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Cite this: DOI: 10.1039/c3cc48293f Received 29th October 2013, Accepted 7th February 2014
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Controlled release of singlet oxygen using diphenylanthracene functionalized polymer nanoparticles† S. Martins, J. P. S. Farinha, C. Baleiza˜o* and M. N. Berberan-Santos*
DOI: 10.1039/c3cc48293f www.rsc.org/chemcomm
Functionalized polymer nanoparticles react and store molecular oxygen for several weeks in the form of endoperoxides. On-demand controlled release of singlet oxygen by the particles is achieved by thermolysis.
Singlet oxygen plays an important role in many oxidative processes, e.g. in natural biological defence processes1 and as a cytotoxic agent in phototherapies.2 Due to its short lifetime and characteristic reactivity,3 a controlled delivery of singlet oxygen in time and space is sometimes necessary.4 This reactive species of oxygen can be generated by the nonradiative excitation of ground-state oxygen via energy transfer, or as a product in a specific chemical reaction.5 In the first case, a photosensitizer is photoexcited and subsequently crosses to the lowest triplet state, then transferring its energy to a neighbouring ground-state oxygen molecule (3O2), which is promoted to the first excited singlet state (1O2).2 The (indirect) photogeneration of 1O2 is used in many fields,4b however, this process requires the simultaneous presence of molecular oxygen and a photosensitizer. Additionally, many photosensitizers are hydrophobic molecules that require elaborate approaches for being used in aqueous media.6 An alternative method for singlet oxygen generation involves the use of specific polycyclic aromatic hydrocarbons (e.g., naphthalene or anthracene derivatives),7 which react reversibly with singlet oxygen to form an endoperoxide, which in some cases can later release 1O2 upon heating, reverting to the original compound.5 Diphenylanthracene (DPA, 1; Scheme 1) is particularly interesting for this application since it can be functionalized for incorporation into different materials, and it forms a stable endoperoxide that can release 1O2 under relatively mild conditions.7 Currently, the development of smart nanoparticles for 1O2 production in photodynamic therapy (PDT) for certain cancer CQFM – Centro de Quı´mica-Fı´sica Molecular and IN – Institute of Nanoscience and Nanotechnology, Instituto Superior Te´cnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available: Experimental synthetic procedure and photophysical characterization of DPA derivatives and functional nanoparticles. See DOI: 10.1039/c3cc48293f
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Scheme 1 Reversible reaction of diphenylanthracenes with oxygen. In the direct reaction, singlet oxygen is generated from ground state oxygen using a photosensitizer in the triplet state, and subsequently adds to the central ring, yielding an endoperoxide. In the reverse reaction, the endoperoxide decomposes upon heating, yielding singlet oxygen.
types is widely explored,8 but most studies focus on the incorporation and delivery of a suitable photosensitizer.4c,6a,9 However, this approach still has several limitations, such as the persistence of the photosensitizers in the body after treatment, and the reduced efficiency for cancer treatment due to the lower oxygen concentration in cancer tissues. Here we report the development of new polymer nanoparticles self-sufficient in oxygen, which allow the thermal release of singlet molecular oxygen in a controlled way. As a proof of concept we prepared different functional polymer nanoparticles (of polystyrene, polybutylmethacrylate and polyethylene glycol acrylate) containing covalently linked endoperoxides. The particles were obtained by emulsion polymerization with DPA polymerisable derivatives, and the endoperoxides were subsequently formed by photooxidation of the DPA using a photosensitizer. The particles were isolated, stored and later used for the thermal release of 1O2, which was followed by fluorescence spectroscopy. Two different DPA derivatives were synthesized for copolymerization with vinyl and acrylate monomers (Scheme S1, ESI†).
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An asymmetrical DPA derivative, 9-(4-phenyl methacrylate)-10(phenyl)anthracene (2), was prepared through a double Suzuki– Miyaura cross-coupling (4-hydroxyphenylboronic acid and phenylboronic acid) and an acylation with methacryloyl chloride in the final step. A second derivative, 9,10-bis(4-vinylphenyl) anthracene (3), was prepared according to a published procedure,10 in one step, by a Suzuki–Miyaura cross-coupling reaction. The DPA derivatives 2 and 3 were purified and fully characterized using NMR, mass spectrometry, absorption and fluorescence spectroscopies. The absorption and fluorescence emission spectra of derivatives 2 and 3, recorded in toluene, show similar shapes to those of pristine DPA (1), exhibiting its characteristic four strong absorption bands (Fig. S1 and Table S1, ESI†). Derivatives 2 and 3 were used to prepare functional polymeric nanoparticles based in poly(butyl methacrylate) (PBMA-2-NP), on a copolymer of di(ethylene glycol) methyl ether methacrylate (DEGA) and poly(ethylene glycol) methyl ether methacrylate (OEGA) (PEGA2-NP) and on polystyrene (PS-3-NP), through emulsion polymerization. Emulsion polymerization not only allows the incorporation of small molecules in the polymeric nanoparticles,11 but it is also a convenient way for surface modification of the particles, through insertion of functional groups, which can then be used for compatibilization with different materials, to bind (bio)molecules for targeting/recognition, etc. Derivative 2 was used to prepare two sets of acrylate based nanoparticles (PBMA-2-NP and PEGA-2-NP) through two-stage seeded semi-continuous starved feed emulsion polymerization.12 In a first stage, a seed particle of butyl metracrylate (BMA) was prepared using ca. 50 mol% ethylene glycol dimethacrylate (EGDMA) as a crosslinker. This particle with (24 7) nm diameter (determined by DLS), was used as a seed to obtain the final particles by copolymerizing BMA or DEGA/OEGA with derivative 2. The PBMA2-NP nanoparticles were prepared using the PBMA seed, BMA as monomer, 13 mol% EGDMA as a crosslinker and 0.1 mol% of derivative 2 (relative to other monomers). The crosslinker amount is extremely important in order to ensure the thermal stability of the final particles at the temperatures required for endoperoxide thermolysis (ca., 90 1C) – with less than 13 mol% of the crosslinker, relative to the total monomer, extensive nanoparticle aggregation at 90 1C was observed. The preparation of PEGA-2-NP followed the same procedure as for PBMA-2-NP, but using di(ethylene glycol) methyl ether methacrylate (DEGA) and poly(ethylene glycol) methyl ether methacrylate (OEGA, Mn = 475), 5 mol% EGDMA as a crosslinking agent and 0.1 mol% of derivative 2 (relative to other monomers). Copolymers of DEGA and OEGA are water-soluble and biocompatible,13 and exhibit thermo-responsive behaviour in water, with a lower critical solution temperature that can be accurately tuned from 28 to 90 1C by adjusting the ratio of the monomers.14 The possibility of controlling the expanded/collapsed state of the chains in water, and thus its hydrophobic/hydrophilic behaviour, opens interesting possibilities for the application of these nanoparticles. The PS-3-NP were prepared in one step by a miniemulsion polymerization procedure, optimized in our research group,15 using styrene as monomer, 13 mol% divinylbenzene (DVB) as a crosslinking agent, and 0.06 mol% of derivative 3 (relative to other monomers).
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Fig. 1 SEM images of PBMA-2-NP ((A) scale bar 1 mm) and PS-3-NP ((B) scale bar 300 nm), and size distributions (C) of the BMA seed, PS-3-NP, PEGA-2-NP and PBMA-2-NP (histogram obtained from several measurements by dynamic light scattering at 20 1C (solid lines), or SEM (dashed lines)).
The nanoparticles were characterized using dynamic light scattering (DLS) and scanning electron microscopy (SEM). DLS diameter distributions (obtained from several measurements at 20 1C) for the different particles and SEM images of PBMA-2-NP and PS-3-NP are presented in Fig. 1. The hydrodynamic diameter obtained from DLS is narrowly distributed, with (24 7) nm for the BMA seed, (73 10) nm for PS-3-NP, (152 64) nm for PEGA-2-NP and (236 37) nm for PBMA-2-NP. The PBMA-2-NP and PS-3-NP particles are spherical and well defined, as can be seen from the SEM image in Fig. 1, with diameters of (249 34) nm for PBMA-2-NP and (59 12) nm for PS-3-NP. For PEGA-2-NP, however, it was not possible to obtained SEM or TEM images due to the poor stability of the particles under the sample preparation and analysis conditions (Fig. S2, ESI†), but AFM and NTA images (Fig. S3 and S4, ESI†) confirm the diameter obtained from DLS. The nanoparticles were also characterized by fluorescence spectroscopy, with the fluorescence emission spectra of the particles (Fig. S5, ESI†) being similar to those of derivatives 2 and 3 with also similar emission maxima (Table S2, ESI†). The fluorescence lifetimes tF of the functionalized nanoparticles (ca. 8 ns in aerated conditions, Table S2, ESI†) are higher than those of the derivatives in aerated solutions (ca. 3 to 5.8 ns, Table S1, ESI†), but similar to the lifetimes obtained in degassed solutions (ca. 8 ns, Table S1, ESI†). This increase can be attributed, at least partially, to a reduction of the quenching by dissolved oxygen, owing to the expected lower diffusion coefficients of both fluorophore and quencher within the particles. The thermal stability of the nanoparticles at high temperatures was evaluated before the photooxidation/thermolysis studies. Water dispersions of the different nanoparticles were kept at 90 1C, the temperature required for thermolysis, and the fluorescence emission was monitored as a function of time. No significant changes were
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Fig. 2 Fluorescence spectra (lexc = 350 nm) of PBMA-2-NP: (A) during irradiation with a mercury lamp (l = 579 nm) in the presence of methylene blue (0 to 6 h in 1 h steps); (B) during thermolysis at 90 1C (0, 6 and 20 h).
observed for PBMA-2-NP after 20 hours, demonstrating that emulsions are stable at this temperature and suitable for thermolysis. However, PEGA-2-NP and PS-3-NP suffer a fluorescence intensity decrease over time (50% and 20% respectively, Fig. S6, ESI†), probably due to loss of nanoparticle stabilization leading to irreversible aggregation and precipitation (no change in intensity upon cooling). At lower temperatures, PEGA-2-NP water dispersions are stable, but DLS measurements at temperatures 20 to 75 1C shows a 15 nm variation in the diameter, with a slight hysteresis in the heating/cooling cycles (Fig. S7, ESI†). To test the endoperoxide production, water dispersions of PBMA2-NP nanoparticles were irradiated in the presence of methylene blue as a photosensitizer, in order to produce singlet oxygen and convert DPA derivatives into endoperoxides. The photosensitizer was excited with a mercury arc lamp at 579 nm (selected using optical filters, I0 = 3.6 10 7 einstein min 1), and the photooxidation of DPA derivatives inside the nanoparticles was followed by fluorescence emission spectroscopy. In Fig. 2A, we show the time evolution of the fluorescence of PBMA-2-NP, with the gradual decrease in fluorescence emission, indicating endoperoxide formation. After irradiation, the methylene blue present in solution was removed using an ion exchange resin to avoid any interference with the thermolysis measurements (methylene blue is known to decompose at high temperatures).16 For the thermolysis, the water dispersion of the clean PBMA-2-NP dispersion was maintained at 90 1C and the fluorescence emission monitored over time (Fig. 2B). The fluorescence of PBMA-2-NP gradually increases indicating a successful DPA
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regeneration with singlet oxygen release. Complete recovery of the initial fluorescence intensity was achieved after 20 hours. The time dependence of the fluorescence intensity for the different nanoparticles during photooxidation and thermolysis are represented in Fig. 3 (normalized fluorescence intensity at 407 nm). The endoperoxide formation rate is similar for PS-3-NP and PBMA-2-NP, but faster in the case of PEGA-2-NP. This is explained by the higher hydrophilicity of the PEGA-2-NP due to the presence of the ethyleneglycol groups. The DPA endoperoxides in the polymer nanoparticles were stable at room temperature for several weeks, with no fluorescence intensity changes being observed (Fig. S8, ESI†). The fluorescence intensity was again monitored during thermolysis at 90 1C (Fig. 3), and for PEGA-2-NP only 40% of the converted DPA was recovered, while for PS-3-NP the recovery was 75%. This is probably due to the lower stability and consequent aggregation of these particles at 90 1C, as mentioned above. In the case of PBMA-2-NP the DPA regeneration is complete. The stable PBMA-2-NP nanoparticles were used in a second irradiation/thermolysis cycle (Fig. S9, ESI†) proving the possibility of using the nanoparticles for several loading–releasing cycles. In summary, we successfully prepared polymeric nanoparticles self-sufficient in oxygen which are stable for at least several weeks, and that can be used for the controlled release of singlet oxygen by thermolysis. Furthermore, the nanoparticles can be recycled and reused. The polymer nanoparticles were prepared by emulsion or miniemulsion polymerization using polymerizable DPA derivatives. The reaction of the DPA derivatives with singlet oxygen (produced by a photosensitizer) leads to the formation of endoperoxides inside the nanoparticles. Later thermolysis of the nanoparticle dispersions in water releases molecular oxygen, opening the possibility for new applications, especially if the endoperoxide functionalized particles/materials are used in combination with gold nanostructures or magnetic nanocrystals, e.g. in the core, allowing us to locally increase the temperature to values higher than 90 1C by an external stimulus (photo irradiation17 or oscillating magnetic field). This light- or magnetic-triggered release of molecular oxygen in aqueous systems should reduce significantly the time needed for complete thermolysis. ˜o para a Cie ˆncia This work was partially supported by Fundaça e a Tecnologia (FCT-Portugal) and COMPETE (FEDER) within projects PEst-OE/CTM/LA0024/2011, PTDC/QUI-QUI/123162/2010 and PTDC/CTM-NAN/115110/2009. S.M. thanks FCT for a PhD grant (SFRH/BD/47660/2008). We thank Dr Aleksander Fedorov (CQFM) for the fluorescence lifetime measurements.
Notes and references 1 2 3 4
Fig. 3 Time dependence of the fluorescence intensity (407 nm) during irradiation (filled symbols) and thermolysis at 90 1C (open symbols) of PBMA-2-NP (circles), PEGA-2-NP (squares) and PS-3-NP (triangles).
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J. R. Kanofsky, Chem.-Biol. Interact., 1989, 70, 1. P. R. Ogilby, Chem. Soc. Rev., 2010, 39, 3181. M. C. DeRosa and R. J. Crutchley, Coord. Chem. Rev., 2002, 233, 351. (a) C. S. Foote, Active Oxygen in Chemistry, Blackie, London U.K., 1995; (b) E. Clo, J. W. Snyder, P. R. Ogilby and K. V. Gothelf, ChemBioChem, 2007, 8, 475; (c) A. L. Harris, Nat. Rev. Cancer, 2002, 2, 38. 5 J. M. Aubry, C. Pierlot, J. Rigaudy and R. Schmidt, Acc. Chem. Res., 2003, 36, 668. 6 (a) M. J. Moreno, E. Monson, R. G. Reddy, A. Rehemtulla, B. D. Ross, M. Philbert, R. J. Schneider and R. Kopelman, Sens. Actuators, B, 2003, 90, 82; (b) S. Z. Wang, R. M. Gao, F. M. Zhou and M. Selke,
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J. Mater. Chem., 2004, 14, 487; (c) J. F. Lovell, T. W. B. Liu, J. Chen and G. Zheng, Chem. Rev., 2010, 110, 2839. N. J. Turro, M. F. Chow and J. Rigaudy, J. Am. Chem. Soc., 1981, 103, 7218. D. Posavec, M. Zabel, U. Bogner, G. Bernhardt and G. Knor, Org. Biomol. Chem., 2012, 10, 7062. (a) D. Zehm, W. Fudickar and T. Linker, Angew. Chem., Int. Ed., 2007, 46, 7689; (b) W. Fudickar and T. Linker, Chem.–Eur. J., 2006, 12, 9276; (c) D. Zehm, W. Fudickar, M. Hans, U. Schilde, A. Kelling and T. Linker, Chem.–Eur. J., 2008, 14, 11429. M. Alvaro, G. Bizzoca, B. Ferrer, H. Garcia, M. de Miguel and L. Teruel, ChemPhysChem, 2010, 11, 3456.
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ChemComm 11 (a) J. F. Zhang, F. F. An, Y. A. Li, C. J. Zheng, Y. L. Yang, X. J. Zhang and X. H. Zhang, Chem. Commun., 2013, 49, 8072; (b) X. Q. Shen, L. Li, H. Wu, S. Q. Yao and Q. H. Xu, Nanoscale, 2011, 3, 5140. 12 X. D. Ye, J. P. S. Farinha, J. K. Oh, M. A. Winnik and C. Wu, Macromolecules, 2003, 36, 8749. 13 J. F. Lutz, Adv. Mater., 2011, 23, 2237. 14 J. F. Lutz and A. Hoth, Macromolecules, 2006, 39, 893. 15 V. Augusto, C. Baleizao, M. N. Berberan-Santos and J. P. S. Farinha, J. Mater. Chem., 2010, 20, 1192. 16 A. Ghauch, A. Tuqan, N. Kibbi and S. Geryes, Chem. Eng. J., 2012, 213, 259. 17 A. M. Asadirad, Z. Erno and N. R. Branda, Chem. Commun., 2013, 49, 5639.
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Cite this: DOI: 10.1039/c3cc48293f Received 29th October 2013, Accepted 7th February 2014
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Controlled release of singlet oxygen using diphenylanthracene functionalized polymer nanoparticles† S. Martins, J. P. S. Farinha, C. Baleiza˜o* and M. N. Berberan-Santos*
DOI: 10.1039/c3cc48293f www.rsc.org/chemcomm
Functionalized polymer nanoparticles react and store molecular oxygen for several weeks in the form of endoperoxides. On-demand controlled release of singlet oxygen by the particles is achieved by thermolysis.
Singlet oxygen plays an important role in many oxidative processes, e.g. in natural biological defence processes1 and as a cytotoxic agent in phototherapies.2 Due to its short lifetime and characteristic reactivity,3 a controlled delivery of singlet oxygen in time and space is sometimes necessary.4 This reactive species of oxygen can be generated by the nonradiative excitation of ground-state oxygen via energy transfer, or as a product in a specific chemical reaction.5 In the first case, a photosensitizer is photoexcited and subsequently crosses to the lowest triplet state, then transferring its energy to a neighbouring ground-state oxygen molecule (3O2), which is promoted to the first excited singlet state (1O2).2 The (indirect) photogeneration of 1O2 is used in many fields,4b however, this process requires the simultaneous presence of molecular oxygen and a photosensitizer. Additionally, many photosensitizers are hydrophobic molecules that require elaborate approaches for being used in aqueous media.6 An alternative method for singlet oxygen generation involves the use of specific polycyclic aromatic hydrocarbons (e.g., naphthalene or anthracene derivatives),7 which react reversibly with singlet oxygen to form an endoperoxide, which in some cases can later release 1O2 upon heating, reverting to the original compound.5 Diphenylanthracene (DPA, 1; Scheme 1) is particularly interesting for this application since it can be functionalized for incorporation into different materials, and it forms a stable endoperoxide that can release 1O2 under relatively mild conditions.7 Currently, the development of smart nanoparticles for 1O2 production in photodynamic therapy (PDT) for certain cancer CQFM – Centro de Quı´mica-Fı´sica Molecular and IN – Institute of Nanoscience and Nanotechnology, Instituto Superior Te´cnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available: Experimental synthetic procedure and photophysical characterization of DPA derivatives and functional nanoparticles. See DOI: 10.1039/c3cc48293f
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Scheme 1 Reversible reaction of diphenylanthracenes with oxygen. In the direct reaction, singlet oxygen is generated from ground state oxygen using a photosensitizer in the triplet state, and subsequently adds to the central ring, yielding an endoperoxide. In the reverse reaction, the endoperoxide decomposes upon heating, yielding singlet oxygen.
types is widely explored,8 but most studies focus on the incorporation and delivery of a suitable photosensitizer.4c,6a,9 However, this approach still has several limitations, such as the persistence of the photosensitizers in the body after treatment, and the reduced efficiency for cancer treatment due to the lower oxygen concentration in cancer tissues. Here we report the development of new polymer nanoparticles self-sufficient in oxygen, which allow the thermal release of singlet molecular oxygen in a controlled way. As a proof of concept we prepared different functional polymer nanoparticles (of polystyrene, polybutylmethacrylate and polyethylene glycol acrylate) containing covalently linked endoperoxides. The particles were obtained by emulsion polymerization with DPA polymerisable derivatives, and the endoperoxides were subsequently formed by photooxidation of the DPA using a photosensitizer. The particles were isolated, stored and later used for the thermal release of 1O2, which was followed by fluorescence spectroscopy. Two different DPA derivatives were synthesized for copolymerization with vinyl and acrylate monomers (Scheme S1, ESI†).
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An asymmetrical DPA derivative, 9-(4-phenyl methacrylate)-10(phenyl)anthracene (2), was prepared through a double Suzuki– Miyaura cross-coupling (4-hydroxyphenylboronic acid and phenylboronic acid) and an acylation with methacryloyl chloride in the final step. A second derivative, 9,10-bis(4-vinylphenyl) anthracene (3), was prepared according to a published procedure,10 in one step, by a Suzuki–Miyaura cross-coupling reaction. The DPA derivatives 2 and 3 were purified and fully characterized using NMR, mass spectrometry, absorption and fluorescence spectroscopies. The absorption and fluorescence emission spectra of derivatives 2 and 3, recorded in toluene, show similar shapes to those of pristine DPA (1), exhibiting its characteristic four strong absorption bands (Fig. S1 and Table S1, ESI†). Derivatives 2 and 3 were used to prepare functional polymeric nanoparticles based in poly(butyl methacrylate) (PBMA-2-NP), on a copolymer of di(ethylene glycol) methyl ether methacrylate (DEGA) and poly(ethylene glycol) methyl ether methacrylate (OEGA) (PEGA2-NP) and on polystyrene (PS-3-NP), through emulsion polymerization. Emulsion polymerization not only allows the incorporation of small molecules in the polymeric nanoparticles,11 but it is also a convenient way for surface modification of the particles, through insertion of functional groups, which can then be used for compatibilization with different materials, to bind (bio)molecules for targeting/recognition, etc. Derivative 2 was used to prepare two sets of acrylate based nanoparticles (PBMA-2-NP and PEGA-2-NP) through two-stage seeded semi-continuous starved feed emulsion polymerization.12 In a first stage, a seed particle of butyl metracrylate (BMA) was prepared using ca. 50 mol% ethylene glycol dimethacrylate (EGDMA) as a crosslinker. This particle with (24 7) nm diameter (determined by DLS), was used as a seed to obtain the final particles by copolymerizing BMA or DEGA/OEGA with derivative 2. The PBMA2-NP nanoparticles were prepared using the PBMA seed, BMA as monomer, 13 mol% EGDMA as a crosslinker and 0.1 mol% of derivative 2 (relative to other monomers). The crosslinker amount is extremely important in order to ensure the thermal stability of the final particles at the temperatures required for endoperoxide thermolysis (ca., 90 1C) – with less than 13 mol% of the crosslinker, relative to the total monomer, extensive nanoparticle aggregation at 90 1C was observed. The preparation of PEGA-2-NP followed the same procedure as for PBMA-2-NP, but using di(ethylene glycol) methyl ether methacrylate (DEGA) and poly(ethylene glycol) methyl ether methacrylate (OEGA, Mn = 475), 5 mol% EGDMA as a crosslinking agent and 0.1 mol% of derivative 2 (relative to other monomers). Copolymers of DEGA and OEGA are water-soluble and biocompatible,13 and exhibit thermo-responsive behaviour in water, with a lower critical solution temperature that can be accurately tuned from 28 to 90 1C by adjusting the ratio of the monomers.14 The possibility of controlling the expanded/collapsed state of the chains in water, and thus its hydrophobic/hydrophilic behaviour, opens interesting possibilities for the application of these nanoparticles. The PS-3-NP were prepared in one step by a miniemulsion polymerization procedure, optimized in our research group,15 using styrene as monomer, 13 mol% divinylbenzene (DVB) as a crosslinking agent, and 0.06 mol% of derivative 3 (relative to other monomers).
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Fig. 1 SEM images of PBMA-2-NP ((A) scale bar 1 mm) and PS-3-NP ((B) scale bar 300 nm), and size distributions (C) of the BMA seed, PS-3-NP, PEGA-2-NP and PBMA-2-NP (histogram obtained from several measurements by dynamic light scattering at 20 1C (solid lines), or SEM (dashed lines)).
The nanoparticles were characterized using dynamic light scattering (DLS) and scanning electron microscopy (SEM). DLS diameter distributions (obtained from several measurements at 20 1C) for the different particles and SEM images of PBMA-2-NP and PS-3-NP are presented in Fig. 1. The hydrodynamic diameter obtained from DLS is narrowly distributed, with (24 7) nm for the BMA seed, (73 10) nm for PS-3-NP, (152 64) nm for PEGA-2-NP and (236 37) nm for PBMA-2-NP. The PBMA-2-NP and PS-3-NP particles are spherical and well defined, as can be seen from the SEM image in Fig. 1, with diameters of (249 34) nm for PBMA-2-NP and (59 12) nm for PS-3-NP. For PEGA-2-NP, however, it was not possible to obtained SEM or TEM images due to the poor stability of the particles under the sample preparation and analysis conditions (Fig. S2, ESI†), but AFM and NTA images (Fig. S3 and S4, ESI†) confirm the diameter obtained from DLS. The nanoparticles were also characterized by fluorescence spectroscopy, with the fluorescence emission spectra of the particles (Fig. S5, ESI†) being similar to those of derivatives 2 and 3 with also similar emission maxima (Table S2, ESI†). The fluorescence lifetimes tF of the functionalized nanoparticles (ca. 8 ns in aerated conditions, Table S2, ESI†) are higher than those of the derivatives in aerated solutions (ca. 3 to 5.8 ns, Table S1, ESI†), but similar to the lifetimes obtained in degassed solutions (ca. 8 ns, Table S1, ESI†). This increase can be attributed, at least partially, to a reduction of the quenching by dissolved oxygen, owing to the expected lower diffusion coefficients of both fluorophore and quencher within the particles. The thermal stability of the nanoparticles at high temperatures was evaluated before the photooxidation/thermolysis studies. Water dispersions of the different nanoparticles were kept at 90 1C, the temperature required for thermolysis, and the fluorescence emission was monitored as a function of time. No significant changes were
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Fig. 2 Fluorescence spectra (lexc = 350 nm) of PBMA-2-NP: (A) during irradiation with a mercury lamp (l = 579 nm) in the presence of methylene blue (0 to 6 h in 1 h steps); (B) during thermolysis at 90 1C (0, 6 and 20 h).
observed for PBMA-2-NP after 20 hours, demonstrating that emulsions are stable at this temperature and suitable for thermolysis. However, PEGA-2-NP and PS-3-NP suffer a fluorescence intensity decrease over time (50% and 20% respectively, Fig. S6, ESI†), probably due to loss of nanoparticle stabilization leading to irreversible aggregation and precipitation (no change in intensity upon cooling). At lower temperatures, PEGA-2-NP water dispersions are stable, but DLS measurements at temperatures 20 to 75 1C shows a 15 nm variation in the diameter, with a slight hysteresis in the heating/cooling cycles (Fig. S7, ESI†). To test the endoperoxide production, water dispersions of PBMA2-NP nanoparticles were irradiated in the presence of methylene blue as a photosensitizer, in order to produce singlet oxygen and convert DPA derivatives into endoperoxides. The photosensitizer was excited with a mercury arc lamp at 579 nm (selected using optical filters, I0 = 3.6 10 7 einstein min 1), and the photooxidation of DPA derivatives inside the nanoparticles was followed by fluorescence emission spectroscopy. In Fig. 2A, we show the time evolution of the fluorescence of PBMA-2-NP, with the gradual decrease in fluorescence emission, indicating endoperoxide formation. After irradiation, the methylene blue present in solution was removed using an ion exchange resin to avoid any interference with the thermolysis measurements (methylene blue is known to decompose at high temperatures).16 For the thermolysis, the water dispersion of the clean PBMA-2-NP dispersion was maintained at 90 1C and the fluorescence emission monitored over time (Fig. 2B). The fluorescence of PBMA-2-NP gradually increases indicating a successful DPA
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regeneration with singlet oxygen release. Complete recovery of the initial fluorescence intensity was achieved after 20 hours. The time dependence of the fluorescence intensity for the different nanoparticles during photooxidation and thermolysis are represented in Fig. 3 (normalized fluorescence intensity at 407 nm). The endoperoxide formation rate is similar for PS-3-NP and PBMA-2-NP, but faster in the case of PEGA-2-NP. This is explained by the higher hydrophilicity of the PEGA-2-NP due to the presence of the ethyleneglycol groups. The DPA endoperoxides in the polymer nanoparticles were stable at room temperature for several weeks, with no fluorescence intensity changes being observed (Fig. S8, ESI†). The fluorescence intensity was again monitored during thermolysis at 90 1C (Fig. 3), and for PEGA-2-NP only 40% of the converted DPA was recovered, while for PS-3-NP the recovery was 75%. This is probably due to the lower stability and consequent aggregation of these particles at 90 1C, as mentioned above. In the case of PBMA-2-NP the DPA regeneration is complete. The stable PBMA-2-NP nanoparticles were used in a second irradiation/thermolysis cycle (Fig. S9, ESI†) proving the possibility of using the nanoparticles for several loading–releasing cycles. In summary, we successfully prepared polymeric nanoparticles self-sufficient in oxygen which are stable for at least several weeks, and that can be used for the controlled release of singlet oxygen by thermolysis. Furthermore, the nanoparticles can be recycled and reused. The polymer nanoparticles were prepared by emulsion or miniemulsion polymerization using polymerizable DPA derivatives. The reaction of the DPA derivatives with singlet oxygen (produced by a photosensitizer) leads to the formation of endoperoxides inside the nanoparticles. Later thermolysis of the nanoparticle dispersions in water releases molecular oxygen, opening the possibility for new applications, especially if the endoperoxide functionalized particles/materials are used in combination with gold nanostructures or magnetic nanocrystals, e.g. in the core, allowing us to locally increase the temperature to values higher than 90 1C by an external stimulus (photo irradiation17 or oscillating magnetic field). This light- or magnetic-triggered release of molecular oxygen in aqueous systems should reduce significantly the time needed for complete thermolysis. ˜o para a Cie ˆncia This work was partially supported by Fundaça e a Tecnologia (FCT-Portugal) and COMPETE (FEDER) within projects PEst-OE/CTM/LA0024/2011, PTDC/QUI-QUI/123162/2010 and PTDC/CTM-NAN/115110/2009. S.M. thanks FCT for a PhD grant (SFRH/BD/47660/2008). We thank Dr Aleksander Fedorov (CQFM) for the fluorescence lifetime measurements.
Notes and references 1 2 3 4
Fig. 3 Time dependence of the fluorescence intensity (407 nm) during irradiation (filled symbols) and thermolysis at 90 1C (open symbols) of PBMA-2-NP (circles), PEGA-2-NP (squares) and PS-3-NP (triangles).
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