temperature variable UV-Vis spectra. The data for NDI-3 are shown in Figure 6a. As such there is no major change in the spectra till 90 oC suggesting no disassembly at


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elevated temperature as the spectrum does not resemble to that in THF representing the monomeric chromophore. However careful analysis reveals subtle change in the absorption spectra and couple of isosbestic points (inset, Figure 6a) implying topological difference in chromophoric arrangement at higher temperature. Absorbance at 380 nm when plotted as a function of temperature, a clear inflection point could be noticed at 47 oC (Figure 6b). Similar observation was observed for NDI-1 and NDI-4 with an inflection point around 41 oC and 45 oC, respectively (Figure S10).

Figure 6. (a) Variable temperature UV-Vis spectroscopy of NDI-3 (inset shows zoomed image of spectral change); (b) Variation of absorption (380 nm) vs. temperature showing inflection point at 47oC. (c) Variation of size (Dh) and zeta potential with increasing temperature of NDI-3; (c) TEM images of aqueous solution of NDI-3 (d) below and (e) above LCST (pH-9; c=0.5mM). Further the particle size increased from 50 nm to 220 nm by elevating the temperature beyond this critical temperature (Figure 6c, S11) and also the zeta potential showed a gradual reduction


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from -34 mV to -7.0 mV (Figure 6c). These changes were also consistent for NDI-1 and NDI-4 (Figure S12). TEM images (Figure 6d-e) of the samples prepared from a hot aqueous solution of NDI-3 showed presence of near spherical particles (size ~ 200 nm) in contrast to hollow spherical morphology at rt indicating thermo-responsive morphology transition. This can be rationalized by considering a lower critical solution temperature (LCST)38-39 of the hydrophilic wedge containing oligo-oxyethylene groups which have been reported in the past to show thermo-responsive phase transition similar to water soluble polymers such as poly(NIPAM). Therefore the inflection point in the absorption vs. temperature plot or for that matter the sharp change in the zeta potential or particle size at similar temperature range can be related to the change in the nature of the amphiphile itself above LCST.

Figure 7. (a) Schematic representation of vesicle to micelle formation above LCST temperature; (b) Comparison of variable temperature plot of emission maxima for ANS dye encapsulated NDI-3 and free ANS dye. While below LCST it behaves like a bola-amphiphile, above LCST, it is converted to an amphiphile with a single ionic head group as the hydrophilic wedge is dehydrated. This leads to the collapse of the vesicular structure to generate compound micelle type large nanoparticle


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(Figure 7a).40-41 In such situation, the water filled lacuna in the vesicle is expected to be converted to a hydrophobic domain. To probe that, NDI-3 vesicle was encapsulated with a polarity sensitive fluorescence dye 8-Anilino-1-naphthalenesulfonic acid (ANS) which is water soluble but given a preference resides in hydrophobic domain which can be characterized by its enhanced fluorescence.42 Variable temperature emission spectra of vesicle entrapped ANS (Figure S13) show almost no change till 40 °C followed by a sharp increase at around the same temperature which was assigned as the LCST of the hydrophilic wedge. A control experiment with ANS dye without the NDI vesicle show almost no change in the emission intensity (Figure S14) as a function of temperature confirming the observed increase is indeed related to the creation of hydrophobic domain by collapse of the vesicular structure above LCST (Figure 7b). NDI-2 (that does not show any self-assembly but contains the same oligo-oxyethylene wedge) does not show any LCST as evident from temperature dependent UV spectra (Figure S15) indicates that LCST is solely exhibited in case of supramolecularly assembled structure not by the monomers. While several examples have been reported on thermo-responsive polymer assembly,38-42 similar phenomenon in supramolecular systems is less explored

43-45

and the

present example of thermo-responsive vesicles could be potentially interesting candidates for stimuli-responsive drug delivery application. 46 CONCLUSION In summary we have demonstrated spontaneous vesicular assembly of a series of unsymmetrical NDI-derived bola-amphiphiles with a similarity in H-bonding driven functional group (anionic group) display, selectively at the outer surface which is highly desirable for molecular recognition, targeting and other important functional applications of vesicles. A structureproperty relationship studies with varying nature of the ionic head groups reveal strong impact of


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the charge delocalization on the membrane curvature and thus size and zeta potential. These anionic vesicles were further explored for enzyme inhibition which revealed an important role of the exact nature of the ionic head group on the inhibition ability. For example NDI-1 and NDI-4 differing only in the position of the phenoxide group with respect to the imide group showed vast difference in size, zeta potential and enzyme inhibition. Furthermore it is revealed that due to the LCST property exhibited by the non-ionic hydrophilic wedge these bola-amphiphiles change its nature at elevated temperature resulting in morphology transition above LCST. In majority examples of polymers or supramolecular systems, a mere precipitation of the material happens above LCST while the present system demonstrates thermo-responsive morphological change from vesicle to nano-particle at LCST associated with drastic reduction in surface charge density. Such stimuli-responsive self-assembled material could be of interest for triggered release in biomedical application and reversible switching of surface properties by thermal control. EXPERIMENTAL Solution preparation: Measured volume of an aliquot from a stock solution of NDI-1, NDI-2, NDI-3 and NDI-4 in THF (0.5 mM) was transferred to another vial; solvent was evaporated and the solid was re-dissolved in measured volume of aqueous NaOH solution (pH = 9.0) by sonication to prepare the solution with a desired concentration. For Calcein encapsulation a stock solution of Calcein in MeOH (20 µL, 1.0 mM) and NDI in THF (100 µL, 5.0 mM) were mixed and solvent was evaporated. To this 500 µL aqueous solution (pH-9) was added and sonicated for few minutes. Subsequently the solution was dialyzed against water (pH = 9.0) for 48 h to remove unencapsulated Calcein. The fluorescence spectrum of the dialyzed solution (after half dilution) was recorded and compared with an absorption-normalized aqueous solution of free Calcein.


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Self-assembly: Solvent dependent UV-Vis absorption were recorded in a Perkin-Elmer Lambda 25 spectrometer with 0.5 mM concentration of each sample in THF and water (pH-9) taken in a quartz cuvette of 0.1 cm path length. HRTEM images were captured in JEOL-2010EX instrument operating at an accelerating voltage of 200 KV. Solutions of NDI (0.5mM) were drop casted on a copper grid and dried at room temperature for 48 h before images were taken. DLS measurements were done at 0.5 mM solution in a Malvern instrument. FT-IR spectra were recorded with 0.5 mM solution in absorbance mode (path length 0. 2 cm) in a Perkin Elmer Spectrum 100 FT-IR spectrometer. Emission spectra for free Calcein and Calcein encapsulated samples were recorded in a FluoroMax-3 spectrophotometer from Horiba. SANS: Small-angle neutron scattering experiments were performed at the SANS diffractometer at Guide Tube Laboratory, Dhruva Reactor, Bhabha Atomic Research Centre, Mumbai, India. 47 In SANS, one measures the coherent differential scattering cross-section (dΣ/dΩ) per unit volume as a function of wave vector transfer Q (= 4π sinθ/λ, where λ is the wavelength of the incident neutrons and 2θ is the scattering angle). It provides information about the shape and size of the scattering particles in the length scale of 10-1000 Å. The mean wavelength of the monochromatized beam from neutron velocity selector is 5.2 Å with a spread of ∆λ/λ ~ 15%. The angular distribution of neutrons scattered by the sample is recorded using a 1 m long onedimensional He3 position sensitive detector. The instrument covers a Q-range of 0.017–0.35 Å-1. The temperature in all the measurements was kept fixed at 30 oC. For analysis part see the supporting information. Enzyme inhibition: All the experiments were performed in 5.0 mM sodium phosphate buffer at pH 9.0 with [Cht] = 3.2 µM and NDI at 0.1mM. The enzymatic hydrolysis reaction was initiated by adding a SPNA stock solution (15 µL) in EtOH: DMSO-D6 (9:1) to a pre-incubated Cht-NDI


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vesicle solution (185 µL) to reach a final SPNA concentration of 2.0 mM and hydrolysis of SPNA was monitored by increasing intensity of the absorption at 405 nm for 1.5 h. Cht at pH = 9.0 in absence of NDI was taken as control experiment. Emission (λex-295 nm) and CD spectra were recorded for a freshly prepared ChT solution (3.2 µM) and the same after incubated with 0.1mM NDI for 24 h while no change in spectral features indicated lack of denaturation. Thermo-responsive morphology transition: For variable temperature experiments, solution of NDI (0.5 mM, 0.2 mL) in water was taken in a 0.1 cm cuvette and the sample was heated with an external temperature controller and spectral measurements were carried out at different temperature after equilibrating for 5 minutes after each temperature is reached. For variable temperature DLS and Zeta potential measurement 1.0 mL, 0.5mM solution was taken in a capped glass cuvette and measurements were carried out at different temperature after equilibrating for 120 seconds after each temperature is reached. TEM images were taken of a drop casted solution of NDI-3 on carbon coated copper grid placed on a hot plate and after it was dried above LCST temperature for 48 h. ASSOCIATED CONTENT Supporting Information: The Supporting Information is available free of charge on the ACS Publications website at DOI: Synthesis, additional information and physical data (PDF) AUTHOR INFORMATION Corresponding Author *Email: [email protected]


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