Abstract
We report a supramolecular process for the synthesis of well-defined fullerene (C60)/polymer colloid nanocomposites in an aqueous solution via complex formation. A biocompatible triblock poly(4-vinylpyridine)-b-polyethylene-b-poly(4-vinylpyridine), P4VP8-b-PEO105-b-P4VP8, was synthesized by atom transfer radical polymerization. The block copolymer formed complexes with C60 in toluene and resulted in fullerene assembly in cluster form. Nanocomposite dispersion in an aqueous solution could be obtained using an aged solution of the polymer/C60/toluene solution by a solvent evaporation technique. The UV-Vis and FTIR spectroscopy confirmed the complex formation of fullerene with the polymer which plays a significant role in controlling the PDI and size of polymer/C60 micelles in the toluene solution. The particle size and morphology of P4VP8-b-PEO105-b-P4VP8 and P4VP8-b-PEO105-b-P4VP8/C60 mixture were studied by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In a cytotoxicity test, both pure polymer and the resulting polymer/C60 composite in water showed more than 90% cell viability at 1 mg/mL concentration.
1. Introduction
The organization of fullerenes (C60) and their derivatives into nanostructures within polymer systems has potential applications in solar cells and biomedicine [1–4]. For example, interesting results on polymer solar cells were reported using a blend of a fullerene derivative and a block copolymer poly(4-vinylpyridine) of poly(3-hexyl thiophene) (P3HT-b-P4VP) [5]. It was also found that a fullerene in an aqueous solution can generate a singlet oxygen under photoirradiation which has implications in the fields of biomedical and environmental science [4, 6]. One of the biologically most relevant features of C60 is the ability to function as a “free radical sponge” and quench various free radicals more efficiently than conventional antioxidants [7]. Fullerenes also have widespread applications ranging from drug-delivery and tissue-scaffolding systems to consumer products [8], and they have been explored in the area of biological chemistry, for activities such as enzyme inhibition, antiviral activity, DNA cleavage, and photodynamic therapy [9].
However, many of C60’s potential applications have been seriously hampered [10, 11] by its extremely low solubility in water. Derivatization of the fullerene molecule with various functional groups and other solubilization procedures using surfactants or long chain polymers [12, 13] through covalent interactions [14–17] may change the properties of C60. It was reported that 1O2 quantum yield decreases with decreasing double bonds in the fullerene molecule due to the modification [18, 19]. Generally, introduction of any substituent into the fullerene core can diminish these properties [18–20]. A more effective and promising approach is to organize the fullerenes by specific noncovalent interactions with amphiphilic block copolymers through charge transfer complexation between fullerenes and block copolymers. Laiho and coworkers fabricated functionally unmodified C60-containing nanostructures via a combination of PS-b-P4VP block copolymer self-assembly and charge-transfer complexation between fullerenes and P4VP segments in an organic solvent [21]. However, synthesis of water-soluble adducts of this spherical carbon allotrope (potential biological applications) became a particularly challenging topic. Therefore, in order to enhance the processability and water solubility of the materials, self-assembly systems of pristine C60 with block copolymers have been explored. For example, water-soluble C60/PS-b-PEO composites obtained through a tedious process exhibited multidistribution of particle sizes [22]. However, supramolecular aggregates with a more uniform size distribution could be obtained. This was done through the aggregation of C60 with PS-b-PDMAEMA in water using a dialysis process, where the fullerene preferably goes to PS region due to hydrophobic interaction [23]. However, in most cases, incorporation of fullerenes into water-soluble supramolecular structures using surfactants or other modifying agent results in the fullerene core being completely covered by the modifying agent. This reduces possibility of contact between water-soluble oxygen and fullerenes, thereby inhibiting some of the desired properties such as the ability to quench singlet oxygen (1O2). Clearly, the preparation of well structure-defined aggregates from C60 with biocompatible polymers in an aqueous medium remains a challenge to overcome.
The present study focuses on the development of a simple method which will allow the assembly of fullerene with an amphiphilic block copolymer P4VP8-b-PEO105-b-P4VP8 in aqueous and organic media. The choice of the P4VP block is due to its more hydrophilic nature as compared to PS which will be more accessible to dissolved oxygen. The P4VP8-b-PEO105-b-P4VP8 amphiphilic block copolymer was synthesized using atom transfer radical polymerization [24, 25].
2. Materials and Methods
2.1. Chemicals and Materials
The hydroxy-end-capped PEO [degree of polymerization (DP): 105; PDI: 1.08] was purchased from Aldrich. 4-Vinylpyridine (VP) (99%) was purified by stirring over CaH2 and distilled under vacuum. 2-Bromoisobutyryl bromide, triethylamine, CuCl (99.99%), N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA 95%), and fullerene (99.5%) were purchased from Aldrich and used without further purification.
2.2. Charge Transfer Complex Formation
The triblock copolymer P4VP8-b-PEO105-b-P4VP8 was synthesized by using the ATRP method described elsewhere [26]. The polymer was dissolved in toluene (1 mg/mL) and kept stirring for 24 hrs. The fullerenes were separately dissolved in toluene with concentration of 1 mg/mL. The color of the solution was purple. The fullerene solution (1 mg/mL) was then added to polymer stock solution, keeping polymer and fullerene ratio of 95 : 5 (w/w). The initial color of solution was purple but with time it gradually turned into brown, indicates complex formation. The formation of the charge transfer complex as a function of time was monitored by UV-Vis spectrophotometry.
2.3. Preparation of Aqueous Colloidal Solution of Fullerene/Polymer Nanocomposite
The aqueous colloidal solution of fullerene/polymer nanocomposite was obtained by pouring 10 mL of a two-month aged solution in toluene into 100 mL of water. The toluene was allowed to evaporate at 80°C for 6 hrs and the final volume of fullerene/polymer composite in aqueous solution was kept at 10 mL. No precipitation was observed.
2.4. In Vitro Cytotoxicity Test
An in vitro cytotoxicity test was performed to study the biocompatibility of the polymer, polymer/C60 nanocomposite, in aqueous solution. For the cytotoxicity test, cell culture experiments were carried out using L929 mouse fibroblasts which were obtained from American Type Culture Collection (ATCC). Cells were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% heat inactivated Fetal Bovine Serum (FBS) (Lonza) and 1% Penicillin-Streptomycin-Glutamine (Antibiotic) (Gibco) maintained at 37°C in 5% CO2 and 95% relative humidity. Cell cytotoxicity test was performed using the Promega CellTiter 96 One Solution Cell Proliferation Assay (MTS). This is a colorimetric method and the solution contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium] (MTS) and an electron coupling reagent (phenazine ethosulfate, PES). The compound is bioreduced by living cells into a colored formazan product that is soluble in tissue culture medium. The quantity of formazan product is measured at 490 nm at which the intensity of absorbance is directly proportional to the number of living cells in culture [27–29]. Cells were seeded at a density of 2 × 104 cells/well in 96-well plates. After 24 hrs, culture medium was replaced by 200 μL serial dilutions of the polymer solutions and the cells were incubated for 24 hrs. Polymer solutions were prepared in serum supplemented DMEM and sterilized by filtration (0.2 μm). An MTS assay was done for four different concentrations, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL. At the end of incubation time period (24 hrs), polymer solutions were aspirated and replaced with 100 μL fresh DMEM, to which 20 μL CellTiter 96 One Solution was added. The cells were then incubated for 4 hrs at 37°C in 5% CO2 incubator. Then the absorbance was measured at 490 nm in a microplate reader (TECAN, Tecan Group Ltd., Switzerland). The cell viability (%) related to control wells containing cell culture medium without polymer was calculated by equation. Each sample was done in triplicate.
2.5. Characterization
The Fourier Transform Infrared (FTIR) spectra were recorded on a Perkin Elmer system 2000. UV Spectrophotometer (UV2501PC, Pharmaspec, Shimadzu) was used to monitor the complex formation. Dynamic light scattering (DLS) [Nano Zetasize ZS90 (Malvern)] and transmission electron microscopy (TEM) (JEOL JEM-2010) were used to monitor the aggregation behavior.
3. Results and Discussion
The block copolymer P4VP8-b-PEO105-b-P4VP8 was prepared via atom transfer radical polymerization (ATRP) and its complex with fullerene as outlined in Figure 1.
Fullerenes (C60) dissolve readily in toluene, which leads to a purple color solution. A fullerene solution in toluene (1 mg/mL) was then added to polymer stock solution (1 mg/mL), keeping polymer fullerene ratio of 95 : 5 by weight. The initial color of solution was purple but with time it gradually turned into brown indicating complex formation as shown in Figure 2.
The color change with time was similar to that for the solution of C60 in pyridine. This agrees with the formation of charge-transfer complexes between the electron-donating pyridine solvent and the electron-accepting fullerenes [30, 31]. The changes in the visible spectrum are shown in Figure 3. The solution shows a broad and weak absorption band ranging from 410 to 650 nm. The absorbance in the region increases with aging. The spectral change (Figure 3(iii)) is attributed to the formation of charge transfer complex [30, 31].
In the solution of P4VP8-b-PEO105-b-P4VP8/C60 (95/5 w/w) the most characteristic absorption peak of fullerenes at ~332 nm was not observed. It is because, due to higher concentration of polymer/C60 in solution, UV-Vis spectroscopy crosses the limit and was not able to pinpoint the most characteristic peak at ~332 nm. But the most characteristic peak at ~332 nm is clearly observed when the polymer/C60 solution concentration was reduced by 50%, as it is shown in Figure 4.
FTIR spectroscopy provides further evidence for the complex formation. Upon aging of P4VP8-b-PEO105-b-P4VP8/C60 (95/5 w/w) in toluene, the pyridine peak at 1603 cm−1 is slowly shifted to 1618 cm−1 as shown in Figure 5. The shifting of the most characteristic peak due to interaction of the P4VP near 1600 cm−1 for C=N stretching indicates that a slight electron transfer took place from the 4-vinylpyridine to C60.
The UV-Vis spectrum of polymer/C60 nanocomposite in an aqueous solution shows a broad peak from the fullerenes at about 340 nm. This peak clearly indicates the presence of fullerene in aqueous solution as shown in Figure 6.
The aggregation behavior of P4VP8-b-PEO105-b-P4VP8 block copolymers was studied by dynamic light scattering using Zetasizer Nano ZS and ZS90 (Malvern). The apparent hydrodynamic radius () can be calculated using the Stokes-Einstein equation:where is the Boltzmann constant, is the absolute temperature, is the solvent viscosity, and is the diffusion coefficient. The DLS was conducted at a scattering angle of 90°. The polymer in toluene solution (1 mg/mL) showed bimodal peaks with mean diameters of ~24.4 nm and ~199 nm, respectively, and the PDI was 0.534 (Figure 7(a)). The particle size ranges from 10 to 400 nm. However, in the presence of C60 the polymer solution aggregates into one type of particle with mean diameter of 28.2 nm (Figure 7(b)) with PDI of 0.205. The particle size varies from 10 to 100 nm. The change in the aggregation behavior is clearly due to the C60 complex formation.
(a)
(b)
(c)
TEM micrographs are shown in Figure 8. Figure 8(a) shows the micelle consisting of a compact P4VP core and PEO chains as the corona. These samples were stained with iodine. On the other hand, the polymer/fullerene composites in toluene and water are shown in Figures 8(b) and 8(c), respectively, without iodine staining. The lack of iodine staining was to avoid confusion between P4VP and fullerene which shows up clearly in the fullerene aggregates. The particle size from TEM studies is in good agreement with the light scattering data for the P4VP8-b-PEO105-b-P4VP8 polymer and C60 complexes.
From the TEM, it was determined that the C60 clusters were aggregated with a size of ~30 nm. In light scattering studies (Figure 7(c)), the polymer/C60 nanocomposite micelle in aqueous solution shows the bimodal distribution with mean diameters of ~30 nm and ~110 nm. This agrees reasonably well with the TEM images. The TEM images confirm the formation of fullerene clusters in both processes. The results confirm that the micelle in solution consists of P4VP in the core and PEO in the corona. The fullerenes are located in the core of the polymer aggregates. A plausible mechanism of the micelle formation of P4VP8-b-PEO105-b-P4VP8 and P4VP8-b-PEO105-b-P4VP8/C60 (95/5 w/w) is shown in Scheme 1.
To evaluate the possibility of using the pure polymer and the polymer/C60 nanocomposite in aqueous solution for bioapplications, in vitro cytotoxicity studies were performed by MTS assay. This is a colorimetric test, based on the selective ability of viable cells to reduce the tetrazolium component of MTS into purple colored formazan crystals. MTS assay was done for four different concentrations, namely, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL. The results (Figure 9) show that there is no significant cytotoxicity of the four different concentrations of the pure polymer and the polymer/C60 nanocomposite. It was found that more than 90% of the cells are viable in all the different concentrations for both cases.
4. Conclusions
This study shows that P4VP8-b-PEO105-b-P4VP8/C60 systems can lead to the formation of self-assembled structures. The C60 complexation process has significant effect on the aggregation behaviour. Upon aging of the solution, a charge transfer complex between vinylpyridine unit and C60 was formed, which leads to the formation of self-assembled fullerene clusters. These results were supported by UV-Vis, TEM, and DLS studies. The formation of biocompatible P4VP8-b-PEO105-b-P4VP8/C60 nanocomposites in water may have potential application in biomedicine, cosmetic (as singlet oxygen generator) or other useful applications. In summary, organic-soluble and water-soluble, structurally well-defined C60/polymer nanocomposites were achieved through the self-assembly of pristine C60 with polymer. Though the rate of complex formation presented in this study was found to be slow, the work has opened the door and enlightened how noncovalent interaction can be used to obtain water-soluble, biocompatible, polymer-fullerene nanoparticles.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
Acknowledgments
The authors acknowledge funding from National Research Foundation of Singapore under a competitive research program (NRF-G-CRP-2007-01). One of the authors (Alok Chaurasia) would like thank Nanyang Technological University for a graduate scholarship.