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Functionalized carbon nanotubes modulate the phase transition behavior of thermoresponsive polymer via hydrophilic-hydrophobic balance
Polymer 178 (2019) 121573
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Functionalized carbon nanotubes modulate the phase transition behavior of thermoresponsive polymer via hydrophilic-hydrophobic balance
T
Ritu Yadav, Pannuru Venkatesu* Department of Chemistry, University of Delhi, Delhi, India
HIGHLIGHTS
the impact of functionalized CNTs on the phase transition behaviour of PNIPAM. • Studied techniques have used to predict the phase transition behaviour. • Biophysical phase transition behaviour of PNIPAM has been altered upon the addition of fCNTs. • The the interactions between thermoresponsive polymer and fCNTs. • Explored • The present study can pave the way for drug delivery and pharmaceutical applications. ARTICLE INFO
ABSTRACT
Keywords: Functionalized carbon nanotubes Poly(N-isopropylacrylamide) Drug delivery Lower critical solution temperature Differential scanning calorimetry
The world of nano-science is getting popularity due to their vast applications in the field of material science, biotechnology and biomedical research. In this context, carbon-nanotubes (CNTs) have provided new horizons because of their use as molecular transporters. Nonetheless, the low solubility in molecular solvents and cytotoxity of CNTs prevent their utility in biological process. Here, we established increasing solubility and dispersion of CNTs by functionalization with –COOH group that overcome these fundamental limitations. In spite of a broad range of applications of CNTs, it is highly desirable to examine the effect of functionalized CNTs (fCNTs) on the thermoresponsive polymers. In this respect, we have examined the effect of fCNTs on the thermoresponsive behavior of aqueous poly(N-isopropylacrylamide) (PNIPAM) solution by using several techniques such as dynamic light scattering (DLS), differential scanning calorimeter (DSC), Fourier transform infrared spectroscopy (FTIR), fluorescence spectroscopy and Field emission scanning electron microscope (FESEM). The present results indicate that the presence of fCNTs causes a small increase in the lower critical solution temperature (LCST) of PNIPAM which becomes more pronounced at lower concentration of fCNTs. It reveals that fCNTs indirectly favors the hydrophilic interactions of surrounded water molecules with PNIPAM that eventually stabilizes the coil state of PNIPAM.
1. Introduction Nanomaterials such as carbon-nanotubes (CNTs) have provided new horizons because of their use as biosensors, ion channel blockers [1], drug delivery, nanoelectronics and therapeutic purposes [2,3] and so on. Carbon nanotubes (CNTs) have received much attention due to their attractive features offered in diverse fields like catalyst support, biomedical, high electrical conductivity etc. CNTs have their nanosize and large surface to mass ratios which contribute towards the interaction of nanoparticles with macromolecules [3,4]. The biocompatibility of CNTs with macromolecules is a key factor in proposing their various bioapplications [5]. The strong van der Waals forces of attractions within
*
the CNTs which limits their dispersion and CNTs tend to agglomerate in aqueous solution and also their further use in medical and materials fields. Studies have been explored to improve their limitations by some alterations in surface morphology of CNTs. Ultimately, the surface modification of CNTs is one of the proposed technique to do these morphological changes in structure of CNTs and reduces their toxicity [6]. Among the various interactions studied, van der Waals interactions, л-л stacking interactions and hydrophobic interactions plays a key role in the binding of CNTs among themselves [3]. These interactions being very strong tend to agglomerate the CNTs in the aqueous solution. Therefore, it is highly advisable to control this aggregation of CNTs in solvents with external stimuli.
Corresponding author. E-mail addresses: [email protected], [email protected] (P. Venkatesu).
https://doi.org/10.1016/j.polymer.2019.121573 Received 13 March 2019; Received in revised form 27 May 2019; Accepted 9 June 2019 Available online 11 June 2019 0032-3861/ © 2019 Elsevier Ltd. All rights reserved.
Polymer 178 (2019) 121573
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By performing the functionalization for CNTs, the hydrophobic interactions within the CNTs become less favourable and the hydrophilicity dominates over the hydrophobicity. Carboxylic groups are introduced for the surface modifications of CNTs which subsequently overcome the problem of CNTs to enhance their applications. The functionalization of CNTs reinforces their solubility [7] and biocompatibility and alters their cellular interaction pathways [8]. The functionalized carbon nanotubes (fCNTs) increase the solubility in biomolecules and dispersion in biocompatible media. Particularly, polymers and fCNTs have received growing attention in biomedical fields. Among various thermoresponsive polymers, poly (N-isopropylacrylamide) (PNIPAM) has been increasingly investigated in medical diagnostics, drug delivery, tissue engineering and many more [9,10]. The polymer PNIPAM shows alteration in properties upon changing the pH, temperature, pressure etc [11]. The water soluble amphiphilic PNIPAM shows its lower critical solution temperature (LCST) in the region of physiological temperature [12–14].The intramolecular hydrogen bonding between –C=O group and –N-H group in PNIPAM chains results in a compact globule conformation which makes it difficult to show the hydrophilic interaction with water molecules at above the LCST of polymer [15]. Henceforth, by increasing the temperature above LCST, shrinkage of microstructure takes place that leads to squeeze out a lot of water [12]. Being more favorable for the water molecules to be expelled from the polymer structure into the bulk water, the polymer PNIPAM exhibits hydrophobicity. In addition to this, LCST have been proved to be entropically driven effect [16]. The protein-CNTs studies are vastly available in literature leading to a great progress and potential applications in various scientific fields [3,17]; however the study on the structural interactions of surface morphology of PNIPAM with nanomaterial is still not well explored. The polymer-fCNTs nanocarrier has the ability to control the hydrophilic-hydrophobic balance of macromolecule at physiological temperature [11]. In this regard, Izumi et al. [18] carried out a detailed analysis of aggregation phenomenon of PNIPAM-single walled carbon nanotubes (SWCNTs) hybrids. From the literature survey, it is well known that PNIPAM grafted onto the sidewalls of CNTs which can function as thermally responsive material for high stability capsule in various drug delivery applications [19]. Tamesue and group [20] emphasized that PNIPAM-multi walled carbon nanotubes (MWCNTs) can be used for photodynamic cancer therapy. To endow the surfaces of CNTs with novel structures, covalently attaching polymers has recently attracted considerable attention [9,21]. Wrapping of polymers is thermodynamically stable coating on the surface of CNTs [19]. The CNTs grafted PNIPAM possessing temperature sensitive shell that have potential applications in various fields like delivery of drug, reusable catalyst, probes, sensors and etc. [15]. The study of fCNTs-based thermoresponsive polymer are considering as nanocarrier for anticancer therapy [22]. The literature appears that covalent bonding interactions occur between CNTs and polymers [23]. However, until now there is no study reported about understanding of the chemical interactions between the fCNTs and thermoresponsive polymers, particularly the influence of fCNTs on the phase transition behavior of thermoresponsive polymer solution. Apparently, PNIPAM is water soluble at below LCST and undergoes phase transition at physiological temperature, hence this polymer can be more useful for drug targeting. Meanwhile fCNTs are highly utilized as nano carriers. Therefore, it is essential to explore the effect of fCNTs on the LCST behavior of PNIPAM which depends on the controlling the hydrophilic – hydrophobic balance of polymer at physiological temperature. Here, we employed fluorescence spectroscopy, dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) to investigate the influence of fCNTs on the phase behavior of PNIPAM solution. Further, to explore the structural morphology of PNIPAM in presence of fCNTs, Field
emission scanning electron microscopy (FESEM) was also performed which shows a clear depiction of structural changes taking place inside the polymer structure. The LCST of PNIPAM is approaching to the body temperature by introducing fCNTs which provides a better insight to explore the study in tissue engineering, drug delivery systems, biosensors and etc. 2. Experimental section 2.1. Materials and sample preparation PNIPAM (Mn = 20,000–25000), 8-anilino-1-naphthalenesulfonic acid (ANS) were purchased from Sigma-Aldrich and used without any further purification. Multiwalled carbon nanotubes (MWCNTs) having diameter 15 nm and length 10–30 μm were purchased from Global Nanotech with a purity more than 97%. Double distilled deionzsed water (Ultra series, Rions India, India) with resistivity 18.3 mΩ was used for sample preparation. Earlier studies show that CNTs cannot be stably dispersed if we keep the concentration of PNIPAM below 5 mg/ ml [24]. Except for FTIR measurements, all sample solutions were prepared by dissolving 5 mg/mL of PNIPAM in double distilled deionized water. In this study, we kept the polymer concentration constant and varied the concentration of fCNTs. PNIPAM solution was prepared in bulk amount followed by the addition of 100 mg PNIPAM in 10 ml water. A completely dispersed solution of fCNTs was prepared by adding 3.3 mg of fCNTs in 10 ml double distilled deionized water. The nanocomposite solution of PNIPAM and fCNTs was prepared by keeping same concentration of PNIPAM and varying concentration of fCNTs from (50–500 μL). Any separation technique like centrifugation was not employed so that the structure of nanocomposite formed (fCNTsPNIPAM) would not disturbed. 2.2. Methods The details of techniques used in the current study are delineated in our earlier papers [3,10] and also provided in the supporting information. Fluorescence emission spectra measurements were ascertained by using a Cary Eclipse fluorescence spectrofluorometer (Varian optical spectroscopy instruments, Mulgrave, Victoria, Australia). All the Fourier transform infrared spectrum were acquired by an iS 50 FT-IR (Thermo- Fischer scientific) spectrometer. MIRA 3 TESCAN electron microscope (operating at 10 kV) was used for FESEM measurements. The Zetasizer Nano ZS90 dynamic light scattering (DLS) instrument (Malvern Instruments Ltd., UK) was used to assess the hydrodynamic diameter (dH) of PNIPAM aqueous solution and PNIPAM in fCNTs solution. DSC measurements of PNIPAM in presence of varying concentrations of fCNTs were performed by NANO DSC from TA Instruments, USA. All the reported results are the average of three experimental measurements and further the spectra described in all techniques are found to be reproducible in all the three measurements. The averaged numerical values of LCST and dH have been already provided in the supplementary information with the error values. 3. Results and discussion 3.1. Synthesis and characterization of functionalized CNTs To synthesize the MWCNTs functionalized with –COOH group, the crude MWCNTs (100 mg) were pretreated with solution of HNO3 and H2SO4 in the ratio 1:3 so that strong van der Waals forces of attractions in MWCNTs would decrease. Scheme 1 represents a schematic depiction of functionalization of MWCNTs. Nitrate and sulphate ions play a key role in intertube and intratube intercalation during pretreatment of CNTs [20]. The mixture was kept for ultrasonication at 40 °C for 4 h. After the completion of sonication, mixture was kept for stirring at 2
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Scheme 1. Synthetic route for the synthesis of functionalized MWCNTs.
presented in Fig. 1. The peaks at 1750 cm−1 in the IR spectrum confirms the presence of carbonyl (-C=O) stretching of COOH group and the absorption band at 3420 cm−1 confirms the presence of –OH group as shown in Fig. 1(a). The absorption band shown at 2940 cm−1 confirms the presence of C–H stretching mode of CNTs. The band appearing at 1530 cm−1 is due to the stretching mode for carboxylate anion. The obtained absorption bands are consistent with the literature value [6,26]. Fig. 1(b) shows the structural morphology of fCNTs individually
room temperature for 24 h. After following these steps, the mixture was centrifuged. Discarding the supernatant, the left over solid sample was collected. The material was washed continuously with distilled water until the material had pH 6–7 and the sample was lyophilized to remove the water content. At the end, we got MWCNTs that were functionalized with –COOH group at their outer surface only [25]. Characterization of the synthesized fCNTs was performed by following techniques like FTIR, FESEM, DLS and DSC and the results are
Fig. 1. The characterization of functionalized MWCNTs (a) FTIR, (b) FESEM micrograph of functionalized MWCNTs. (c) represents hydrodynamic diameter of pure fCNTs. (d) DSC heating curve of pure fCNTs. 3
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obtained through FESEM micrographs. Tube like structure of fCNTs is observed in the FESEM image. Furthermore, the aggregation tendency for fCNTs can be depicted through DLS technique which shows intensity distribution of various sized particles present in the solution mixture. Fig. 1 (c) represents the average hydrodynamic diameter (dH) of fCNTs ~ 161 nm which is consistent with literature value [27]. Furthermore, DSC heating curve of fCNTs does not show any transition in the temperature range of 20–100 °C as shown in Fig. 1 (d).
3.2.2. Fourier transform infrared spectroscopy (FTIR) analysis of PNIPAM in presence of fCNTs The molecular interaction changes induced among PNIPAM by fCNTs studies were further carried out by FTIR. To exclude the –OH band due to water, D2O was used as a solvent for prepare the samples. Fig. 3 depicts the FTIR spectra of PNIPAM in fCNTs as a function of the concentration of fCNTs. The spectrum of PNIPAM in aqueous solution shows two stretching bands, one at 1625 cm−1 another one at 1480 cm−1. The two obtained bands at different stretching frequencies are amide I, amide II bands, respectively. The band at 1625 cm−1 corresponds to the carbonyl –C=O—D-O-D hydrogen bonds and the amide II band occurring at 1480 cm−1 is related to N-D deformations caused by D2O molecules around the microstructure of PNIPAM [10]. Fig. 3 clearly depicts that with increase in concentration of fCNTs, the intensity or absorbance also increases however, the intensity is still less than that of the intensity of pure PNIPAM. Moreover, the darkness of MWCNT also can lead to an increasing of FTIR absorbance. It reveals that at higher concentration of fCNTs, the interactions among CNTs are more effective leading to their only a small interference with PNIPAM. At lower concentration of fCNTs, the interactions among –COOH group and amide group is much noticed. Hydrogen bonding interactions are observed among –C=O bond of PNIPAM and –OH group of fCNTs that causes a decrease in intensity of the peak at 1625 cm−1. This indicates that the formation of hydrogen bonds between polymer and fCNTs lead to rearrangement of solvent molecules around polymer structure.
3.2. Spectroscopic analysis of phase transition behavior of PNIPAM in presence of varying concentration of fCNTs 3.2.1. Steady state fluorescence spectroscopy analysis of PNIPAM in presence of fCNTs Steady state fluorescence spectroscopy is assessed to investigate the effect of fCNTs on the phase transition temperature of PNIPAM aqueous solution. ANS is used as an external probe in the present study. The excitation at 360 nm shows emission maxima at 513 nm and also very low fluorescence intensity in water [10]. For this study, we have observed the changes in fluorescence intensity of ANS in PNIPAM in varying concentrations of fCNTs within the range of 420–600 nm at the interval of 1 nm. It can be seen in Fig. 2 (a) that there is no any influence on the fluorescence intensity of fCNTs in ANS. As shown in Fig. 2 (a) the fluorescence intensity of ANS in PNIPAM is ~250 a.u at wavelength maxima (λmax) 513 nm and this value is consisting with the literature value i.e 510 nm [10]. It is found that intensity continuously decreases with the increasing concentration of fCNTs. This decrease in fluorescence intensity may be due to the presence of fCNTs which alter the water quantity close to the ANS in PNIPAM solution.The presence of fCNTs is basically altering the water structure around PNIPAM which facilitates the variation in ANSpolymer interactions that leads to decrease in the fluorescence intensity. It is noticed that there is λmax shift from 513 to 503 nm is due to the presence of fCNTs. As ANS prominently responds to hydrophobic environment around the polymer, the presence of fCNTs containing COOH group provide more hydrophilicity to the solution mixture. The overall polarity of the solution increases with increasing concentration of fCNTs which is due to –COOH group attached on the CNTs surface. The hydrophilicity around the microenvironment dominates over hydrophobicity and results a decrease in intensity of ANS in PNIPAM as the concentration of fCNTs increases. Fig. 2(b) shows the trend for fluorescence intensity decrease is quiet regular with increase in the concentration of fCNTs. Moreover, for the maximum concentration of fCNTs (500 μL), the intensity approaches to ~58 a.u.
3.2.3. Dynamic light scattering (DLS) studies of PNIPAM in presence of fCNTs To investigate more appropriately the information regarding the association behavior of macromolecular assemblies by estimating their size distribution, DLS analysis was performed at 25 °C. The size distribution of fCNTs and fCNTs-PNIPAM has been presented in Fig. 4 (a) fCNTs showed an average dH of ~161 nm which is consistent with the literature value [27]. Fig. 4 (a) shows that the average dH value of 28 nm for PNIPAM in aqueous solution. The results in Fig. 4 (a) explicitly elucidate that the dH values for fCNTs in PNIPAM are appreciably changed as a function of fCNTs concentration. For example, the dH values are 161, 168, 177, 182, 184, 186 and 188 for 0, 50, 100, 200, 300, 400 and 500 μL fCNTs, respectively at 25 °C. Table 1S represents the dH values of PNIPAM in presence of fCNTs at room temperature. With the increasing concentration of fCNTs, shift in intensity and size of peak of fCNTs has been observed. The hydrophobic association of fCNTs is playing a vital role in the enhancement of dH values of PNIPAM. At lower concentration of fCNTs, the effect is less pronounced, however at
Fig. 2. (a) Steady state fluorescence spectroscopy of ANS in PNIPAM aqueous solution containing varying concentrations of fCNTs. Pure ANS in water (black) and Pure fCNTs in ANS (red), pure PNIPAM (blue), 50 μL fCNTs (dark cyan), 100 μL fCNTs (pink), 200 μL fCNTs (dark yellow), 300 μL fCNTs (navy blue), 400 μL fCNTs (dark brown), 500 μL fCNTs (magenta). (b) The maximum fluorescence intensity of ANS in PNIPAM as a function of concentrations of fCNTs. 4
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Fig. 3. Fourier transform infrared spectroscopy (FTIR) analysis of PNIPAM in D2O with varying concentrations of fCNTs. Pure PNIPAM (black), 50 μL fCNTs (red), 100 μL fCNTs (blue), 200 μL fCNTs (dark cyan), 300 μL fCNTs (pink), 400 μL fCNTs (dark yellow), 500 μL fCNTs (navy blue). The inset shows the FTIR spectra PNIPAM of amide I in D2O containing varying amounts of fCNTs.
higher concentration, the shift in dH values is more prominent which directly correlates with the hydrophobic collapse of fCNTs. The probable reason for this increased dH values may be the large size aggregates formed by the fCNTs at higher concentrations that leads to a shift in the dH values towards the maximum. The results of Fig. 4 (a) suggest that the monomers of polymer changes relatively from hydrophilic to hydrophobic when the concentration of fCNTs increases. This further indicates that as the concentration of fCNTs increases, there is a shift in interaction forces, at higher concentration of fCNTs hydrophobic interactions are more prominent due to increase in fCNTs amount and hence PNIPAM acquires collapsed state ideal for hydrophobic interaction. Furthermore, correlation function in the presence of noise can be employed to extract the time dependence of a signal [10]. The decay time of correlation coefficient gives the information about the mean diameter of aggregated species. The contribution due to multiple scattering in the solution will be neglected if the value of correlation coefficient is approx one [10]. Fig. 4 (b) represents correlation coefficients signal values as a function of time for PNIPAM in fCNTs. The signal for pure fCNTs decays at a fast rate as compared to the decay in presence of PNIPAM. At the maximum concentration of fCNTs i.e 500 μL, the decay time of signal is found to be maximum which reflects the large size of fCNTs agglomerates. On the other hand, 50 μL concentration of fCNTs is observed to form small agglomerates leading to fast decay of signal. The larger aggregation formation is consistent with the dH as shown in Fig. 4 (a).
3.2.4. FESEM analysis of PNIPAM in presence of fCNTs The structural morphology of PNIPAM, fCNTs and PNIPAM-fCNTs nanocomposites are investigated using FESEM. Fig. 5 shows the variation in surface morphologies of PNIPAM, fCNTs and PNIPAM-fCNTs nanocomposites. All the reported images are taken at EHT voltage of 5.00 Kilovolt (KV). Images taken at different magnifications of freezedried sample are shown for better understanding of the ongoing interaction. Fig. 5(a and b) shows the morphology of PNIPAM and very clearly depicts characteristic globular microstructures of polymer. Fig. 5(c and d) illustrates the micrograph of fCNTs showing a typical tubular morphology of CNT with some rough surface indicating functionalization of CNTs. The micrographs of nanocomposite PNIPAMfCNTs in Fig. 5(e and f) shows flattened polymer surface morphology with fCNTs extruding out from it. Interestingly, fCNTs are not found to be adsorbed on the surface of PNIPAM however they are directly extending out from the hydrophobic core of the polymer, further confirming that molecular interactions are playing the key role in bringing the morphological changes in PNIPAM surface as shown in Fig. 5(e and f). The direct interaction of PNIPAM and fCNTs has been observed in the micrographs of PNIPAM-fCNTs in Fig. 5(e and f). In the micrographs of PNIPAM-fCNTs nanocomposites, most of the surface of PNIPAM has been interacted with carbon nanotubes as the fCNTs are partially extending out from the PNIPAM surface. Furthermore, fCNTs in Fig. 5(c and d), are mostly founded in aggregated form however, here after interaction with PNIPAM a uniformly distributed fCNTs can be observed, even single fCNT coming out of the modified PNIPAM surface
Fig. 4. (a) Hydrodynamic diameter of fCNTs in presence of thermoresponsive polymer PNIPAM. Pure PNIPAM (black), pure fCNTs (red), 50 μL fCNTs (blue), 100 μL fCNTs (dark cyan), 200 μL fCNTs (pink), 300 μL fCNTs (dark yellow), 400 μL fCNTs (navy blue), 500 μL fCNTs (dark brown). (b) Represents DLS correlation coefficient plots of varying concentration of fCNTs at 25 °C. 5
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Fig. 5. FESEM micrographs of pure PNIPAM (a and b), the micrographs of fCNTs (c and d), (e and f) represents micrographs of fCNTS-PNIPAM nanocomposite.
Thermal fluorescence studies reflect that LCST values of PNIPAM are reaching at its maximum limit for the lowest concentration (50 μL) of fCNTs whereas, the LCST starts to decrease with increasing the concentration of fCNTs. For the maximum concentration of fCNTs i.e 500 μL, the increase in LCST is less pronounced as compared to that on addition of 50 μL of fCNTs. Interestingly, LCST value in fCNTs is still higher than the LCST of pure PNIPAM in aqueous solution as shown in Fig. 6 (b). It may be due to the predominant hydrophobic association among the fCNTs that leads to its lesser interaction with the amide moieties on the PNIPAM surface. At higher concentrations of fCNTs, hydrophobic collapse of fCNTs leads to large size agglomerates of fCNTs, which in turn available for more prominent interaction with PNIPAM chains. Furthermore, at the lower concentrations of fCNTs, interactions between –COOH group of fCNTs and amide group of PNIPAM takes place to a better extent causing a shift towards the higher values of transition temperature of polymer PNIPAM. Fig. 6 (b) shows a shift in the LCST values of PNIPAM with increase in the concentration of fCNTs. Practically, our thermal fluorescence results explicitly elucidates that a relatively small amount of the fCNTs in PNIPAM is capable of significantly changing the LCST value of PNIPAM.
can be seen in Fig. 5(f). It can be concluded that due to interaction of fCNTs with hydrophobic core of PNIPAM, fCNTs modifies the surface morphology of PNIPAM, while PNIPAM helps in uniform distribution of fCNTs and preventing their agglomeration in aqueous medium. Therefore, sufficient interactions are occurring among PNIPAM and fCNTs causing conformational changes in the polymer structure. 3.3. The temperature responsive characterization of PNIPAM in presence of fCNTs 3.3.1. Thermal fluorescence analysis of PNIPAM in presence of fCNTs To estimate the transition behaviour of PNIPAM in the presence of fCNTs, thermal fluorescence studies are carried out as a function of temperature. The temperature at which the polymer switches from hydrophilic to hydrophobic environment is named as a lower critical solution temperature (LCST). With the onset of temperature, the LCST can be determined as the first break point of the fluorescence intensity. The fluorescence intensity measurements of ANS in PNIPAM in the presence of fCNTs were performed for the temperature range 31–37 °C in the interval of 0.1 °C. Fig. 6 (a) demonstrates the thermal fluorescence emission spectra of ANS in PNIPAM in presence of fCNTs. The inset images in the Fig. 6 (a) show the thermoresponsive behavior of PNIPAM aqueous solution in the presence of fCNTs. At a temperature lower than the LCST of PNIPAM, the solution is very clear however, at the LCST of PNIPAM due to some conformational changes, solution became slightly turbid in appearance. Attaining the complete globular conformation by PNIPAM, the solution became completely turbid after reaching a temperature higher than the LCST of polymer. From the present study, it is very clear that PNIPAM aqueous solution shows its LCST at 33.0 °C which is in corroborate of the studies that have been explored earlier [10]. Moreover, the LCST of PNIPAM is found to increase in the presence of fCNTs.
3.3.2. Dynamic light scattering (DLS) studies of PNIPAM in presence of fCNTs as a function of temperature To investigate further studies on the phase transition of polymer PNIPAM under the influence of fCNTs, DLS is also performed. The change in coil to globular state depicts the hydrodynamic diameter of the aggregated polymer. At a particular temperature, polymer size changes abruptly, this can be accredited to conformational changes of polymer. The temperature at which conformational change takes place is called LCST of polymer. Fig. 7 (a) shows the variation of (dH) of PNIPAM with different fCNTs concentration as a function of temperature. The polymer 6
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Fig. 6. (a) Temperature dependent fluorescence spectroscopy of ANS in PNIPAM with varying concentration of fCNTs. Pure PNIPAM (black), 50 μL fCNTs (red), 100 μL fCNTs (blue), 200 μL fCNTs (dark cyan), 300 μL fCNTs (pink), 400 μL fCNTs (dark yellow), 500 μL fCNTs (navy blue). (b) Represents LCST values of PNIPAM in varying concentrations of fCNTs.
PNIPAM is fairly soluble in water which is shown by the low dH values at below the LCST of polymer. The DLS results revealed that heating of PNIPAM in fCNTs from room temperature to LCST of PNIPAM, the size of polymer in fCNTs surroundings does not change which indicates that these fCNTs do not allow the polymer to aggregate in its surroundings due to hydrophilic interaction. At the phase transition temperature, the dH increases drastically because of large size of agglomerates and it further decreases caused by settling down of agglomerates that are not able to scatter the light further to large extent. A dramatic change in dH values has been observed over a particular temperature which is probed as the LCST of the polymer. The LCST of PNIPAM shifts towards the higher value in presence of fCNTs which is in favor of the results shown by thermal fluorescence spectroscopy technique. Fig. 7 shows that for PNIPAM solution, increasing the amount of fCNTs leads to interaction between fCNTs and PNIPAM resulted in larger sized particle and slightly larger LCST values. The dH values of PNIPAM in the presence of fCNTs are about 110, 242, 282, 337, 381, 550 and 584 nm for 0, 50, 100, 200, 300, 400 and 500 μL of fCNTs, respectively at their respective LCST values. The LCST values of PNIPAM in the presence of 0, 50, 100, 200, 300, 400 and 500 μL of fCNTs are located to be 33.0, 34.4, 34.2, 34.0, 33.9, 33.8 and 33.8 °C, respectively. Therefore, the LCST of PNIPAM is found to decrease with increasing in the concentration of fCNTs, interestingly, their LCST values are more than the LCST of pure PNIPAM aqueous solution. At higher concentration of fCNTs, the interaction within the CNTs are favorable leading to only a small shift in the LCST value of PNIPAM towards the higher values.
At lower concentrations of fCNTs, the interactions between –COOH group of fCNTs and amide groups of polymer PNIPAM are more effective and it stabilizes the coil form of polymer up to the higher value of transition temperature. Therefore, the LCST of PNIPAM being more pronounced towards higher values at lower concentration of fCNTs which approaches the human body temperature as compared to that at higher concentration. Clearly, the presence of fCNTs in temperature induced phase transition of PNIPAM was observed. The LCST of PNIPAM is more sensitive to changes in the dH values induced by the phase transition. The dH values become larger (above the LCST) when the fCNTs content is enhanced which indicates polymer-fCNTs interactions and cause expansion of monomers in the bulk solution. 3.3.3. Differential scanning calorimetry (DSC) studies of PNIPAM in presence of fCNTs as a function of temperature To appraise the phase transition behavior of PNIPAM in varying concentrations of fCNTs, differential scanning calorimetry (DSC) measurements are also carried out. The DSC heating curves of pure PNIPAM as well as PNIPAM with different concentrations of fCNTs are presented in Fig. 8 (a). The heating curve of pure PNIPAM without fCNTs shows a phase transition temperature at 34.6 °C which is consistent with the value reported in literature [27]. From the heating curves presented in Fig. 8 (a), it is clear that fCNTs have little influence on the phase transition behavior of PNIPAM. It is worth noting that at the highest concentration of fCNTs, the thermoresponsive behavior of PNIPAM is very less affected. However, at the lowest concentration of fCNTs i.e 50 μL, the LCST of PNIPAM has been shifted up to 1 °C. Based on the
Fig. 7. (a) (dH) of PNIPAM in presence of varying concentration of fCNTs. 50 μL fCNTs (black), 100 μL fCNTs (red), 200 μL fCNTs (blue), 300 μL fCNTs (dark cyan), 400 μL fCNTs (pink), 500 μL fCNTs (dark yellow). Inset is the phase transition of pure PNIPAM aqueous solution. (b) Represents LCST values of PNIPAM in presence of varying concentration of fCNTs. 7
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Fig. 8. Differential scanning calorimetry of PNIPAM in presence of varying concentrations of fCNTs, 0 μL fCNTs (black), 50 μL (red), 100 μL fCNTs (blue), 200 μL fCNTs (dark cyan), 300 μL fCNTs (pink), 400 μL fCNTs (dark yellow), 500 μL fCNTs (navy blue). (b) Represents LCST values of PNIPAM in presence of varying concentration of fCNTs.
of significantly changing the LCST value of PNIPAM. This could be explained that the alteration of surroundings of fCNTs due to hydrophilic–hydrophobic balance of PNIPAM transformation. In other words, the incorporation of fCNTs into the PNIPAM chains may be responsible for the alteration of LCST of PNIPAM which is mainly due to the formation of covalent bonds with the amide linkage of PNIPAM. Scheme 2 represents a schematic depiction of binding of PNIPAM onto the sidewalls of fCNTs. On the other hand, it is also known from the literature that fCNTs have more surface roughness which promotes the covalent bonding interactions at the interface of fCNTs-matrix formed of poly(furfuryl alcohol) [21]. Moreover, molecular dynamic simulations by Mavrantzas and his group demonstrate that the formation of CNT composites with poly(methyl methacrylate) involved strong binding interactions and hence results in higher tendency of polymer to entre inside the CNTs [32].
DSC heating curves shown in the Fig. 8 (a), a preliminary conclusion can be made that at higher concentration of fCNTs, large size agglomerates of CNTs forms which further disrupts the interactions among the –COOH group of fCNTs and –C=O bond of PNIPAM. Moreover, at lower concentration of fCNTs, –COOH group of fCNTs interacts up to a large extent with the amide bond of polymer and shows a shift towards higher values of transition temperature. Fig. 8 (b) shows the shift in the LCST values of PNIPAM in varying concentration of fCNTs. The results are consistent with the results obtained from all the biophysical techniques used in this study. In addition to the shift in LCST values of PNIPAM, it is more interesting that the endothermic peak values of PNIPAM have also been shifted in the presence of fCNTs. The change from coil to globule conformation of PNIPAM in aqueous solution is an endothermic process [28]. A complete shift of the process from endothermic to exothermic has been observed at lower concentration (50 μL) of fCNTs which indicating the interactions are taking place in fCNTs- PNIPAM complex. The results obtained from current study reveals that PNIPAM has the ability to change its molecular structure and interactions in response to the nanostructures i.e fCNTs. Addition of fCNTs to PNIPAM aqueous solution leads to PNIPAM/fCNTs composite and thus results in higher LCST of PNIPAM. Earlier studies show that addition of hydrophobic moieties decrease the LCST of polymer [29,30]. In this context, LCST of PNIPAM has been found to increase in all of biophysical techniques which attributed to the hydrophilic character provided by –COOH groups of fCNTs. Additionally, the strong van der Waals interactions within the CNTs structures makes them to interact less strongly with the amide groups of PNIPAM structure at higher concentrations which results in only a slight increase in the LCST of PNIPAM. The LCST of PNIPAM in the presence of fCNTs was observed at 35.5 °C in DSC measurements however in DLS, LCST of PNIPAM was observed at 34.8 °C. Literature survey shows that slight difference occurs in the results obtained from various techniques [31]. A much profound effect of fCNTs on the LCST of polymer is observed during the measurements and hence the LCST of PNIPAM in the presence of fCNTs (~34.0 °C) is close to the LCST of PNIPAM aqueous solution i.e 33.0 °C. Thermal analysis results show that a slight increment in LCST behaviour of PNIPAM in presence of fCNTs that compare to pure PNIPAM solution. Interestingly, the lower concentration is showing much more enhancement in LCST value of PNIPAM. The results obtained from various biophysical techniques shows a clear depiction of less enhancement of LCST of PNIPAM in presence of fCNTs. Virtually, our thermal fluorescence, DLS and DSC results (Table 2S) explicitly elucidates that a relatively small amount of the fCNTs in PNIPAM is capable
Scheme 2. Schematic depiction of binding of PNIPAM onto the sidewalls of fCNTs.
4. Conclusion Scientific communities already reported the role of fCNTs in modulating the biocatalaytic activity of proteins [3,17]. Further, we have extended our current study to tune the thermoresponsive behavior of PNIPAM which is accepted as a model protein in presence of fCNTs. The functionality in the MCNTs was added by introducing the –COOH group to its surface. Experimental analysis reveals significant interactions 8
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between the polymer and fCNTs, which is observed to be absent when unmodified CNTs were used together with the polymer. Moreover, only the fCNTs formed stable homogeneously dispersed suspensions in the polymer solution. This observance, we propose is due to the existence of hydrophilic character provided by the COOH groups in fCNTs. Thereby, fCNTs indirectly favors the hydrophilic interactions of surrounded water molecules with macromolecular system that eventually stabilizes the coil state of polymer PNIPAM at temperature higher to its LCST more at lower concentration than at higher concentration. This tuning of the thermoresponsive nature of the PNIPAAM with fCNTs finds wide applications in areas such as drug delivery, tissue engineering or in biosensors where various functionalities are simultaneous needed. PNIPAM-fCNTs nanocomposites are interesting and fascinating material from the viewpoint of designing new composite biomaterials.
[12]
[13] [14] [15] [16] [17]
Acknowledgments
[18]
We would like to acknowledge the financial grant from the SERB, Department of Science and Technology, New Delhi, India (Grant No. EMR/2016/001149). R.Y. is thankful to UGC, New Delhi for providing JRF.
[19]
[20]
Appendix A. Supplementary data
[21]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.polymer.2019.121573.
[22]
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Functionalized carbon nanotubes modulate the phase transition behavior of thermoresponsive polymer via hydrophilic-hydrophobic balance
T
Ritu Yadav, Pannuru Venkatesu* Department of Chemistry, University of Delhi, Delhi, India
HIGHLIGHTS
the impact of functionalized CNTs on the phase transition behaviour of PNIPAM. • Studied techniques have used to predict the phase transition behaviour. • Biophysical phase transition behaviour of PNIPAM has been altered upon the addition of fCNTs. • The the interactions between thermoresponsive polymer and fCNTs. • Explored • The present study can pave the way for drug delivery and pharmaceutical applications. ARTICLE INFO
ABSTRACT
Keywords: Functionalized carbon nanotubes Poly(N-isopropylacrylamide) Drug delivery Lower critical solution temperature Differential scanning calorimetry
The world of nano-science is getting popularity due to their vast applications in the field of material science, biotechnology and biomedical research. In this context, carbon-nanotubes (CNTs) have provided new horizons because of their use as molecular transporters. Nonetheless, the low solubility in molecular solvents and cytotoxity of CNTs prevent their utility in biological process. Here, we established increasing solubility and dispersion of CNTs by functionalization with –COOH group that overcome these fundamental limitations. In spite of a broad range of applications of CNTs, it is highly desirable to examine the effect of functionalized CNTs (fCNTs) on the thermoresponsive polymers. In this respect, we have examined the effect of fCNTs on the thermoresponsive behavior of aqueous poly(N-isopropylacrylamide) (PNIPAM) solution by using several techniques such as dynamic light scattering (DLS), differential scanning calorimeter (DSC), Fourier transform infrared spectroscopy (FTIR), fluorescence spectroscopy and Field emission scanning electron microscope (FESEM). The present results indicate that the presence of fCNTs causes a small increase in the lower critical solution temperature (LCST) of PNIPAM which becomes more pronounced at lower concentration of fCNTs. It reveals that fCNTs indirectly favors the hydrophilic interactions of surrounded water molecules with PNIPAM that eventually stabilizes the coil state of PNIPAM.
1. Introduction Nanomaterials such as carbon-nanotubes (CNTs) have provided new horizons because of their use as biosensors, ion channel blockers [1], drug delivery, nanoelectronics and therapeutic purposes [2,3] and so on. Carbon nanotubes (CNTs) have received much attention due to their attractive features offered in diverse fields like catalyst support, biomedical, high electrical conductivity etc. CNTs have their nanosize and large surface to mass ratios which contribute towards the interaction of nanoparticles with macromolecules [3,4]. The biocompatibility of CNTs with macromolecules is a key factor in proposing their various bioapplications [5]. The strong van der Waals forces of attractions within
*
the CNTs which limits their dispersion and CNTs tend to agglomerate in aqueous solution and also their further use in medical and materials fields. Studies have been explored to improve their limitations by some alterations in surface morphology of CNTs. Ultimately, the surface modification of CNTs is one of the proposed technique to do these morphological changes in structure of CNTs and reduces their toxicity [6]. Among the various interactions studied, van der Waals interactions, л-л stacking interactions and hydrophobic interactions plays a key role in the binding of CNTs among themselves [3]. These interactions being very strong tend to agglomerate the CNTs in the aqueous solution. Therefore, it is highly advisable to control this aggregation of CNTs in solvents with external stimuli.
Corresponding author. E-mail addresses: [email protected], [email protected] (P. Venkatesu).
https://doi.org/10.1016/j.polymer.2019.121573 Received 13 March 2019; Received in revised form 27 May 2019; Accepted 9 June 2019 Available online 11 June 2019 0032-3861/ © 2019 Elsevier Ltd. All rights reserved.
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By performing the functionalization for CNTs, the hydrophobic interactions within the CNTs become less favourable and the hydrophilicity dominates over the hydrophobicity. Carboxylic groups are introduced for the surface modifications of CNTs which subsequently overcome the problem of CNTs to enhance their applications. The functionalization of CNTs reinforces their solubility [7] and biocompatibility and alters their cellular interaction pathways [8]. The functionalized carbon nanotubes (fCNTs) increase the solubility in biomolecules and dispersion in biocompatible media. Particularly, polymers and fCNTs have received growing attention in biomedical fields. Among various thermoresponsive polymers, poly (N-isopropylacrylamide) (PNIPAM) has been increasingly investigated in medical diagnostics, drug delivery, tissue engineering and many more [9,10]. The polymer PNIPAM shows alteration in properties upon changing the pH, temperature, pressure etc [11]. The water soluble amphiphilic PNIPAM shows its lower critical solution temperature (LCST) in the region of physiological temperature [12–14].The intramolecular hydrogen bonding between –C=O group and –N-H group in PNIPAM chains results in a compact globule conformation which makes it difficult to show the hydrophilic interaction with water molecules at above the LCST of polymer [15]. Henceforth, by increasing the temperature above LCST, shrinkage of microstructure takes place that leads to squeeze out a lot of water [12]. Being more favorable for the water molecules to be expelled from the polymer structure into the bulk water, the polymer PNIPAM exhibits hydrophobicity. In addition to this, LCST have been proved to be entropically driven effect [16]. The protein-CNTs studies are vastly available in literature leading to a great progress and potential applications in various scientific fields [3,17]; however the study on the structural interactions of surface morphology of PNIPAM with nanomaterial is still not well explored. The polymer-fCNTs nanocarrier has the ability to control the hydrophilic-hydrophobic balance of macromolecule at physiological temperature [11]. In this regard, Izumi et al. [18] carried out a detailed analysis of aggregation phenomenon of PNIPAM-single walled carbon nanotubes (SWCNTs) hybrids. From the literature survey, it is well known that PNIPAM grafted onto the sidewalls of CNTs which can function as thermally responsive material for high stability capsule in various drug delivery applications [19]. Tamesue and group [20] emphasized that PNIPAM-multi walled carbon nanotubes (MWCNTs) can be used for photodynamic cancer therapy. To endow the surfaces of CNTs with novel structures, covalently attaching polymers has recently attracted considerable attention [9,21]. Wrapping of polymers is thermodynamically stable coating on the surface of CNTs [19]. The CNTs grafted PNIPAM possessing temperature sensitive shell that have potential applications in various fields like delivery of drug, reusable catalyst, probes, sensors and etc. [15]. The study of fCNTs-based thermoresponsive polymer are considering as nanocarrier for anticancer therapy [22]. The literature appears that covalent bonding interactions occur between CNTs and polymers [23]. However, until now there is no study reported about understanding of the chemical interactions between the fCNTs and thermoresponsive polymers, particularly the influence of fCNTs on the phase transition behavior of thermoresponsive polymer solution. Apparently, PNIPAM is water soluble at below LCST and undergoes phase transition at physiological temperature, hence this polymer can be more useful for drug targeting. Meanwhile fCNTs are highly utilized as nano carriers. Therefore, it is essential to explore the effect of fCNTs on the LCST behavior of PNIPAM which depends on the controlling the hydrophilic – hydrophobic balance of polymer at physiological temperature. Here, we employed fluorescence spectroscopy, dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) to investigate the influence of fCNTs on the phase behavior of PNIPAM solution. Further, to explore the structural morphology of PNIPAM in presence of fCNTs, Field
emission scanning electron microscopy (FESEM) was also performed which shows a clear depiction of structural changes taking place inside the polymer structure. The LCST of PNIPAM is approaching to the body temperature by introducing fCNTs which provides a better insight to explore the study in tissue engineering, drug delivery systems, biosensors and etc. 2. Experimental section 2.1. Materials and sample preparation PNIPAM (Mn = 20,000–25000), 8-anilino-1-naphthalenesulfonic acid (ANS) were purchased from Sigma-Aldrich and used without any further purification. Multiwalled carbon nanotubes (MWCNTs) having diameter 15 nm and length 10–30 μm were purchased from Global Nanotech with a purity more than 97%. Double distilled deionzsed water (Ultra series, Rions India, India) with resistivity 18.3 mΩ was used for sample preparation. Earlier studies show that CNTs cannot be stably dispersed if we keep the concentration of PNIPAM below 5 mg/ ml [24]. Except for FTIR measurements, all sample solutions were prepared by dissolving 5 mg/mL of PNIPAM in double distilled deionized water. In this study, we kept the polymer concentration constant and varied the concentration of fCNTs. PNIPAM solution was prepared in bulk amount followed by the addition of 100 mg PNIPAM in 10 ml water. A completely dispersed solution of fCNTs was prepared by adding 3.3 mg of fCNTs in 10 ml double distilled deionized water. The nanocomposite solution of PNIPAM and fCNTs was prepared by keeping same concentration of PNIPAM and varying concentration of fCNTs from (50–500 μL). Any separation technique like centrifugation was not employed so that the structure of nanocomposite formed (fCNTsPNIPAM) would not disturbed. 2.2. Methods The details of techniques used in the current study are delineated in our earlier papers [3,10] and also provided in the supporting information. Fluorescence emission spectra measurements were ascertained by using a Cary Eclipse fluorescence spectrofluorometer (Varian optical spectroscopy instruments, Mulgrave, Victoria, Australia). All the Fourier transform infrared spectrum were acquired by an iS 50 FT-IR (Thermo- Fischer scientific) spectrometer. MIRA 3 TESCAN electron microscope (operating at 10 kV) was used for FESEM measurements. The Zetasizer Nano ZS90 dynamic light scattering (DLS) instrument (Malvern Instruments Ltd., UK) was used to assess the hydrodynamic diameter (dH) of PNIPAM aqueous solution and PNIPAM in fCNTs solution. DSC measurements of PNIPAM in presence of varying concentrations of fCNTs were performed by NANO DSC from TA Instruments, USA. All the reported results are the average of three experimental measurements and further the spectra described in all techniques are found to be reproducible in all the three measurements. The averaged numerical values of LCST and dH have been already provided in the supplementary information with the error values. 3. Results and discussion 3.1. Synthesis and characterization of functionalized CNTs To synthesize the MWCNTs functionalized with –COOH group, the crude MWCNTs (100 mg) were pretreated with solution of HNO3 and H2SO4 in the ratio 1:3 so that strong van der Waals forces of attractions in MWCNTs would decrease. Scheme 1 represents a schematic depiction of functionalization of MWCNTs. Nitrate and sulphate ions play a key role in intertube and intratube intercalation during pretreatment of CNTs [20]. The mixture was kept for ultrasonication at 40 °C for 4 h. After the completion of sonication, mixture was kept for stirring at 2
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Scheme 1. Synthetic route for the synthesis of functionalized MWCNTs.
presented in Fig. 1. The peaks at 1750 cm−1 in the IR spectrum confirms the presence of carbonyl (-C=O) stretching of COOH group and the absorption band at 3420 cm−1 confirms the presence of –OH group as shown in Fig. 1(a). The absorption band shown at 2940 cm−1 confirms the presence of C–H stretching mode of CNTs. The band appearing at 1530 cm−1 is due to the stretching mode for carboxylate anion. The obtained absorption bands are consistent with the literature value [6,26]. Fig. 1(b) shows the structural morphology of fCNTs individually
room temperature for 24 h. After following these steps, the mixture was centrifuged. Discarding the supernatant, the left over solid sample was collected. The material was washed continuously with distilled water until the material had pH 6–7 and the sample was lyophilized to remove the water content. At the end, we got MWCNTs that were functionalized with –COOH group at their outer surface only [25]. Characterization of the synthesized fCNTs was performed by following techniques like FTIR, FESEM, DLS and DSC and the results are
Fig. 1. The characterization of functionalized MWCNTs (a) FTIR, (b) FESEM micrograph of functionalized MWCNTs. (c) represents hydrodynamic diameter of pure fCNTs. (d) DSC heating curve of pure fCNTs. 3
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obtained through FESEM micrographs. Tube like structure of fCNTs is observed in the FESEM image. Furthermore, the aggregation tendency for fCNTs can be depicted through DLS technique which shows intensity distribution of various sized particles present in the solution mixture. Fig. 1 (c) represents the average hydrodynamic diameter (dH) of fCNTs ~ 161 nm which is consistent with literature value [27]. Furthermore, DSC heating curve of fCNTs does not show any transition in the temperature range of 20–100 °C as shown in Fig. 1 (d).
3.2.2. Fourier transform infrared spectroscopy (FTIR) analysis of PNIPAM in presence of fCNTs The molecular interaction changes induced among PNIPAM by fCNTs studies were further carried out by FTIR. To exclude the –OH band due to water, D2O was used as a solvent for prepare the samples. Fig. 3 depicts the FTIR spectra of PNIPAM in fCNTs as a function of the concentration of fCNTs. The spectrum of PNIPAM in aqueous solution shows two stretching bands, one at 1625 cm−1 another one at 1480 cm−1. The two obtained bands at different stretching frequencies are amide I, amide II bands, respectively. The band at 1625 cm−1 corresponds to the carbonyl –C=O—D-O-D hydrogen bonds and the amide II band occurring at 1480 cm−1 is related to N-D deformations caused by D2O molecules around the microstructure of PNIPAM [10]. Fig. 3 clearly depicts that with increase in concentration of fCNTs, the intensity or absorbance also increases however, the intensity is still less than that of the intensity of pure PNIPAM. Moreover, the darkness of MWCNT also can lead to an increasing of FTIR absorbance. It reveals that at higher concentration of fCNTs, the interactions among CNTs are more effective leading to their only a small interference with PNIPAM. At lower concentration of fCNTs, the interactions among –COOH group and amide group is much noticed. Hydrogen bonding interactions are observed among –C=O bond of PNIPAM and –OH group of fCNTs that causes a decrease in intensity of the peak at 1625 cm−1. This indicates that the formation of hydrogen bonds between polymer and fCNTs lead to rearrangement of solvent molecules around polymer structure.
3.2. Spectroscopic analysis of phase transition behavior of PNIPAM in presence of varying concentration of fCNTs 3.2.1. Steady state fluorescence spectroscopy analysis of PNIPAM in presence of fCNTs Steady state fluorescence spectroscopy is assessed to investigate the effect of fCNTs on the phase transition temperature of PNIPAM aqueous solution. ANS is used as an external probe in the present study. The excitation at 360 nm shows emission maxima at 513 nm and also very low fluorescence intensity in water [10]. For this study, we have observed the changes in fluorescence intensity of ANS in PNIPAM in varying concentrations of fCNTs within the range of 420–600 nm at the interval of 1 nm. It can be seen in Fig. 2 (a) that there is no any influence on the fluorescence intensity of fCNTs in ANS. As shown in Fig. 2 (a) the fluorescence intensity of ANS in PNIPAM is ~250 a.u at wavelength maxima (λmax) 513 nm and this value is consisting with the literature value i.e 510 nm [10]. It is found that intensity continuously decreases with the increasing concentration of fCNTs. This decrease in fluorescence intensity may be due to the presence of fCNTs which alter the water quantity close to the ANS in PNIPAM solution.The presence of fCNTs is basically altering the water structure around PNIPAM which facilitates the variation in ANSpolymer interactions that leads to decrease in the fluorescence intensity. It is noticed that there is λmax shift from 513 to 503 nm is due to the presence of fCNTs. As ANS prominently responds to hydrophobic environment around the polymer, the presence of fCNTs containing COOH group provide more hydrophilicity to the solution mixture. The overall polarity of the solution increases with increasing concentration of fCNTs which is due to –COOH group attached on the CNTs surface. The hydrophilicity around the microenvironment dominates over hydrophobicity and results a decrease in intensity of ANS in PNIPAM as the concentration of fCNTs increases. Fig. 2(b) shows the trend for fluorescence intensity decrease is quiet regular with increase in the concentration of fCNTs. Moreover, for the maximum concentration of fCNTs (500 μL), the intensity approaches to ~58 a.u.
3.2.3. Dynamic light scattering (DLS) studies of PNIPAM in presence of fCNTs To investigate more appropriately the information regarding the association behavior of macromolecular assemblies by estimating their size distribution, DLS analysis was performed at 25 °C. The size distribution of fCNTs and fCNTs-PNIPAM has been presented in Fig. 4 (a) fCNTs showed an average dH of ~161 nm which is consistent with the literature value [27]. Fig. 4 (a) shows that the average dH value of 28 nm for PNIPAM in aqueous solution. The results in Fig. 4 (a) explicitly elucidate that the dH values for fCNTs in PNIPAM are appreciably changed as a function of fCNTs concentration. For example, the dH values are 161, 168, 177, 182, 184, 186 and 188 for 0, 50, 100, 200, 300, 400 and 500 μL fCNTs, respectively at 25 °C. Table 1S represents the dH values of PNIPAM in presence of fCNTs at room temperature. With the increasing concentration of fCNTs, shift in intensity and size of peak of fCNTs has been observed. The hydrophobic association of fCNTs is playing a vital role in the enhancement of dH values of PNIPAM. At lower concentration of fCNTs, the effect is less pronounced, however at
Fig. 2. (a) Steady state fluorescence spectroscopy of ANS in PNIPAM aqueous solution containing varying concentrations of fCNTs. Pure ANS in water (black) and Pure fCNTs in ANS (red), pure PNIPAM (blue), 50 μL fCNTs (dark cyan), 100 μL fCNTs (pink), 200 μL fCNTs (dark yellow), 300 μL fCNTs (navy blue), 400 μL fCNTs (dark brown), 500 μL fCNTs (magenta). (b) The maximum fluorescence intensity of ANS in PNIPAM as a function of concentrations of fCNTs. 4
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Fig. 3. Fourier transform infrared spectroscopy (FTIR) analysis of PNIPAM in D2O with varying concentrations of fCNTs. Pure PNIPAM (black), 50 μL fCNTs (red), 100 μL fCNTs (blue), 200 μL fCNTs (dark cyan), 300 μL fCNTs (pink), 400 μL fCNTs (dark yellow), 500 μL fCNTs (navy blue). The inset shows the FTIR spectra PNIPAM of amide I in D2O containing varying amounts of fCNTs.
higher concentration, the shift in dH values is more prominent which directly correlates with the hydrophobic collapse of fCNTs. The probable reason for this increased dH values may be the large size aggregates formed by the fCNTs at higher concentrations that leads to a shift in the dH values towards the maximum. The results of Fig. 4 (a) suggest that the monomers of polymer changes relatively from hydrophilic to hydrophobic when the concentration of fCNTs increases. This further indicates that as the concentration of fCNTs increases, there is a shift in interaction forces, at higher concentration of fCNTs hydrophobic interactions are more prominent due to increase in fCNTs amount and hence PNIPAM acquires collapsed state ideal for hydrophobic interaction. Furthermore, correlation function in the presence of noise can be employed to extract the time dependence of a signal [10]. The decay time of correlation coefficient gives the information about the mean diameter of aggregated species. The contribution due to multiple scattering in the solution will be neglected if the value of correlation coefficient is approx one [10]. Fig. 4 (b) represents correlation coefficients signal values as a function of time for PNIPAM in fCNTs. The signal for pure fCNTs decays at a fast rate as compared to the decay in presence of PNIPAM. At the maximum concentration of fCNTs i.e 500 μL, the decay time of signal is found to be maximum which reflects the large size of fCNTs agglomerates. On the other hand, 50 μL concentration of fCNTs is observed to form small agglomerates leading to fast decay of signal. The larger aggregation formation is consistent with the dH as shown in Fig. 4 (a).
3.2.4. FESEM analysis of PNIPAM in presence of fCNTs The structural morphology of PNIPAM, fCNTs and PNIPAM-fCNTs nanocomposites are investigated using FESEM. Fig. 5 shows the variation in surface morphologies of PNIPAM, fCNTs and PNIPAM-fCNTs nanocomposites. All the reported images are taken at EHT voltage of 5.00 Kilovolt (KV). Images taken at different magnifications of freezedried sample are shown for better understanding of the ongoing interaction. Fig. 5(a and b) shows the morphology of PNIPAM and very clearly depicts characteristic globular microstructures of polymer. Fig. 5(c and d) illustrates the micrograph of fCNTs showing a typical tubular morphology of CNT with some rough surface indicating functionalization of CNTs. The micrographs of nanocomposite PNIPAMfCNTs in Fig. 5(e and f) shows flattened polymer surface morphology with fCNTs extruding out from it. Interestingly, fCNTs are not found to be adsorbed on the surface of PNIPAM however they are directly extending out from the hydrophobic core of the polymer, further confirming that molecular interactions are playing the key role in bringing the morphological changes in PNIPAM surface as shown in Fig. 5(e and f). The direct interaction of PNIPAM and fCNTs has been observed in the micrographs of PNIPAM-fCNTs in Fig. 5(e and f). In the micrographs of PNIPAM-fCNTs nanocomposites, most of the surface of PNIPAM has been interacted with carbon nanotubes as the fCNTs are partially extending out from the PNIPAM surface. Furthermore, fCNTs in Fig. 5(c and d), are mostly founded in aggregated form however, here after interaction with PNIPAM a uniformly distributed fCNTs can be observed, even single fCNT coming out of the modified PNIPAM surface
Fig. 4. (a) Hydrodynamic diameter of fCNTs in presence of thermoresponsive polymer PNIPAM. Pure PNIPAM (black), pure fCNTs (red), 50 μL fCNTs (blue), 100 μL fCNTs (dark cyan), 200 μL fCNTs (pink), 300 μL fCNTs (dark yellow), 400 μL fCNTs (navy blue), 500 μL fCNTs (dark brown). (b) Represents DLS correlation coefficient plots of varying concentration of fCNTs at 25 °C. 5
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Fig. 5. FESEM micrographs of pure PNIPAM (a and b), the micrographs of fCNTs (c and d), (e and f) represents micrographs of fCNTS-PNIPAM nanocomposite.
Thermal fluorescence studies reflect that LCST values of PNIPAM are reaching at its maximum limit for the lowest concentration (50 μL) of fCNTs whereas, the LCST starts to decrease with increasing the concentration of fCNTs. For the maximum concentration of fCNTs i.e 500 μL, the increase in LCST is less pronounced as compared to that on addition of 50 μL of fCNTs. Interestingly, LCST value in fCNTs is still higher than the LCST of pure PNIPAM in aqueous solution as shown in Fig. 6 (b). It may be due to the predominant hydrophobic association among the fCNTs that leads to its lesser interaction with the amide moieties on the PNIPAM surface. At higher concentrations of fCNTs, hydrophobic collapse of fCNTs leads to large size agglomerates of fCNTs, which in turn available for more prominent interaction with PNIPAM chains. Furthermore, at the lower concentrations of fCNTs, interactions between –COOH group of fCNTs and amide group of PNIPAM takes place to a better extent causing a shift towards the higher values of transition temperature of polymer PNIPAM. Fig. 6 (b) shows a shift in the LCST values of PNIPAM with increase in the concentration of fCNTs. Practically, our thermal fluorescence results explicitly elucidates that a relatively small amount of the fCNTs in PNIPAM is capable of significantly changing the LCST value of PNIPAM.
can be seen in Fig. 5(f). It can be concluded that due to interaction of fCNTs with hydrophobic core of PNIPAM, fCNTs modifies the surface morphology of PNIPAM, while PNIPAM helps in uniform distribution of fCNTs and preventing their agglomeration in aqueous medium. Therefore, sufficient interactions are occurring among PNIPAM and fCNTs causing conformational changes in the polymer structure. 3.3. The temperature responsive characterization of PNIPAM in presence of fCNTs 3.3.1. Thermal fluorescence analysis of PNIPAM in presence of fCNTs To estimate the transition behaviour of PNIPAM in the presence of fCNTs, thermal fluorescence studies are carried out as a function of temperature. The temperature at which the polymer switches from hydrophilic to hydrophobic environment is named as a lower critical solution temperature (LCST). With the onset of temperature, the LCST can be determined as the first break point of the fluorescence intensity. The fluorescence intensity measurements of ANS in PNIPAM in the presence of fCNTs were performed for the temperature range 31–37 °C in the interval of 0.1 °C. Fig. 6 (a) demonstrates the thermal fluorescence emission spectra of ANS in PNIPAM in presence of fCNTs. The inset images in the Fig. 6 (a) show the thermoresponsive behavior of PNIPAM aqueous solution in the presence of fCNTs. At a temperature lower than the LCST of PNIPAM, the solution is very clear however, at the LCST of PNIPAM due to some conformational changes, solution became slightly turbid in appearance. Attaining the complete globular conformation by PNIPAM, the solution became completely turbid after reaching a temperature higher than the LCST of polymer. From the present study, it is very clear that PNIPAM aqueous solution shows its LCST at 33.0 °C which is in corroborate of the studies that have been explored earlier [10]. Moreover, the LCST of PNIPAM is found to increase in the presence of fCNTs.
3.3.2. Dynamic light scattering (DLS) studies of PNIPAM in presence of fCNTs as a function of temperature To investigate further studies on the phase transition of polymer PNIPAM under the influence of fCNTs, DLS is also performed. The change in coil to globular state depicts the hydrodynamic diameter of the aggregated polymer. At a particular temperature, polymer size changes abruptly, this can be accredited to conformational changes of polymer. The temperature at which conformational change takes place is called LCST of polymer. Fig. 7 (a) shows the variation of (dH) of PNIPAM with different fCNTs concentration as a function of temperature. The polymer 6
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Fig. 6. (a) Temperature dependent fluorescence spectroscopy of ANS in PNIPAM with varying concentration of fCNTs. Pure PNIPAM (black), 50 μL fCNTs (red), 100 μL fCNTs (blue), 200 μL fCNTs (dark cyan), 300 μL fCNTs (pink), 400 μL fCNTs (dark yellow), 500 μL fCNTs (navy blue). (b) Represents LCST values of PNIPAM in varying concentrations of fCNTs.
PNIPAM is fairly soluble in water which is shown by the low dH values at below the LCST of polymer. The DLS results revealed that heating of PNIPAM in fCNTs from room temperature to LCST of PNIPAM, the size of polymer in fCNTs surroundings does not change which indicates that these fCNTs do not allow the polymer to aggregate in its surroundings due to hydrophilic interaction. At the phase transition temperature, the dH increases drastically because of large size of agglomerates and it further decreases caused by settling down of agglomerates that are not able to scatter the light further to large extent. A dramatic change in dH values has been observed over a particular temperature which is probed as the LCST of the polymer. The LCST of PNIPAM shifts towards the higher value in presence of fCNTs which is in favor of the results shown by thermal fluorescence spectroscopy technique. Fig. 7 shows that for PNIPAM solution, increasing the amount of fCNTs leads to interaction between fCNTs and PNIPAM resulted in larger sized particle and slightly larger LCST values. The dH values of PNIPAM in the presence of fCNTs are about 110, 242, 282, 337, 381, 550 and 584 nm for 0, 50, 100, 200, 300, 400 and 500 μL of fCNTs, respectively at their respective LCST values. The LCST values of PNIPAM in the presence of 0, 50, 100, 200, 300, 400 and 500 μL of fCNTs are located to be 33.0, 34.4, 34.2, 34.0, 33.9, 33.8 and 33.8 °C, respectively. Therefore, the LCST of PNIPAM is found to decrease with increasing in the concentration of fCNTs, interestingly, their LCST values are more than the LCST of pure PNIPAM aqueous solution. At higher concentration of fCNTs, the interaction within the CNTs are favorable leading to only a small shift in the LCST value of PNIPAM towards the higher values.
At lower concentrations of fCNTs, the interactions between –COOH group of fCNTs and amide groups of polymer PNIPAM are more effective and it stabilizes the coil form of polymer up to the higher value of transition temperature. Therefore, the LCST of PNIPAM being more pronounced towards higher values at lower concentration of fCNTs which approaches the human body temperature as compared to that at higher concentration. Clearly, the presence of fCNTs in temperature induced phase transition of PNIPAM was observed. The LCST of PNIPAM is more sensitive to changes in the dH values induced by the phase transition. The dH values become larger (above the LCST) when the fCNTs content is enhanced which indicates polymer-fCNTs interactions and cause expansion of monomers in the bulk solution. 3.3.3. Differential scanning calorimetry (DSC) studies of PNIPAM in presence of fCNTs as a function of temperature To appraise the phase transition behavior of PNIPAM in varying concentrations of fCNTs, differential scanning calorimetry (DSC) measurements are also carried out. The DSC heating curves of pure PNIPAM as well as PNIPAM with different concentrations of fCNTs are presented in Fig. 8 (a). The heating curve of pure PNIPAM without fCNTs shows a phase transition temperature at 34.6 °C which is consistent with the value reported in literature [27]. From the heating curves presented in Fig. 8 (a), it is clear that fCNTs have little influence on the phase transition behavior of PNIPAM. It is worth noting that at the highest concentration of fCNTs, the thermoresponsive behavior of PNIPAM is very less affected. However, at the lowest concentration of fCNTs i.e 50 μL, the LCST of PNIPAM has been shifted up to 1 °C. Based on the
Fig. 7. (a) (dH) of PNIPAM in presence of varying concentration of fCNTs. 50 μL fCNTs (black), 100 μL fCNTs (red), 200 μL fCNTs (blue), 300 μL fCNTs (dark cyan), 400 μL fCNTs (pink), 500 μL fCNTs (dark yellow). Inset is the phase transition of pure PNIPAM aqueous solution. (b) Represents LCST values of PNIPAM in presence of varying concentration of fCNTs. 7
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Fig. 8. Differential scanning calorimetry of PNIPAM in presence of varying concentrations of fCNTs, 0 μL fCNTs (black), 50 μL (red), 100 μL fCNTs (blue), 200 μL fCNTs (dark cyan), 300 μL fCNTs (pink), 400 μL fCNTs (dark yellow), 500 μL fCNTs (navy blue). (b) Represents LCST values of PNIPAM in presence of varying concentration of fCNTs.
of significantly changing the LCST value of PNIPAM. This could be explained that the alteration of surroundings of fCNTs due to hydrophilic–hydrophobic balance of PNIPAM transformation. In other words, the incorporation of fCNTs into the PNIPAM chains may be responsible for the alteration of LCST of PNIPAM which is mainly due to the formation of covalent bonds with the amide linkage of PNIPAM. Scheme 2 represents a schematic depiction of binding of PNIPAM onto the sidewalls of fCNTs. On the other hand, it is also known from the literature that fCNTs have more surface roughness which promotes the covalent bonding interactions at the interface of fCNTs-matrix formed of poly(furfuryl alcohol) [21]. Moreover, molecular dynamic simulations by Mavrantzas and his group demonstrate that the formation of CNT composites with poly(methyl methacrylate) involved strong binding interactions and hence results in higher tendency of polymer to entre inside the CNTs [32].
DSC heating curves shown in the Fig. 8 (a), a preliminary conclusion can be made that at higher concentration of fCNTs, large size agglomerates of CNTs forms which further disrupts the interactions among the –COOH group of fCNTs and –C=O bond of PNIPAM. Moreover, at lower concentration of fCNTs, –COOH group of fCNTs interacts up to a large extent with the amide bond of polymer and shows a shift towards higher values of transition temperature. Fig. 8 (b) shows the shift in the LCST values of PNIPAM in varying concentration of fCNTs. The results are consistent with the results obtained from all the biophysical techniques used in this study. In addition to the shift in LCST values of PNIPAM, it is more interesting that the endothermic peak values of PNIPAM have also been shifted in the presence of fCNTs. The change from coil to globule conformation of PNIPAM in aqueous solution is an endothermic process [28]. A complete shift of the process from endothermic to exothermic has been observed at lower concentration (50 μL) of fCNTs which indicating the interactions are taking place in fCNTs- PNIPAM complex. The results obtained from current study reveals that PNIPAM has the ability to change its molecular structure and interactions in response to the nanostructures i.e fCNTs. Addition of fCNTs to PNIPAM aqueous solution leads to PNIPAM/fCNTs composite and thus results in higher LCST of PNIPAM. Earlier studies show that addition of hydrophobic moieties decrease the LCST of polymer [29,30]. In this context, LCST of PNIPAM has been found to increase in all of biophysical techniques which attributed to the hydrophilic character provided by –COOH groups of fCNTs. Additionally, the strong van der Waals interactions within the CNTs structures makes them to interact less strongly with the amide groups of PNIPAM structure at higher concentrations which results in only a slight increase in the LCST of PNIPAM. The LCST of PNIPAM in the presence of fCNTs was observed at 35.5 °C in DSC measurements however in DLS, LCST of PNIPAM was observed at 34.8 °C. Literature survey shows that slight difference occurs in the results obtained from various techniques [31]. A much profound effect of fCNTs on the LCST of polymer is observed during the measurements and hence the LCST of PNIPAM in the presence of fCNTs (~34.0 °C) is close to the LCST of PNIPAM aqueous solution i.e 33.0 °C. Thermal analysis results show that a slight increment in LCST behaviour of PNIPAM in presence of fCNTs that compare to pure PNIPAM solution. Interestingly, the lower concentration is showing much more enhancement in LCST value of PNIPAM. The results obtained from various biophysical techniques shows a clear depiction of less enhancement of LCST of PNIPAM in presence of fCNTs. Virtually, our thermal fluorescence, DLS and DSC results (Table 2S) explicitly elucidates that a relatively small amount of the fCNTs in PNIPAM is capable
Scheme 2. Schematic depiction of binding of PNIPAM onto the sidewalls of fCNTs.
4. Conclusion Scientific communities already reported the role of fCNTs in modulating the biocatalaytic activity of proteins [3,17]. Further, we have extended our current study to tune the thermoresponsive behavior of PNIPAM which is accepted as a model protein in presence of fCNTs. The functionality in the MCNTs was added by introducing the –COOH group to its surface. Experimental analysis reveals significant interactions 8
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between the polymer and fCNTs, which is observed to be absent when unmodified CNTs were used together with the polymer. Moreover, only the fCNTs formed stable homogeneously dispersed suspensions in the polymer solution. This observance, we propose is due to the existence of hydrophilic character provided by the COOH groups in fCNTs. Thereby, fCNTs indirectly favors the hydrophilic interactions of surrounded water molecules with macromolecular system that eventually stabilizes the coil state of polymer PNIPAM at temperature higher to its LCST more at lower concentration than at higher concentration. This tuning of the thermoresponsive nature of the PNIPAAM with fCNTs finds wide applications in areas such as drug delivery, tissue engineering or in biosensors where various functionalities are simultaneous needed. PNIPAM-fCNTs nanocomposites are interesting and fascinating material from the viewpoint of designing new composite biomaterials.
[12]
[13] [14] [15] [16] [17]
Acknowledgments
[18]
We would like to acknowledge the financial grant from the SERB, Department of Science and Technology, New Delhi, India (Grant No. EMR/2016/001149). R.Y. is thankful to UGC, New Delhi for providing JRF.
[19]
[20]
Appendix A. Supplementary data
[21]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.polymer.2019.121573.
[22]
References [23]
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