Striking Effect of Polymer End-Group on C60 Nanoparticle Formation

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Letter Cite This: ACS Macro Lett. 2019, 8, 172−176

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Striking Effect of Polymer End-Group on C60 Nanoparticle Formation by High Shear Vibrational Milling with Alkyne-Functionalized Poly(2-oxazoline)s Joachim F. R. Van Guyse,† Victor R. de la Rosa,† Reidar Lund,‡ Michiel De Bruyne,§,∥ Riet De Rycke,§,∥ Sergey K. Fillipov,⊥ and Richard Hoogenboom*,†

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†

Supramolecular Chemistry Group, Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281-S4, 9000 Ghent, Belgium ‡ Department of Chemistry, University of Oslo, P.O. Box 1033 Blindern, 0371 Oslo, Norway § Ghent University, Department of Biomedical Molecular Biology, 9052 Ghent, Belgium and VIB Center for Inflammation Research, 9052 Ghent, Belgium ∥ Ghent University Expertise Centre for Transmission Electron Microscopy and VIB BioImaging Core, 9052 Ghent, Belgium ⊥ Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, 162 06 Prague 6, Czech Republic S Supporting Information *

ABSTRACT: Buckminsterfullerene (C60) has a large potential for biomedical applications. However, the main challenge for the realization of its biomedical application potential is to overcome its extremely low water solubility. One approach is the coformulation with biocompatible water-soluble polymers, such as poly(2-oxazoline)s (PAOx), to form water-soluble C60 nanoparticles (NPs). However, uniform and defined NPs have only been obtained via a thin film hydration method or using cyclodextrin-functionalized PAOx. Here, we report the mechanochemical preparation of defined and stable C60:PAOx NPs by the introduction of a simple alkyne group as a polymer endgroup. The presence of this alkyne bond is proven to be crucial in the mechanochemical synthesis of stable, defined sub-100 nm C60:PAOx NPs, with high C60 content up to 8.9 wt %. uckminsterfullerene (C60) and other fullerenes were first discovered by Kroto et al.1 in 1985 and later made accessible in large quantities by Krätschmer et al.2 Ever since, fullerenes have been the subject of extensive research due to their unique properties for material science and biomedicine.3−5 In biomedicine, in particular, the antioxidant and photosensitizer properties of C60 have been used for the treatment and prevention of cancer and several other degenerative diseases linked to oxidative stress.6−12 Other than the treatment of degenerative diseases, fullerenes have also been applied in the areas of diagnostics, radioprotection, HIV-1 protease binding, and antibacterials.13−15 Despite this biomedical application potential of C60, its negligible water solubility leads to extremely low bioavailability.16 Several approaches to overcome this issue have already been developed, namely, utilizing chemical modification, cosolvent evaporation or intermolecular complexation to generate watersoluble fullerene derivatives, dispersions, or (nano)composites.17−19 In general, intermolecular complexation offers the most elegant solution, as it allows the synthesis of functional fullerene composites with a high degree of complexity, without generating a new C60 derivative with unknown toxicological profile nor having to cope with the

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complex regio- and stereoselectivity during chemical modification of C60.17,20,21 A supramolecular complexation approach can rely on host− guest complexation, whereby the guest, C60, is accommodated in the hydrophobic cavity of the host. Most well-known hosts are calixarenes and cyclodextrins (CD), yielding bicapped sandwich-like complexes or giving rise to nanoparticle assemblies.22−25 Other complexation examples rely on the formation of a supramolecular electron donor−acceptor complex, with C60 as the electron acceptor. The electron donor can be a highly unsaturated moiety such as a porphyrin,26,27 a “Bucky catcher” or “Bucky bowl”,28,29 all of which have been exploited in the synthesis of polymeric C60 hosts.30,31 Alternatively, the electron donor can also contain multiple polarized bonds, such as the tertiary amide bonds found in poly(N-vinylpyrrolidone) (PVP) and poly(2-alkyl-2oxazoline)s (PAOx).9,32 PAOx constitute an interesting class of complexation candidates since they possess high physical and Received: December 23, 2018 Accepted: January 16, 2019

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DOI: 10.1021/acsmacrolett.8b00998 ACS Macro Lett. 2019, 8, 172−176

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prepared with a DP of 100. All polymerizations were terminated with a methanolic solution of potassium hydroxide to install a hydroxyl terminal group. Full details of the synthesis and characterization of the polymers, that all have a dispersity below 1.30, is provided in the Supporting Information. Next, these polymers were used for the mechanochemical (HSVM) preparation of C60 NPs. In short, a mixture of C60 and the polymer were milled for 10 min at 50 Hz, resulting in a homogeneous brown powder. In order to redisperse the powder, Milli-Q water was added, followed by a 2 min HSVM treatment at 50 Hz. Upon opening the grinding vessel, a clear difference was observed between the mixtures obtained with alkyne-PEtOx or methyl-PEtOx (mPEtOx). When using the alkyne-PEtOx, a brown foamy mixture was obtained, while mPEtOx yielded a brown suspension. In a final step, the C60 complexes were recovered from the reaction vessel by extraction with water, followed by filtration over a 0.2 μm pore size filter. This pore size was chosen, as the biological relevance of NPs decreases with increasing size and, generally, a particle size below 200 nm in diameter is preferred for efficient cellular internalization.41,42 After filtration of the brown solution, the alkyne-PEtOx complex with C60 yielded a dark brown filtrate, clearly indicating the presence of C60 containing NPs in solution. In contrast, the mPEtOx-C60 complex yielded a clear filtrate, indicating that larger particles were obtained that could not pass the 0.2 μm filter. These observations provide clear evidence of the importance of the alkyne group for the synthesis of sub-200 nm NPs via HSVM. A step-by-step video illustrating the HSVM process and the difference between using alkyne-PEtOx and mPEtOx is included in the Supporting Information. While the exact role of the alkyne group is still unclear, it may be attributed to the electron donor−acceptor interaction between the alkyne and C60, in addition to the amide-C60 interactions. Apparently, this additional interaction dramatically affects the supramolecular assembly, yielding sub-200 nm NPs instead of micron-sized aggregates. The supramolecular nature of the interactions in the formed NPs was confirmed, as refluxing the formulation in toluene resulted in a purple solution, characteristic of molecularly dissolved C60, as was also confirmed by mass spectrometry. Further analysis via dynamic light scattering (DLS) of a nonfiltered sample confirmed that the formulation of C60 with mPEtOx resulted in ill-defined, micron-sized particles, while alkyne-PEtOx yielded well-defined, sub-100 nm particles (Figure 2A).

chemical stability, combined with large structural variability that may be exploited for the synthesis of stimuli-responsive and multifunctional nanoparticles (NPs).33,34 Traditionally, the supramolecular complexation of C60 is performed in the presence of organic solvents, often with large dilution yielding large solvent waste streams.35 In addition, the often limited compatibility of the organic solvent with the water-soluble host can obstruct efficient complexation. In this context, mechanochemistry, and more specifically high speed vibration milling (HSVM), is gaining significant attention as a facile complexation route for C60 allowing faster, higher yielding, and greener, that is, does not involve the use of toxic organic solvents, formulation of C60. Furthermore, the scalability of HSVM has been recently demonstrated, as the 20−50 kg synthesis of a drug was achieved by Vectorpharma,36 and other reports have shown the successful translation of HSVM synthesis to a continuous twin extruder process.37,38 Therefore, the mechanochemical complex formation of C60 has already been explored with calixarenes, cyclodextrins, calixazulenes, and biocompatible polymers.8,22,39,40 While the mechanochemical preparation of several biocompatible polymer C60 complexes/NPs has been reported, the mechanochemical preparation of well- defined PAOx C60 NPs remains unexplored. To the authors’ knowledge, well-defined C60 PAOx NPs have only been obtained via a thin film hydration method9 and, in our recent work, via HSVM and the use of PAOx having a CD on the α-terminus of the polymer.25 However, in the absence of the CD on the polymer chain, the mechanochemical preparation of C60 PAOx NPs led to the formation of ill-defined NPs with sizes over 200 nm, limiting their biomedical usefulness.41,42 In this work, we report the mechanochemical production of stable, well-defined C60:PAOx NPs through the use of a greatly simplified polymer having a single alkyne group at the αterminus as stabilizer, hypothesized to enhance the donor− acceptor interactions between the polymer and C60, leading to the formation of stable C60-PAOx NPs (Figure 1). We report

Figure 1. Schematic representation of the mechanochemical preparation of well-defined C60 PAOx NPs with alkyne-PAOx.

the complexation of C60 with alkyne-poly(2-ethyl-2-oxazoline) (PEtOx) of varying degrees of polymerization (DP) and poly(2-methyl-2-oxazoline) (PMeOx) and assessed the C60 content, size, and long-term stability in water of the obtained nanoparticles. Finally, we investigated the supramolecular structure of these NPs via small-angle X-ray scattering (SAXS). The synthesis of alkyne-PAOx was performed by cationic ring opening polymerization of the 2-oxazolines utilizing propargyl benzenesulfonate as the initiator, following literature reports.43−45 Alkyne-PEtOx was prepared with a degree of polymerization (DP) of 20, 50, and 100, while PMeOx was

Figure 2. (A) Size distribution obtained by DLS from the obtained particles after HSVM, C60 with alkyne-PEtOx (red) and mPEtOx (black) unfiltered; intensity-weighted plots (dashed line) and volumeweighted plots (solid line). (B) Corresponding UV−vis spectra of the formed particles, mPEtOx in black at 1 mg/mL and alkyne-PEtOx in red at 0.1 mg/mL. 173

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different polymers. Here it can be seen that up to a molar ratio of 1:1, all NPs show a relatively high weight content of C60 up to 8.9 wt % with narrow standard deviations while forming well-defined NPs that are stable in time in water, as was confirmed by DLS (Figure S6 and Table S1) and TEM (Figures S12 and S13). From the data it is evident that the longer polymer chains outperform the shorter polymer chains at higher molar ratios in terms of obtained yields and, thus, provide improved stabilization, which may be ascribed to the increased number of amide groups that can interact with C60. This trend was amplified when the C60:PAOx molar ratio was further increased to 2:1. Here a drop in C60 content and yield was observed for the alkyne-PEtOx DP50 and alkyne-PMeOx DP100, indicating their less-efficient interaction with C60 compared to alkyne-PEtOx DP100, either due to the lower number of amide groups of alkyne-PEtOx DP50 or due to the higher hydrophilicity of alkyne-PMeOx DP100, both compared to alkyne-PEtOx DP100. The formulation of C60 with alkynePEtOx DP50 only led to the formation of stable NPs in twothirds of the preparation attempts. Alkyne-PMeOx DP100, on the other hand, consistently yielded NPs, even though the C60 content showed larger variations. However, DLS data revealed that NPs with alkyne-PMeOx DP100 were not stable, resulting in the formation of larger aggregates in time (Figure S7). In contrast, alkyne-PEtOx DP100 consistently yielded stable, well-defined NPs with minor variations in C60 content, even at this 2:1 molar ratio of C60 to PAOx. The formed particles showed comparable stability as the particles with lower C60 content. Finally, the structure of the NPs formed by formulation of C60 with alkyne-PEtOx DP100 at a 0.5:1 molar ratio of C60 to polymer was investigated by SAXS. The data obtained from different concentrations (Figure S9) was extrapolated to zero concentration of solute to remove the influence of interparticle interactions (Figure 4).

In addition, the characteristic absorption bands of C60 are more pronounced in the UV−vis spectrum of the NPs of C60 with alkyne-PEtOx than for the ill-defined microparticles of C60 with mPEtOx (Figure 2B). In a next step, the role of the polymer structure on the mechanochemical preparation of C60 NPs was investigated using alkyne-PEtOx with a DP of 20, 50, and 100 and alkynePMeOx with DP 100 while varying the molar ratio of C60 to polymer chains from 0.5:1 to 1:1, and 2:1. The formed NPs were isolated via lyophilization followed by redispersion in water and evaluated on their C60 content via UV−vis spectroscopy, utilizing the Lambert−Beer law and the known molar absorption coefficient of C60 of ε(340 nm) = 49000 M−1 cm−1.9,46 Additionally, the size and stability of the formed C60 NPs was followed in time via DLS. The results of the C60 NPs obtained with alkyne-PEtOx DP 50 and 100 and alkynePMeOx with DP 100 are shown in Figure 3. The HSVM

Figure 3. DLS plots of redispersed C60 NPs (1 mg/mL) with PEtOx DP50 (top), PEtOx DP100 (middle), and PMeOx DP100 (bottom) using a C60:PAOx molar ratio of 0.5:1, obtained directly after redispersion (fresh, black) and after 4 weeks (red).

treatment of C60 with alkyne-PEtOx with DP 20 did not yield any NPs in the used molar ratios, indicating that the same number of shorter polymer chains does not provide enough stabilization of the hydrophobic C60. In fact, this indicates that the successful formation of NPs relies on a combination of both the alkyne-C60 interactions as well as the amide-C60 interactions, as having less amide groups impairs the formation of NPs. Figure 3 shows that NPs with a C60:alkyne-PEtOx molar ratio of 0.5:1 maintain a sub-100 nm size upon redispersion (see Figure 2A vs Figure3, middle) and are stable in aqueous solutions for at least 4 weeks. Table 1 shows the weight percentage of C60 in function of the molar ratio for the

Figure 4. Mathematical extrapolation to zero concentration of SAXS data obtained from the NPs obtained after HSVM treatment of C60 with alkyne-PEtOx DP 100 at a 0.5:1 molar ratio of C60:PAOx. Inset: Kratky plot.

Table 1. Overview of Obtained Weight Percentages for C60 Formulations with a Given Polymer in Function of the Applied Molar Ratio in the HSVM Preparation

The Kratky plot (I(q)q2 vs q) demonstrates that these C60alkyne-PEtOx DP100 NPs have a compact structure (Figure 4, inset). The obtained data fits well with the Benoit model in combination with a mass fractal aggregate form factor, indicating that the NPs can be described by a hyperbranched structure, where C60 may act as physical branching points by interaction with multiple polymer chains via both the alkyne and amide groups of the polymers (Figure 4).

polymer C60 weight percentage (max wt %) molar ratio (C60:PAOx)

PEtOx DP 50

PEtOx DP 100

PMeOx DP 100

0.5:1 1:1 2:1

3.3 ± 0.5 (6) 6.9 ± 0.5 (11.3) 2.9 ± 4.1 (20.5)

2.0 ± 0.5 (3.1) 5.5 ± 0.1 (6) 8.9 ± 0.1 (11.3)

3.4 ± 0.2 (4.1) 5.2 ± 1.2 (7.8) 3.8 ± 2.0 (14.5) 174

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(5) Endo, M.; Hayashi, T.; Kim, Y. A.; Terrones, M.; Dresselhaus, M. S. Applications of Carbon Nanotubes in the Twenty-First Century. Philos. Trans. A. Math. Phys. Eng. Sci. 2004, 362, 2223−2238. (6) Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T. D.; Mazur, M.; Telser, J. Free Radicals and Antioxidants in Normal Physiological Functions and Human Disease. Int. J. Biochem. Cell Biol. 2007, 39, 44−84. (7) Valko, M.; Rhodes, C. J.; Moncol, J.; Izakovic, M.; Mazur, M. Free Radicals, Metals and Antioxidants in Oxidative Stress-Induced Cancer. Chem.-Biol. Interact. 2006, 160, 1−40. (8) Misirkic, M. S.; Todorovic-Markovic, B. M.; Vucicevic, L. M.; Janjetovic, K. D.; Jokanovic, V. R.; Dramicanin, M. D.; Markovic, Z. M.; Trajkovic, V. S. The Protection of Cells from Nitric OxideMediated Apoptotic Death by Mechanochemically Synthesized Fullerene (C(60)) Nanoparticles. Biomaterials 2009, 30, 2319−2328. (9) Tong, J.; Zimmerman, M. C.; Li, S.; Yi, X.; Luxenhofer, R.; Jordan, R.; Kabanov, A. V. Neuronal Uptake and Intracellular Superoxide Scavenging of a Fullerene (C60)-Poly(2-oxazoline)s Nanoformulation. Biomaterials 2011, 32, 3654−3665. (10) Wang, I. C.; Tai, L. A.; Lee, D. D.; Kanakamma, P. P.; Shen, C. K. F.; Luh, T. Y.; Cheng, C. H.; Hwang, K. C. C60 and Water-Soluble Fullerene Derivatives as Antioxidants Against Radical-Initiated Lipid Peroxidation. J. Med. Chem. 1999, 42, 4614−4620. (11) Chistyakov, V. A.; Smirnova, Y. O.; Prazdnova, E. V.; Soldatov, A. V. Possible Mechanisms of Fullerene C60 Antioxidant Action. BioMed Res. Int. 2013, 2013, 821498. (12) Zhu, X.; Quaranta, A.; Bensasson, R.; Sollogoub, M.; Zhang, Y. Secondary-Rim γ-Cyclodextrin Functionalization to Conjugate with C60: Improved Efficacy as a Photosensitizer. Chem. - Eur. J. 2017, 23, 9462−9466. (13) Hovland, R.; Gløgård, C.; Aasen, A. J.; Klaveness, J. Gadolinium DO3A Derivatives Mimicking Phospholipids; Preparation and In Vitro Evaluation as pH Responsive MRI Contrast Agents. J. Chem. Soc. Perkin Trans. 2 2001, 929−933. (14) Tzoupis, H.; Leonis, G.; Durdagi, S.; Mouchlis, V.; Mavromoustakos, T.; Papadopoulos, M. G. Binding of Novel Fullerene Inhibitors to HIV-1 Protease: Insight Through Molecular Dynamics and Molecular Mechanics Poisson−Boltzmann Surface Area Calculations. J. Comput.-Aided Mol. Des. 2011, 25, 959−976. (15) Daroczi, B.; Kari, G.; McAleer, M. F.; Wolf, J. C.; Rodeck, U.; Dicker, A. P. In vivo Radioprotection by the Fullerene Nanoparticle DF-1 as Assessed in a Zebrafish Model. Clin. Cancer Res. 2006, 12, 7086−7091. (16) Heymann, D. Chemistry of Fullerenes on the Earth and in the Solar System: A 1995 Review. Lunar Planet. Sci. 1996, 27, 543−544. (17) Semenov, K. N.; Charykov, N. A.; Keskinov, V. N. Fullerenol Synthesis and Identification. Properties of the Fullerenol Water Solutions. J. Chem. Eng. Data 2011, 56, 230−239. (18) Deguchi, S.; Alargova, R. G.; Tsujii, K. Stable Dispersions of Fullerenes, C60 and C70, in Water. Preparation and Characterization. Langmuir 2001, 17, 6013−6017. (19) Badamshina, E.; Gafurova, M. Polymeric Nanocomposites Containing Non-Covalently Bonded Fullerene C60: Properties and Applications. J. Mater. Chem. 2012, 22, 9427−9438. (20) Kolosnjaj, J.; Szwarc, H.; Moussa, F. Toxicity Studies of Fullerenes and Derivatives. Adv. Exp. Med. Biol. 2007, 620, 168−180. (21) Rajagopalan, P.; Wudl, F.; Schinazi, R. F.; Boudinot, F. D. Pharmacokinetics of a Water-Soluble Fullerene in Rats. Antimicrob. Agents Chemother. 1996, 40, 2262−2265. (22) Komatsu, K.; Fujiwara, K.; Murata, Y.; Braun, T. Aqueous Solubilization of Crystalline Fullerenes by Supramolecular Complexation with γ-Cyclodextrin and Sulfocalix[8]arene under Mechanochemical High-Speed Vibration Milling. J. Chem. Soc., Perkin Trans. 1 1999, 105, 2963−2966. (23) Anderson, T.; Nilsson, K.; Sunda, M.; Westman, G.; Wennerstrom, O. C60 Embedded in γ-Cyclodextrin: A Water-Soluble Fullerene. J. Chem. Soc., Chem. Commun. 1992, 604−606. (24) Iohara, D.; Hirayama, F.; Higashi, K.; Yamamoto, K.; Uekama, K. Formation of Stable Hydrophilic C60 Nanoparticles by 2-

In summary, we report a simple and scalable approach to obtain water-soluble C60 NPs, a major challenge in unlocking C60’s biomedical potential.4,47,48 Our approach relies on the introduction of a simple alkyne moiety on the polymer αterminus, which is essential in the mechanochemical synthesis of well-defined C60 PAOx NPs, as micron-sized aggregates were obtained with a corresponding nonfunctional polymer. Other determining factors in the nanoparticle synthesis were polymer length and hydrophobicity, both affecting C60 loading and nanoparticle stability. Under optimal conditions, the formed particles remained stable for at least 4 weeks and had a high fullerene content, up to ±8.9 wt %, a major improvement over earlier C60 polymer composites with biocompatible polymers that had a C60 content of ∼1 wt %.9,19 SAXS data implies that the NPs have a hyperbranched internal structure, with C60 molecules forming a star-like aggregate that assemble into a mass fractal object. This study demonstrates the importance of simple unsaturated PAOx termini in the complexation of C60. Future work will focus on the effect of different unsaturated chain termini as well as the use of other polymer backbones.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.8b00998. Polymer characterization and further nanoparticle characterizations (PDF). C60 formulation with alkyne PEtOx (AVI) C60 formulation with methyl PEtOx (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Reidar Lund: 0000-0001-8017-6396 Sergey K. Fillipov: 0000-0002-4253-5076 Richard Hoogenboom: 0000-0001-7398-2058 Notes

The authors declare the following competing financial interest(s): RH, JVG and VR are listed as inventors on patent WO2016170100A1 that is based on parts of this work. RH and VR are the founders of Avroxa BVBA that commercializes poly(2-oxazoline)s as Ultroxa. The other authors have no conflicts to declare.

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ACKNOWLEDGMENTS S.F. and R.H. acknowledge the support of the mobility project AV Č R − FWO (FWO-17-05). REFERENCES

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