CNT Induced β-Phase in Polylactide: Unique Crystallization

Apr 22, 2013 - CNTs having remarkable optical, electrical, and mechanical ..... (49, 50) It is worth mentioning that Proteinase K exhibits greater act...
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CNT Induced β‑Phase in Polylactide: Unique Crystallization, Biodegradation, and Biocompatibility Narendra K. Singh,†,§ Sunil K. Singh,‡ Debabrata Dash,‡ Prasad Gonugunta,§ Manjusri Misra,*,§,∥ and Pralay Maiti*,† †

School of Materials Science and Technology, Indian Institute of Technology (Banaras Hindu University), Varanasi 221 005, India Department of Biochemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India § School of Engineering, Thornbrough Building and ∥Department of Plant Agriculture, Bioproducts Discovery and Development Centre, University of Guelph, Guelph, Ontario N1G2W1, Canada ‡

S Supporting Information *

ABSTRACT: The effect of multi-walled carbon nanotube (MWCNT) on the crystal structure, unique crystallization, mechanical behavior, enzymatic degradation, and significant improvement in biocompatibility of polylactide (PLA) nanohybrid has been reported. Functionalization of carbon nanotube using stearyl alcohol has been carried out and has been confirmed through FTIR and Raman spectroscopy. PLA nanohybrids have been synthesized using functionalized and neat MWCNT through solution route, and the improved level of dispersion of MWCNT has been achieved in PLA matrix. Highmagnification transmission electron microscope images indicate the unique adsorption of PLA chain leading to the crystallization of β-phase structure on the surface of the functionalized MWCNT against the usual crystallized α-form of pure PLA. The presence of β phase in nanohybrids has been confirmed through electron diffraction pattern, differential scanning calorimetry thermograms, and X-ray diffraction patterns. The improved and diverse mechanical, thermal properties, and crystallization kinetics have been explored with the special emphasis on the relaxation behavior of β phase in dynamic mechanical analysis. The cause of these developments has been appraised from the interaction point of view as calculated from the interaction parameter (χ) using melting-point depression technique. The rate of biodegradation has been studied in detail with plausible mechanism in Proteinase K enzyme media showing their specificity and tuning of biodegradation rate followed by their optimization. For biomedical applications, the effect of pure polymer and nanohybrids on circulating blood cells has been evaluated in detail, and the hemocompatible nature of the nanohybrids has been revealed, suppressing the cellular toxicity of MWCNT.

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INTRODUCTION Eco-friendly polylactide (PLA) has attracted increasing interest in the recent past. PLA is derived from renewable resources,1 and it is biocompatible,2,3 biodegradable,4,5 nontoxic to the human body and the environment, having unique mechanical properties. Biomedical and pharmaceutical applications of PLA cover a wide range from sutures and tissue engineering to biologically active controlled release devices. 6,7 Recent innovation on the production technology has lowered the production cost significantly, which further stimulates the potential use requiring specific performances1 (mechanical, physical, and biodegradation/biocompatible properties). For further improving the aforementioned properties, several approaches have been applied by introduction of nanofiller in polymer matrix to prepare nanohybrids. They exhibit improvement of various properties with respect to neat polymer. For this purpose, various types of nanofillers, that is, organically modified nanoclay,8−11 layered double hydroxide,12 fibers,13 and recently carbon nanotubes (CNTs)14−16 have received considerable attention. CNTs having remarkable optical, © XXXX American Chemical Society

electrical, and mechanical properties with large aspect ratio17−19 may lead to excellent reinforcing filler to substitute or complement the conventional nanofillers in the fabrication of advanced multifunctional polymer nanohybrids,20−22 which is expected to tailor-make the enhanced physical properties of the lightweight materials, especially for biological applications.23−25 However, to realize the full potential of CNTs for developing high-performance polymer−CNT nanohybrids, there is a need to disperse CNT homogenously in the polymer matrix. Considering that the PLA is a semicrystalline polymer,26,27 its mechanical and physical properties are governed by the supramolecular morphology, which in turn is controlled by the crystallization process. Reinforcement at the nanoscale is expected to change the PLA crystallization behavior to improve the polymer/nanohybrid properties.28−30 Zhang et al. studied Received: January 27, 2013 Revised: April 2, 2013

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been achieved by sonication for 5−10 min. The nanohybrids of PLA were prepared through solution route by dissolving PLA in the dispersion of MWCNT/FMWCNT in chloroform, followed by removing the solvent at a fast rate. The solution was sonicated for 30 min to ensure proper mixing of MWCNT/FMWCNT and PLA. The nanohybrids were dried under vacuum for 24 h. Henceforth, we will term the nanohybrids as PLA-MWCNT and PLA-FMWCNT using MWCNT and FMWCNT as the nanofiller, respectively. X-ray Diffraction. X-ray diffraction (XRD) experiments were performed using a Bruker AXS D8 Advance wide-angle Xray diffractometer with Cu Kα radiation and a graphite monochromator (wavelength, λ = 0.154 nm). The generator was operated at 40 kV and 20 mA. Thin polymer films were placed on a quartz sample holder at room temperature and were scanned at diffraction angle 2θ from 1 to 40° at the scanning rate of 1°/min. Differential Scanning Calorimetry. Melting, crystallization temperature, and corresponding enthalpy of fusion and crystallization of the pure PLA and its nanohybrids were determined via differential scanning calorimetry (DSC) using Mettler 832 at a scan rate of 10 °C min−1 (both heating and cooling rate). The DSC was calibrated with indium before use. Mechanical Properties. Dynamic mechanical properties of the samples were studied on thick films of pure PLA and its nanohybrids with dynamic mechanical analyzer Q 800 (TA instrument) in the tensile mode. The dynamic response was from 10 to 140 °C at a frequency of 1 Hz with strain amplitude of 15 μm and at a heating rate of 3 °C/min. The sample with the rectangular cross section having the dimension of 65 × 5 × 1 mm3 was used for DMA testing. Morphological Investigation. TEM was used to observe the nanoscale dispersion of MWCNT/FMWCNT in the PLA matrix. TEM images were obtained using a Tecnai-G2-20 operated at an accelerating voltage of 200 kV. A thin layer was sectioned using a Leica ultra cut UCT equipped with a diamond knife. Selected-area electron diffraction patterns were taken from specific area focusing on the particular area, as shown in the ED figures (on MWCNT or away from MWCNT). The surface morphology of pure PLA and its nanohybrids before and after enzymatic degradation was examined with a Supra Zeiss scanning electron microscope operated at an accelerating voltage of 5 kV. All of the gold coating of samples was done by sputtering apparatus under vacuum before observation in SEM. Enzymatic Degradation. Enzymatic degradation of PLA and its nanohybrids was performed at 37 °C in potassium phosphate buffer (pH 7.4) containing 0.2 mg/mL enzyme Proteinase K. Initial film dimensions of 5 × 5 × 0.1 mm3 were placed in a small vial having 5 mL of phosphate buffer at 37 °C with constant shaking. Enzyme degradation was carried out for 24, 48, 72, and 96 h. Samples were removed, washed with water, and vacuum-dried to constant weight before analyses.

the morphology, thermal properties, electronic transport, and biocompatibility of PLA multiwalled CNT.31 The effect of functionalized CNTs on crystallization kinetics, hydrolytic degradation,32 mechanical properties, and crystal structure of PLA has also been investigated,33 showing the chain conformations of α- and β-phase left-handed 10/3 and 3/1 helices, respectively.34 Recently, CNT-induced crystallization of PLA, especially in early stages, and change of conformational ordering of PLA have only been investigated, concluding CNTbased templates for the alteration of conformational ordering of PLA by providing reactive surfaces where strong noncovalent binding with polymer main chains35 exists, but there is no report of β-phase formation in the presence of CNT as the template. In this work, we have studied the effect of multi-walled carbon nanotube (MWCNT) on the structure, crystallization behavior, physical and mechanical properties and enzymatic degradation of PLA with the whole range of biocompatibility for its use in biomedical applications. The β-phase has been established for the first time using MWCNT as the template. Appropriate functionalization of MWCNT using stearyl alcohol and thereafter preparation of nanohybrids help improving the properties. The functionalization has been confirmed using IR and Raman spectroscopy and level of dispersion of MWCNT in PLA matrix through transmission electron microscopy (TEM). The unique crystallization of PLA on top of MWCNT particles through selected area electron diffraction has been revealed. The improved and diverse mechanical, thermal properties, and crystallization kinetics have been explored. The relaxation behavior through dynamic measurement has been discovered for different phases in the templated system. The comparative higher rates of biodegradation in nanohybrids have been studied in detail with plausible mechanism in enzyme media. Furthermore, we have explored the hemocompatibility of PLA hybrids at different concentrations in circulating blood cells and platelet activation.

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EXPERIMENTAL SECTION Materials. PLA biomer L9000 (Mw = 200 000 g/mol; Mn =101 000 g/mol, 2% D-content) was procured from Biomer, Germany, and MWCNT (20−30 nm outer diameter, 1 to 2 nm thickness with 0.5 to 2 μm length), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), and other reagents were purchased from Sigma Aldrich. Preparation of Functionalized MWCNT. Carboxylfunctionalized MWCNT was prepared by oxidation of MWCNT with a mixture of concentrated nitric acid and sulphuric acid (1:3 v/v) in ultrasonic bath for 5 min and subsequently stirring at 100 °C for 24 h. Vacuum filtration of resulting solution was made through polycarbonate Millipore membrane, and the excess acid was washed thoroughly with deionized water until the pH of the solution reached ∼7. Carboxyl-functionalized MWCNT sample was dried at 80 °C in vacuum oven overnight and ground into powder with a mortar vessel. Carboxyl-functionalized MWCNT (0.5g) was dispersed in dry DMF (30 mL) and was mixed with stearyl alcohol (1.5g) and 1 g dicyclohexyl carbodiimide (DCC), and the mixture was stirred for 48 h. Subsequently, stearyl alcohol was grafted onto CNT at room temperature under the stirring condition for 48 h.36 Henceforth, we will term the stearyl-functionalized MWCNT as FMWCNT. Nanohybrid Preparation. Good dispersion of MWCNT/ FMWCNT with predetermined amount in chloroform has

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BIOCOMPATIBILITY Platelet Preparation. Platelets preparation from human blood was followed as per previous publications.37−39 In brief, peripheral venous blood in citrate phosphate dextrose adenine was centrifuged at 180g for 20 min. The platelet-rich plasma (PRP) was incubated with acetylsalicylic acid (1 mM) for 15 min at 37 °C, followed by ethylenediaminetetraacetate (EDTA) at 5 mM and centrifuged (600g for 15 min) to pellet the cells. Platelets were then washed in buffer A (20 mM HEPES, 138 B

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mM NaCl, 2.9 mM KCl, 1 mM MgCl2, 0.36 mM NaH2PO4, 1 mM ethylene glycol tetraacetic acid (EGTA), 5 mM glucose, and 0.6 ADPase units of apyrase/mL, pH 6.2). Cells were finally suspended in buffer B (pH ∼7.4), which was similar to buffer A but lacks EGTA. Cell count was adjusted to (0.5 to 0.8) × 109 platelets/mL in final suspension. Platelet Aggregation Studies. Platelet aggregation was monitored turbidimetrically using an optical lumi-aggregometer (Chrono-log model 700−2, Wheecon Instruments, India). Platelets were incubated at 37 °C for 1 min under constant stirring (1200 rpm) before the addition of PLA, PLAMWCNT, or PLA-FMWCNT. Aggregation was recorded as percent of light transmitted through the sample as a function of time, while the blank represented 100% light transmission. In Vitro Hemolysis Assay. In vitro hemolysis assay was performed following the literature report.38 In brief, fresh EDTA-stabilized human whole blood samples from healthy volunteers were used. Typically, 2 mL PBS containing 1 mL whole blood was centrifuged at 500g for 10 min to separate red blood corpuscle (RBCs). The purification process was repeated four times, and washed RBCs were diluted to 10 mL in PBS. To test hemolytic activity of PLA, PLA-MWCNT, and PLAFMWCNT, we exposed 1 mL of RBC suspension (∼0.4 × 108 cells/mL) to different concentrations of PLA, PLA-MWCNT, and PLA-FMWCNT in PBS. To obtain positive and negative controls, we suspended RBCs in deionized water and PBS, respectively. Samples were incubated in a rocking shaker at 37 °C for 4 h, followed by centrifugation at 10 000g for 10 min. Absorbance of Hemoglobin was measured at 540 nm, with 655 nm as a reference, in a microplate spectrophotometer (BioTek model Power Wave XS2, Medispec India) at 37 °C. Percent hemolysis was calculated as described elsewhere.39 MTT Assay. MTT assay measures the reduction of a tetrazolium component (MTT) into an insoluble dark-blue formazan initiated by the mitochondria of viable cells. Cytotoxicity of pure polymer/nanohybrids against platelets was assessed after 2 h of exposure of platelets to different concentrations (2−100 μg/mL) of PLA, PLA-MWCNT, and PLA-FMWCNT. After treatment, the cells were incubated with 50 μM MTT for an additional 4 h at 37 °C. The soluble formazan formed after the reduction of MTT was dissolved in 200 μL of DMSO, and absorbance was measured at 570 nm with microplate spectrophotometer (BioTek model Power Wave XS2, Medispec India) at 37 °C. Untreated cells were used as positive control (100% viable) in the study.

Figure 1. Raman spectra of pristine MWCNT, stearyl-alcoholfunctionalized MWCNT (FMWCNT), and their corresponding PLA nanohybrids (PLA-MWCNT, PLA-FMWCNT).

There is no shifting of both of the peaks after nanohybrid formation, suggesting only physical adsorption of PLA on the surface of MWCNT. The value of the D/G band intensity ratio (ID/IG) provides an estimation of the degree of functionalization. The ID/IG ratio of 1.2 for pristine MWCNT increases to 2.4 for stearyl-alcohol-functionalized FMWCNT, and these values remain constant for respective nanohybrids, suggesting physical adsorption of PLA onto CNT surfaces in the nanohybrids. Furthermore, the structural integrity of the CNT walls was fully preserved after nanohybrids formation. FTIR spectra of pure MWCNT, MWCNT-COOH, and FMWCNT (Figure S1 in the Supporting Information) also confirm the functionalization of stearyl alcohol on MWCNT by the presence of additional C−H stretching peaks at 2922 and 2850 cm−1 of the long alkyl chain of stearyl alcohol. Adsorption and Crystallization on MWCNT. The PLAMWCNT and PLA-FMWCNT nanohybrids with 1 wt % loading were prepared through solution route. Figure 2a presents the TEM images of the ultrathin section of the nanohybrids showing fine dispersion of FMWCNT in the PLA matrix, while aggregation is clearly observed in PLA-MWCNT nanohybrid. TEM observations indicate that the dispersion is much better with functionalized MWCNT (FMWCNT) than that of pristine MWCNT as a result of better interaction through organic moiety of stearyl group on top of MWCNT surface and PLA molecules. Interestingly, high-resolution TEM micrographs, focusing single MWCNT, revealed a clear adsorption of PLA chains onto the surface of the MWCNT/ FMWCNT as a distinct white zone on top of black MWCNT/ FMWCNT due to low density of ordered crystalline polymer. It is worth mentioning that there is considerable difference in density of amorphous and purely crystalline phase of PLA.42 Figure 2b represents the selected-area electron diffraction patterns of the crystallized nanohybrids in different indicated locations marked with red circles in the bright-field image. It is obvious that the predominantly β phase of PLA is present in both PLA-MWCNT and PLA-FMWCNT on or around the vicinity of MWCNTs. The crystalline spots/arcs are due to (350)β, (253)β, and (003)β34,43 planes obtained on the top of MWCNT/FMWCNT (marked as I), while pure α phase (216)α (marked as III)43 is present far away from the location

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RESULTS AND DISCUSSION Functionalization of MWCNT. Raman spectroscopic technique was used to characterize the functionalization of MWCNT walls arising out of the introduction of defects caused by the attachment of different chemical species.40 Figure 1 shows the Raman spectra of pristine MWCNT, stearyl-alcoholfunctionalized FMWCNT, and their subsequent PLA nanohybrids (PLA-MWCNT, PLA-FMWCNT). The peak at 1304 cm−1 (D-band) is assigned to the disordered graphite structure or sp3-hybridized carbons of the CNT, and the high-frequency peak at 1600 cm−1 is tangential mode, also known as G-band, and reflects the structural intensity of the sp2-hybridized carbon atoms.41 Both characteristic bands (D and G) have shifted to higher wavenumber in the case of FMWCNT (1310 and 1608 cm−1 for D and G bands, respectively), which indicates that the system needs more energy to vibrate the individual tube or, in other words, each tube became bulkier due to functionalization. C

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Figure 2. (a) Bright-field transmission electron micrographs of PLA-MWCNT and PLA-FMWCNT nanohybrids showing the dispersion of MWCNT in the polymer matrix and (b) electron diffraction patterns showing (I) β phase in PLA-MWCNT, (II) β phase in PLA-FMWCNT, and (III) only α-phase in PLA, corresponding to the specified selected regions as indicated in high-magnification TEM image with red circles.

functionalized nanohybrid (PLA-FMWCNT) as compared with PLA-MWCNT, suggesting a greater amount of β-phase formation in the presence of functionalized MWCNT (Figure 3a) due to stronger interaction with the organic modifier on top of MWCNT. However, the intensity of the β-phase peak is relatively weak as compared with the predominant α phase in the crystallized sample peaks primarily due to their relative abundance. It is worth mentioning that quenched pristine PLA and its nanohybrids did not show the sharp crystalline peaks corresponding to β phase (Figure S3a,b in the Supporting Information). DSC thermograms also support this crystallization behavior of PLA molecules on MWCNT surface in β phase, as evident from the presence of one additional small peak marked as a star at the lower melting point ∼155 °C, about 17 °C below the melting temperature of the α-phase structure (172 °C), as compared with pristine PLA having only α-phase melting peak at 174 °C (Figure 3b).34 β structure is somewhat less stable than α structure corresponding to a certain degree of disorder in the crystals. The chains are forced into a somewhat less favorable packing, which prevents chains from crystallizing in folded-chain crystals. Therefore, the β phase has a lower melting point than that of the α-phase structure. The absence of the β melting peak in quenched nanohybrids further supports the β-phase formation only after crystallization, as observed in XRD studies (Figure S3b in the Supporting Information). The greater ordering phenomena during high-temperature crystallization favors the oriented β-

of MWCNT/FMWCNT. Electron diffraction patterns for pure MWCNTs are observed for (100) and (002) planes.44 These results indicate that PLA crystallizes in the β phase on top of MWCNT surfaces, while it crystallizes in the α form away from the MWCNT particles. This is the first ever instance of forming β-phase of PLA on the surface of CNT. Hu et al.15 showed that the CNT surface induced conformational ordering of PLA chains at lower temperature, while disorder-to-order (α/- to α-) transition has been reported33 through XRD measurement, but there was no indication of β-phase formation in the presence of MWCNT. Similar epitaxial crystallization of nylon 6 on top of 2D layered silicate has been reported with the change in crystal structure of the polymer, causing high heat distortion temperature with respect to pure polymer.45 This structural transformation of PLA in the presence of MWCNT has also been confirmed through XRD and DSC measurements (Figure 3a,b). XRD patterns of pristine PLA and its nanohybrids, after crystallization at 130 °C, show characteristic α-peaks at 2θ ≈ 14.4, 16, 18.2, and 28.4° corresponding to (010), (110)/(200), (203), and (105) planes (Figure S2 in the Supporting Information), respectively, while an additional peak appears at higher 2θ ≈ 29.8° corresponding to the (003) plane of the β phase46 exclusively for nanohybrids (PLA-MWCNT and PLA-FMWCNT), presumably due to suitable interaction between PLA and MWCNT/FMWCNT, where the MWCNT nanoparticles act as template to crystallize the β phase. Furthermore, the intensity of the β phase increases for D

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suggests the interaction between PLA and MWCNT/ FMWCNT, and the extent is stronger for FMWCNT as compared with pristine MWCNT. The enhancement of enthalpy of fusion in nanohybrids is presumably due to the nucleation effect and subsequent growth of crystal in the presence of MWCNT/FMWCNT, which is further revealed from the cold crystallization as well during heating with the heat of crystallization value of 3, 11, and 16 J g−1 for pristine PLA, PLA-MWCNT, and PLA-FMWCNT, respectively. The decrease in cold crystallization temperature for nanohybrids also supports the nucleating power of MWCNT/FMWCNT. The sharp and strong double endothermic peaks of PLA and its nanohybrids (Figure S4b in the Supporting Information) are due to melt recrystallization,47 while the cold crystallization disappears in nanohybrids due to strong nucleating effect of MWCNT/FMWCNT. Similar changes in cold crystallization and glass-transition temperature have been reported for PLA nanocomposites with layered silicate.48 Moreover, the nucleating phenomena have also been observed in polarizing optical micrographs (Figure 5). The average spherulitic diameters are 246, 100, and 58 μm for pure PLA, PLA-MWCNT and PLAFMWCNT, respectively. Both the pure and functionalized MWCNTs act as nucleating agents, and relatively functionalized MWCNT is the better nucleating agent for PLA crystallization as compared with pristine MWCNT. Thermogravimetric analyses for PLA and its nanohybrids exhibit the higher decomposition temperature of nanohybrids by 10 °C as compared with pristine PLA by acting as a better mass transport barrier in the functionalized MWCNT distributed uniformly in the polymer matrix (Figure 6). However, the addition of MWCNTs enhances the thermal performance of PLA, and it acts as good nucleating agent while functionalization of MWCNT further improves the properties. Mechanical Responses. Figure 7 shows the dynamic mechanical behavior of PLA and its nanohybrids (PLAMWCNT, PLA-FMWCNT) in the temperature range of 10 to 140 °C measured at 1 Hz. The significant enhancement of storage modulus has been observed for both of the nanohybrids in the entire range of temperature, and functionalized MWCNT exhibits the highest increment (Table S1 in the Supporting Information) as compared with the PLA-MWCNT, indicating that MWCNTs have a strong reinforcing effect on the elastic properties of pure PLA. The enhancement of storage modulus is obvious for all nanohybrids by 215 and 276% for PLA-MWCNT and PLA-FMWCNT, respectively, at room temperature, below the glass-transition temperature. Pure PLA and its nanohybrids become soft beyond their glass-transition temperatures, while PLA-FMWCNT nanohybrid shows a greater increase in storage modulus by 338% at high temperature (75 °C) as compared with that of PLA matrix due to greater mechanical reinforcement of FMWCNT arising out of better interactions between organically modified MWCNT and PLA. Furthermore, the peaks ∼55 °C of tan δ curves, which represent the glass-transition temperature (Tg) of both of the nanohybrids, are more or less the same as compared with pure PLA. Interestingly, the nanohybrids show a small hump at ∼30 °C, below the glass-transition temperature, which is categorically absent in pure PLA and is presumably due to the relaxation behavior of the β phase at the interface of the polymer and MWCNT/FMWCNT. The β phase is less thermally stable than the α phase; as previously discussed, it relaxes at lower temperature. This type of hump was not observed with layered silicate nanocomposites of PLA, where

Figure 3. (a) XRD patterns and (b) DSC thermograms of pristine PLA and its indicated nanohybrids crystallized at 130 °C for 6 h showing assigned β peaks using * marks. The major peak corresponds to α-phase.

phase formation on top of MWCNT wall (Scheme 1), whereas the higher nucleation rate at lower crystallization temperature (large undercooling) does not allow the ordering incident to take place; instead, direct α-phase formation is facilitated for quenched sample on MWCNT surface. The β-phase fractions have been calculated from the deconvoluted DSC peaks and have been found to be to 8 and 11% for PLA-MWCNT and PLA-FMWCNT, respectively, indicating a little more β-phase content in PLA-FMWCNT as compared with PLA-MWCNT. However, from electron diffraction, XRD, and DSC studies, we conclude that MWCNT/FMWCNT provide templates for the crystallization of PLA chain in the β phase by providing reactive surfaces for strong noncovalent bonding with polymer chains; furthermore, the extent of β phase is more in functionalized MWCNT, providing a better template as compared with pure MWCNT. Thermal Behavior. The thermal behavior of pristine PLA and its nanohybrids (PLA-MWCNT, PLA-FMWCNT has been investigated using DSC. The glass-transition temperature increases for nanohybrids by 5 °C as compared with pristine PLA due to restricted chain movement in the presence of MWCNT/FMWCNT (Figure 4a), and this shifting is slightly higher for PLA-FMWCNT. First heating thermograms exhibit distinct endothermic melting peaks (Tm) at 170.4, 169, and 165 °C with the enthalpy of fusion 28, 34, and 36 J g1− for pristine PLA, PLAMWCNT, and PLA-FMWCNT nanohybrids, respectively (Figure S4 in the Supporting Information). The depression of melting point of the nanohybrids strongly E

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Scheme 1. Schematic Representation of β-Phase PLA on top of MWCNT Surface through Conformational Ordering during Crystallization at High Temperature against the α-Phase Crystallization upon Quenching

gradation in comparison with pure PLA, and among the nanohybrids, PLA-FMWCNT exhibits higher degradation rate due to enhanced interaction between PLA matrix and FMWCNT as well as the smallest spherulitic dimension in PLA-FMWCNT, which in turn has the greater amorphous zone (interspherulitic region) susceptible to hydrolysis by enzyme for biodegradation.49,50 It is worth mentioning that Proteinase K exhibits greater activity for L-PLA and DL-PLA, and, in general, the enzyme activity depends on its binding capability on the substrate. The adsorption of Proteinase K on the nanoscopic dimension of CNT is high;51 favoring higher effective enzyme loading in nanohybrids lead to greater biodegradation in PLA-MWCNT nanohybrids as compared with pure PLA. Furthermore, stearyl modification on MWCNT presumably enhances the binding activity of proteinase K auxiliary, an increase in the biodegradation rate of PLAFMWCNT vis-à-vis PLA-MWCNT. Hence, the combined effect of large interspherulitic amorphous region and higher enzyme loading in PLA-MWCNT nanohybrid exhibits greater biodegradation. The morphology as observed in SEM of pure PLA and its nanohybrids before and after biodegradation has been presented to compare the relative rates of biodegradation (Figure 8b). There is a gradual change of surface roughening with time caused by biodegradation, and severe roughening takes place in the PLA-FMWCNT system, further supporting the relative rates of biodegradation as observed in the kinetics of biodegradation. The fibrous morphology appears only after the degradation, presumably due to the fact that most degradation occurs away from the MWCNT, leaving the MWCNT zone intact, and the dispersion is finer in FMWCNT as compared with MWCNT, causing more fibrous pattern in PLA/FMWCNT nanohybrid. However, enzyme−CNT con-

Figure 4. DSC thermograms of PLA and its indicated nanohybrids showing glass-transition temperatures .

there was no transformation of structure in the presence of layered silicate even though it showed 150% improvement in storage modulus as compared with pure PLA. 9 Hence, the relaxation behavior of β phase is clearly observed for nanohybrids whose relative extent is more in the case of FMWCNT composite because of its greater abundance arising from good dispersion. Enzymatic Degradation. Biodegradation tests have been carried out at 37 °C with enzyme media (Proteinase K from T. album) in buffer solution of pH ∼7.4 for pure PLA and its nanohybrids (Figure 8a). The weight loss of 30 and 60% occurs for PLA-MWCNT and PLA-FMWCNT, respectively, after 96 h of enzymatic degradation against minimum weight loss of only 25% for pure PLA. The nanohybrids exhibit faster biodeF

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Figure 5. Polarizing optical micrographs of PLA and its indicated nanohybrids. The samples were crystallized at 130 °C up to full solidification.

Figure 6. TGA thermograms of PLA and its indicated nanohybrids.

jugates provide a route for regulating biodegradation, especially for PLA. Interactions in Nanohybrids. In the previous sections, we have observed improvement in thermal and mechanical properties including biodegradation of the nanohybrids with greater enhancement in functionalized nanohybrids due to the stronger interaction between matrix PLA and MWCNT/ FMWCNT. To quantify the interactions between polymer and MWCNT/FMWCNT, we have taken the interaction parameter (χ) into account by using the depression of melting point of PLA in the presence of CNT/FCNT following the equation.52,53 RVPLA 1 1 − o =− ϕ2 χ o TNC TPLA ΔHuVMWCNT MWCNT

ToNC

Figure 7. Dynamic mechanical responses of pure PLA and its indicated nanohybrids as a function of temperature in tensile mode (a) storage modulus, and (b) tan δ curves. The arrow indicates the position of the relaxation temperature of β-PLA.

(1)

ToPLA

where and are the equilibrium melting points of the nanohybrid and that of the pure PLA, respectively. VPLA and TMWCNT are the corresponding molar volumes of the repeating unit, respectively. ϕMWCNT is the volume fraction of MWCNT, and ΔHu is the enthalpy of fusion per mole of repeating unit of PLA. The extrapolation technique of the Tm−Tc plot to the Tm = Tc line has been used to measure the equilibrium melting point. Figure 9a shows the representative plot of melting temperature against the heat of fusion of pure PLA crystallized at different temperatures mentioned for various times. The melting temperature increases with increasing the crystallization

temperature (Tc), and this phenomenon is followed for the nanohybrids as well (Figure S5 in the Supporting Information). The representative Tm−Tc plot is shown in Figure 9b. The equilibrium melting points, Tm0 , of pure PLA and its nanohybrids have been evaluated after extrapolation to the Tm = Tc line, and the values are 201, 192, and 187 °C for neat PLA, PLA-MWCNT, and PLA-FMWCNT, respectively. The equilibrium melting temperatures of the nanohybrids decreased as a result of considerable interaction between matrix and G

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Figure 8. (a) Percentage weight loss of PLA and its indicated nanohybrids during enzymatic (Proteinase K from T. album) degradation at 37 °C. (b) FE-SEM images of PLA and its indicated nanohybrids before and after degradation. The ‘0’ indicates the before degradation.

MWCNT/FMWCNT, and the nature of interaction is primarily dipolar, arising from the π cloud of MWCNTs and stearyl moiety with the >CO group of PLA. The calculated values of χ (using eq 1) are thus obtained as (−4.32 and −6.79) × 10−5 for PLA-MWCNT and PLA-FMWCNT, respectively. The lower value of χ for PLA-FMWCNT indicates stronger interaction between its components as compared with PLAMWCNT nanohybrid, and the superior interaction with FMWCNT is mainly due to additional interaction with organic modification (stearyl group) attached to the MWCNT in FMWCNT. Nanohybrids as Biomaterial. When any material is intravenously injected for biomedical applications like biosensors, cell imaging, and drug delivery, it is the surface of the material that comes into contact with or exposes itself to circulating blood cells, much before the material reaches target tissues. Biomedical application of materials implies that it should be compatible with blood cells, particularly platelets, which are known to be highly sensitive to external stimuli and RBCs, cells present in abundance in blood. Because the use of materials is also on the rise each passing day, it becomes pertinent to study their effect on platelets, known for their role

in blood coagulation and hemostasis as well as RBCs. Therefore, we sought here to determine the effect of functionalized polymer nanohybrids on platelets and RBCs. Recently, we have demonstrated that graphene-induced cell aggregation in a concentration-dependent manner is even stronger than that elicited by thrombin, one of the most potent platelet agonists.38 To check the effect of polymer or its nanohybrids on human platelets, we have performed platelet aggregation studies at different concentrations under stirring condition at 37 °C (Figure 10). The addition of PLA (2 and 5 μg/mL) to a suspension of freshly isolated human platelets failed to induce platelet aggregation (Figure 10a,b, tracing 2), while at 10 times higher concentration (50 μg/mL) it could evoke only a minor wave of transmittance (amplitude 17 ± 0.5%) (Figure 10c, tracing 2). However, at identical concentration (50 μg/mL), aggregation elicited by PLAMWCNT and PLA-FMWCNT increased to 30 ± 2 and 34 ± 4%, respectively, (Figure 10c, tracing 3 and 4), while at a lower concentrations they showed minimal aggregation (Figure 10a,b, tracing 3 and 4). We have also studied the adhesion of platelets on coated polymer matrix/nanohybrids (Figure 11). As expected, platelets underwent shape change or spreading on H

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Figure 10. Effect of differentially modified polymer nanohybrids on platelet function. Platelet aggregation induced by samples indicated as 0, 1, 2, 3, and 4 corresponding to Thrombin (control), DMSO (as vehicle), PLA, PLA-MWCNT, and PLA-FMWCNT, respectively, at a different concentration of (a) 2, (b) 5, and (c) 50 μg/mL.

Figure 9. (a) Representative plot of melting temperatures (Tm) versus heat of fusion (ΔH) of pure PLA at the indicated crystallization temperatures (Tc). (b) Tm−Tc plot at 5% ΔH for indicated specimens. The numbers show the corresponding equilibrium melting temperatures (Tm0) in degrees Celsius.

Figure 11. Adhesion and spreading of platelets on immobilized matrices BSA, collagen on pure PLA, and its nanohybrids (PLAMWCNT, PLA-FMWCNT). Scale bars = 10 μm. The collagen fiber is observed in positive control.

adhesion to collagen matrix, which have been shown in positive control. However, PLA and its nanohybrids (PLA-MWCNT and PLA-FMWCNT) did not exhibit any change in platelet shape, which was more similar to negative control with BSA, consistent with minimal platelet activation. It is worth mentioning that pure MWCNT appears to be an incredibly toxic material that initiates strong platelet activation,54,55 while rapping with a biodegradable polymer like PLA in the form of nanohybrid eliminates its toxicity effect completely and promotes the hybrid materials as a potential biomaterial. In a recent report, we showed that graphene oxide sheets induce significant break down of RBC membrane, leading to hemolysis.38 Contrasting this, the nanohybrids of MWCNT/ FMWCNT exhibited hemolytic activity less than 5%, even when concentration was increased to 100 μg/mL, which appears well within the permissible limit56 (Figure 12). Results showed that both pure polymer and its nanohybrids are highly hemocompatible material at moderate concentrations, main-

taining the circulating blood cells (both RBCs as well as platelets) in resting/normal state. Cytotoxicity Assessment of Polymer Nanohybrids. To confirm the biocompatibility of PLA and its nanohybrids, we assessed their cytotoxicity through MTT assay. MTT is known to be reduced to formazan in viable cells by mitochondrial reductase, exhibiting a purple color. Formazan production was measured after 2 h of exposure of platelets to different concentrations (2−100 μg/mL) of PLA, PLA-MWCNT, and PLA-FMWCNT against a control of platelets without any polymer/nanohybrids. None of the polymer nanohybrids induce cell death, even when incubated at the concentration of 100 μg/mL (Figure 13). SDS (1%)-treated platelets were used as positive control. The results supported the conclusion that both of the nanohybrids do not exhibit any cellular toxicity, facilitating the use of PLA-MWCNT/FMWCNT nanohybrids as potential biomaterial. I

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MWCNT-based polymer hybrid materials as potential biomaterial.

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ASSOCIATED CONTENT

S Supporting Information *

Storage modulus of pure PLA and its nanohybrids at different temperature range, FTIR spectra of pure MWCNT and functionalized FMWCNT showing presence of organic group attached to MWCNT, XRD patterns of pristine PLA and its nanohybrids, XRD patterns and DSC thermograms of quenched pristine PLA and its indicated nanohybrids, DSC thermograms of PLA and its indicated nanohybrids, and representative plot of melting temperatures versus heat of fusion of PLA-MWCNT and PLA-FMWCNT nanohybrids. This material is available free of charge via the Internet at http://pubs.acs.org.

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Figure 12. Effect of polymer nanohybrids on erythrocyte membrane integrity. RBC suspensions were exposed to varying concentrations (2, 5, 50, and 100 μg/mL) of PLA or PLA-MWCNT or PLA-FMWCNT for 4 h, followed by centrifugation. Red color of supernatant indicates hemolysis. (+) and (−) symbols represent positive (RBCs suspended in deionized water) and negative (RBCs suspended in phosphatebuffered saline, PBS) controls, respectively.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (P.M.); [email protected] (M.M.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the receipt of research funding from Department of Biotechnology (DBT), New Delhi, Ministry of Science and Technology, Government of India (Project No. BT/PR-6929/BCE/08/433/2005). We acknowledge the receipt of research funding from Department of Foreign Affairs and International Trades (DFAIT) Canadian Bureau for International Education (CBIE), Canada2009, Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA), Canada − New Directions & Alternative Renewable Fuels Research Program Project number SR9225. N.K.S. acknowledges the receipt of funding for his fellowship from CSIR. We acknowledge Dr. D. K. Avasthi and Pawan K. Kulriya of IUAC, New Delhi, for XRD measurements.

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Figure 13. MTT assay of differentially functionalized polymernanohybrid-treated platelets. All data are representative of three independent experiments and are presented as mean ± SEM. SDS indicates the controlled experiment.

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CONCLUSIONS In the present study, we have functionalized MWCNTs through stearyl alcohol by using DCC dehydrating agents. PLA nanohybrids were prepared through solution route using chemically modified MWCNT. The dispersion of CNT in the PLA matrix has been investigated by TEM, showing better dispersion with functionalized MWCNT. Crystallization of PLA in the β phase on the surface of nanotubes has been revealed through TEM, XRD, and DSC for the first time. Good dispersion of nanotubes improves the thermal and mechanical properties of the nanohybrids and also indicates the separate relaxation behavior of β-phase on top of MWCNT surfaces in dynamic measurement. The rate of biodegradation of PLA increases in the presence of MWCNT and further improves with functionalized MWCNT. PLA nanohybrids are found to be hemocompatible nanomaterial as a solid matrix, which does not affect the biology of circulating blood cells and maintains both RBCs as well as platelets in resting state against the highly toxic nature of pure MWCNT, increasing the scope of using J

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