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Nanoengineering of a Supramolecular Gel by Copolymer Incorporation: Enhancement of Gelation Rate, Mechanical Property, Fluorescence and Conductivity Priyadarshi Chakraborty, Sujoy Das, Sanjoy Mondal, Partha Bairi, and Arun K. Nandi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04714 • Publication Date (Web): 02 Feb 2016 Downloaded from http://pubs.acs.org on February 2, 2016
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Nanoengineering of a Supramolecular Gel by Co-polymer Incorporation: Enhancement of Gelation Rate, Mechanical Property, Fluorescence and Conductivity Priyadarshi Chakraborty, Sujoy Das, Sanjoy Mondal, Partha Bairi and Arun K. Nandi* Polymer Science Unit, Indian Association for the Cultivation of Science, Jadavpur Kolkata-700 032, INDIA *For Correspondance: A.K.Nandi, email:
[email protected], Telephone No. 913324734971
Abstract: In the quest to engineer the nanofibrillar morphology of folic acid (F) gel poly(4vinylpyridine-co-styrene)(PVPS) is judiciously integrated as a polymeric additive because of its potential to form H-bonding and π-stacking with F. The hybrid gels are designated as F-PVPSx gels where x denotes the amount of PVPS (mg) added in 2 ml of F gel (0.3%, w/v). The assistance of PVPS in the gelation of F is manifested from the drop in critical gelation concentration and increased fiber diameter and branching of F-PVPSx gels compared to that of F gel. PVPS induces a magnificent improvement of mechanical properties; a 500 times increase of storage modulus and ~62 times increase of yield stress in the F-PVPS5 gel compared to the F gel. The complex modulus also increases with increasing PVPS concentration with a maximum in F-PVPS5 gel. Creep recovery experiments suggest PVPS induced elasticity in the otherwise viscous F gel. The fluorescence intensity of F-PVPSx gels at first increases with increasing PVPS concentration showing maxima at F-PVPS5 gel and then slowly decreases. Gelation is monitored by time dependent fluorescence spectroscopy and it is observed that F and F-PVPSx gels exhibit perfectly opposite trend; the former shows a sigmoidal decrease in fluorescence intensity during gelation but the later shows a sigmoidal increase. The gelation rate constants calculated from Avrami treatment on the time dependent fluorescence data manifests that PVPS effectively enhances the gelation rate showing a maximum for F-PVPS5 gel. The hybrid gel
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exhibit five orders increase of dc-conductivity than that of F-gel showing semiconducting nature in the current-voltage plot. The Nyquist plot in impedance spectra of F-PVPS5 xerogel exhibit a depressed semicircle with a spike at lower frequency region and the equivalent circuit represent a complex combination of resistance–capacitance circuits attributed to the hybrid morphology of the gel fibers. Introduction Supramolecular gels,1-5 formed through a variety of non-covalent interactions,6-8 posses exciting properties of responding to external stimuli like heat, light, ultrasound, mechanical shear etc.9-15 The stimuli responsive property along with the soft nature of these supramolecular gels make them suitable candidates for diverse applications in medicine,16,
17
sensing,18 remediation,19,
20
soft lithography,21 tissue engineering,22, 23 and designing different microarray kits.24 In spite of these fascinating vistas, the practical applications of supramolecular gels are limited because of their weak mechanical properties. Their mechanical stability is exclusively dependent on noncovalent interactions, and they can be easily converted to the sol state by a small mechanical force. One most simple way to avoid this problem is to engineer hybrid gels by combining supramolecular gels with covalent polymers. Covalent polymers are mechanically robust, and a suitable combination of supramolecular gels and covalent polymers can yield smart gels with excellent mechanical integrity still retaining the dynamic nature of supramolecular systems. The hybrid gels can also enhance other physical properties of the parent gels. The topic though intriguing, has not been investigated meticulously, and only a few reports are available in the literature hither to.25-32 However, in all these reports, enhancement of mechanical properties and changes in morphology of the gels are mainly enlightened. The effect of polymer additives on gelation rate 2 ACS Paragon Plus Environment
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has scarcely been investigated. The key objective of this work is, therefore, to investigate whether a covalent polymer can assist in the gelation of a small molecule by enhancing the gelation rate. Also, we are curious to inspect the changes in mechanical, fluorescence and conducting properties of the native gels after addition of the polymer. For this purpose a judicious choice of the gelator molecule and the complementary polymer is indispensible. Here, we have elegantly engineered a hybrid gel of Folic acid (F) with poly(4-vinylpyridine)-costyrene)(PVPS) (Scheme 1). F has been reported as a gelator in mixed solvent31,
32
and it
possesses two -COOH groups which can act as H bond donor (Scheme 1). PVPS is capable of forming H-bond with -COOH groups of F and acts as a good H bond acceptor through its nitrogen atom (Scheme 1). Also there is a fine probability of π-stacking interactions between PVPS and F. Based on these intuitions it may be anticipated that PVPS can co-assemble with F, hence can influence the gel morphology, gelation rate, mechanical property etc. To our expectations, PVPS has magnificently improved the mechanical properties of the native F gel and also renders adequate elasticity to it. It also reduces the critical gelation concentration (CGC) of F. The fluorescence intensity of the F gel has also improved after addition of PVPS. Avrami treatment carried on the native and hybrid gels proves that PVPS augments the gelation rate of F, thereby playing a significant role in the gel formation. Moreover, a dramatic improvement of dc-conductivity is observed in the hybrid F-PVPS5 gel compared to the native F gel. So, this work nicely demonstrates the enhancement of gelation rate, improvement of mechanical property, enhancement of fluorescence intensity and conductivity of the native gel by incorporation of a non-conducting polymer which is essentially a new and exciting report in supramolecular gel systems.
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Experimental Materials. Folic acid (F) was purchased from Sisco Research Laboratories Pvt. Ltd. Mumbai, India. Poly(4-vinylpyridine-co-styrene)(PVPS) was purchased from Aldrich Chemical Co., USA. N, N-dimethylformamide (DMF) (HPLC grade) was purchased from RFCL, New Delhi, India. Water was double distilled before use. Preparation of F and F-PVPS hybrid gels. Required quantity of F (0.3% w/v) was dissolved in DMF-water mixture at a volume ratio of 1:1 by heating in a capped tube and was subsequently cooled at room temperature to produce F gel. Gelation was confirmed by cessation of flow on inverting the tube. A stock solution of PVPS (1% w/v) was prepared by dissolving required quantity of PVPS in DMF by stirring overnight. Required amounts of this PVPS solution was added to a solution of F (0.3% w/v) in DMF-water mixture (1:1) and the mixed solution was heated to 80 oC and cooled at 30 oC to obtain the hybrid F-PVPS gels. The gels were designated as F-PVPSx where x denotes the amount of PVPS (mg) added in 2 ml of F gel (0.3%, w/v). The concentrations (% w/v) of F and PVPS in the F and F-PVPSx gels are presented in Table S1. The
Self Assembly
Folic acid Co-Assembly
F-PVPSx gel
F gel PVPS
Scheme 1. Chemical structures of Folic acid and PVPS. Digital images of F and F-PVPSx gels showing diminishing transparency. 4 ACS Paragon Plus Environment
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xerogels were prepared by drying F and F-PVPSx gels on a glass slide in open air followed by drying in vacuum at 30 oC for three days. Measurement of gel–sol transition temperature (Tgm). The gel melting temperature (Tgm) of the gels were measured using “dropping ball method”. The F and F-PVPSx gels were filled into a screw capped vial, sealed and kept at room temperature for 6 hour. A steel ball (diameter: 2.1 mm, weight: 45 mg) was put on the top of the gel, and the vial was heated at the rate 1 oC/min in a thermostatic water bath. Tgm was determined as the temperature at which the steel ball reaches to the bottom of the vial and average of four such measurements was taken as Tgm. The temperature was measured in a reference vial filled with two ml of DMF-water mixture (1:1). Characterization techniques. To study the morphology of the gels small portion of the diluted F and F-PVPSx gels were drop casted on a carbon coated copper grid (300 mesh) and dried at room temperature. It was then observed through a TEM instrument (JEOL, model 2010EX) directly under a voltage of 200 kV. Mechanical properties of the F and F-PVPSx gels were studied with an advanced rheometer (AR 2000, TA Instrument, USA) using cone plate geometry on a peltier plate having a diameter of 40 mm, cone angle of 4 degree and a plate gap of 121 µm. The UV-vis spectra of F assembly (in DMF /water 1:1 mixture) and F solution (in DMF) were recorded with a Hewlett-Packard UV-vis spectrophotometer (model 8453) in a cuvette of 0.1 cm path length. Fluorescence study of the F-PVPSx gels were carried out in a Horiba Jobin Yvon Fluoromax 3 instrument. Each gel samples were prepared in a quartz cell of 1 cm path length and was excited at 350 nm. The emission scans were recorded from 370 to 800 nm using a slit width of 5 nm with a 1 nm wavelength increment having an integration time of 0.1 s. Fluorescence lifetime values of the F and F-PVPS6 gels were measured using a time-correlated single photon
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counting fluorometer (Fluorecule, Horiba Jobin Yvon). The fluorescence lifetime (τf) was calculated from the decay time (τi) and the relative amplitude (ai) using the following equation
τf =
(1)
a1τ1+ a2τ2+ a3τ3
where a1, a2, a3 are relative amplitudes and τ1, τ2, τ3 are their respective lifetimes. The FT-IR spectra of pure components and the xerogels were recorded using KBr pellets in a Perkin Elmer FT-IR instrument (FT-IR-8400S). Wide angle X-ray scattering (WAXS) experiments of F and F-PVPSx xerogels were performed in a Bruker AXS diffractomer (model D8 Advance) using a Lynx Eye detector. The instrument was operated at a 40 KV voltage and at a 40 mA current. Samples were placed on glass slides and were scanned in the range of 2θ = 4-40o at the scan rate of 0.5 sec/step with a step width of 0.02o. Conductivity. The dc-conductivity of the F and F-PVPS5 xerogel films was measured by twoprobe method at 25 oC. At first the respective gels were drop casted on the conducting strip (1 mm width) of an indium tin oxide (ITO) coated glass and the xerogel films were prepared by drying at ambient temperature. The conductivity was measured by taking the sample between two ITO conducting strips of 1 mm width placed perpendicularly. The area of the sample was 0.01cm2 and a screw gauge was used to measure the thickness of the samples. The conductivities of the sandwiched samples were measured by an electrometer (Keithley, model 617) at 25 oC using the equation:
σ=
1 l × R a
(2)
Where ‘R’ is the resistance, ‘a’ is the area, ‘l’ is the thickness of the samples. The I−V characteristics of the F and F-PVPS5 xerogel films were recorded at 25 oC by scanning from (i) 0 to +5 V and then (ii) +5 to -5 V followed by (iii) a reverse scan from -5 to 0 V. The impedance 6 ACS Paragon Plus Environment
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spectra of the F and F-PVPS5 xerogel films were measured using a Solarton SI 1260 impedance analyzer (Solarton, London, U.K.). The impedance spectra of the xerogel films were recorded at 25 °C over the frequency range from 1Hz to 32 MHz with ac perturbation of 25 mV at 0 V dc level. Result and discussion F forms a bright yellow colored transparent gel in DMF-water mixture (1:1) and it is very weak in nature. However, the transparency decreases with addition of PVPS and the gel becomes mechanically robust as evident from the digital images shown in Scheme 1. The thermal stability of the gels is obtained from the gel melting temperature (Tgm) values which increase with increase in PVPS concentration in the F gel (Figure S1). The highest increase is observed in FPVPS5 gel suggesting highest co-assembly formation between F and PVPS in this particular concentration. So, PVPS enhances the thermal stability of F gel and it might be due to the increase of fibrillar network density with increase of PVPS concentration yielding larger surface force to entrap the solvent molecules (cf. morphology section). The decrease of Tgm above 0.25 % (w/v) PVPS may come from the formation of disordered network structure arising from the onset of self-aggregation of the polymer chains disturbing the growth of co-assembled fibers. The critical gelation concentration (CGC) at 30 oC of pure F in DMF-water mixture (1:1, v/v) is measured to be 0.25 % (w/v) for 24 hrs of gelation time, but in the presence of one mg of PVPS it’s CGC reduces to 0.07 %, crowning F-PVPS system, the title of a supergelator. Morphology and structure. TEM studies on the F and F-PVPS2 gels (Figure 1) are performed to enlighten the morphological metamorphosis of the F gel with addition of PVPS. It is evident from Figure 1a that F gel exhibits three dimensional fibrous network morphology, with average fibrillar diameter of 7.3 ± 0.6 nm. It is apparent from the TEM images of F-PVPS2 (Figure 1b) 7 ACS Paragon Plus Environment
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gels that the fibers are more frequent with an increase in average fiber diameter (20.5 ± 2.5 nm) and increased branching, causing an increase of fiber density. It suggests that PVPS plays a crucial role in the aggregation process and this type of increase in fiber diameter of a small molecular gel after addition of a non-gelling polymer is not common, rather a decrease in fiber diameters are reported.28, 31
(a)
(b)
Figure 1. TEM images of (a) F and (b) F-PVPS2 gels. To elucidate the supramolecular organization of F molecules with themselves and with PVPS it is necessary to shed light on the structures of the complexes. FTIR spectra of pure F powder, F and F-PVPSx xerogels (Figure S2) are carried out to envisage the H-bonding interactions present in the gels. The sharp vibrational peaks of pure F powder, for -OH and –NH2 groups in the region 3000-3600 cm-1 merge together generating a broad band at about 3399 cm-1 indicating the involvement of all these complementary groups in the intermolecular H-bonding interaction to produce the F gel. The peak at 1694 cm-1 in the F powder belongs to the >C=O bond stretching vibration which shifts to 1699 cm-1 in the F gel indicating H bonding interactions. To ascertain the H-bonding interactions between PVPS and F, FTIR spectra of pure PVPS is carried out and a selected portion of the spectra is presented in Figure 2a. The band at 995 cm-1 is ascribed to the vibration of the free pyridine groups33 and in the F-PVPS5 gel (Figure 2b) the intensity of this
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band has drastically reduced whereas new bands at the region 1000-1020 cm-1 are originated. These bands at 1000-1020 cm-1 are due to H bonded pyridine groups and their presence in the FPVPS5 gel confirms H-bonding between N atom of pyridine rings of PVPS with the –COOH group of F (Figure 2c). This type of H-bonding is also apparent from the shifting of the >C=O band in the F gel at 1699 cm-1 to 1692 cm-1 in the F-PVPS5 gel (Figure S2).
a
c
b
Figure 2. FTIR spectra of selected portions of (a) pure PVPS and (b) F-PVPS5 xerogels. (c) Schematic representation of the H-bonding between F and PVPS. WAXS patterns of the xerogels (Figure S3) reflect the amorphous nature of the samples. A single diffraction peak at 2θ = 27.1o (dhkl = 3.3 Ǻ) present in all the gels corresponds to π-π stacking distances. However, it is important to note that the intensity of the π-π stacking peak increases gradually making the peak sharper after addition of PVPS to the F gel indicating better π-π stacking interaction. Here no prominent crystalline peak(s) is observed suggesting both the F and F-PVPS gels are amorphous in nature. From these studies we can envisage the gelation mechanisms of F and F-PVPSx gels (Scheme 2). It is well known that the pterin ring of F forms disc like H-bonds through tetramerization of F.34 Gottarelli et al. have proved the tetramer formation by H-bonding using small-angle neutron9 ACS Paragon Plus Environment
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scattering, circular-dichroism, and NMR techniques.35 They determined the columnar length from the characteristic X-ray diffraction peak (dhkl = 3.4 Ǻ) corresponding to the tetramer stacking. The disk like arrays produced from H-bonds, through the tetramerization of F molecules, act as a precursor of the fibers.36,
37
In the WAXS patterns of the F and F-PVPSx
xerogels the diffraction peak at 2θ = 27.1o (dhkl = 3.3 Ǻ) can be ascribed to the π-stacking of disk like arrays of the F tetramers. These discs gradually form columnar structures via π-stacking which further aggregate to yield fibrils. These fibrils then entangle resulting in the 3D fibrillar nanostructure. However, a different scenario is expected to occur in the case of F-PVPSx gels. Because of the presence of PVPS, the π-stacking of the disc shaped F tetramers is hindered to some extent causing a smaller columnar structure. PVPS gets adsorbed on the growing tip of the fibrils and exerts a certain degree of structural mismatch with respect to the crystallographic orientation of the parent fibrils (Scheme 2). This phenomena is termed as “crystallographic mismatch branching” which causes the formation of new daughter fibers on the tips of the parent fibers.26, 31 So, the fibers become more branched compared to that of the F gel causing higher density of fibers in the F-PVPSx gels. The PVPS molecules get attached to the outer surface of the fibers through H-bonding as evident from the FTIR spectra, causing further lateral growth of the fibers, thereby increasing the fiber diameter. Here, apart from the full polymer chain, a part of the polymer chain may also be involved together for the variation of morphology of F-fibers for both branching and thickening processes. Thus thicker fibers with high branching and high density are produced. Mechanical properties. Rheological studies of the F and F-PVPSx gels are carried out to confirm the solid like nature of the gel samples, and also to investigate the change in mechanical
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Scheme 2. Schematic representation of gelation mechanism of F and F-PVPSx (the structure of F is depicted in a simplified way to avoid clumsiness) properties of the F gel after addition of PVPS. Frequency sweep experiments carried on the F and F-PVPS5 gels (Figure 3a) exhibit a wide linear viscoelastic region (LVR) in each case, and the storage modulus (G’) is higher than the loss modulus (G”) confirming their gel nature. Both the storage and loss modulus values have magnificently improved (~500 times) in the F-PVPS5 gel compared to the F gel. Increased branching density facilitates easy storage and dissipation of energy through its surfaces increasing the modulus values. Also, the increased fiber density helps in entrapping the solvent molecules more tightly which causes the increase of Tgm. To elucidate the variation in mechanical properties of the F-PVPSx gels, complex modulus (G* = (G'2 + G''2)1/2) values are calculated and are presented in a bar diagram against PVPS concentration (Figure 3b). G* values increase with increase in PVPS concentration initially and shows a maximum value at a PVPS concentration of 0.25% (F-PVPS5 gel). The maximum complex 11 ACS Paragon Plus Environment
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modulus, indicating the maximum ratio of stress to strain under steady state vibratory condition, is suggestive of maximum elastic property of the material and it probably arises due to the best co-assembly between F and PVPS chains at this composition. At PVPS concentrations higher than >0.25% it decreases continuously which may be ascribed to the self aggregation of PVPS producing somewhat disordered network structure.31 Stress sweep experiments (Figure S4) reveal that the yield stress value, measured from the crossing of G’ and G’’ curves, increases from 3.2 Pa in the F gel to 199 Pa in the F-PVPS5 gel. This type of spectacular (~62 times) increase in yield stress is attributed to the highly branched network formation in the F-PVPS5 gels increasing the number of physical junctions yielding maximum surface force than that in F gel. To have a better understanding of long-term viscoelastic behavior of gels, time-dependent evolution of compliance is investigated from creep and creep recovery experiments.38 Various mammalian cells exert stress on hydrogel scaffolds and display different behaviors in response to the compliance of the gel scaffold.39 From this perspective, the characterization of compliance of the gels is very important. Here, we have performed creep and creep recovery experiments on F, and F-PVPS5 gels in a consecutive order (Figure 3c, d.). At the first step, there is an instantaneous step rise of stress, from 0 to 0.5 Pa. The stress is kept constant from t0 to t1 in the creep phase. After that, the stress is completely removed (0 Pa) in the recovery phase. The strain resulting in both the stages is monitored against time (t0
