Polypyrrole Nanoparticles
Multifunctional Polypyrrole@Fe3O4 Nanoparticles for Dual-Modal Imaging and In Vivo Photothermal Cancer Therapy Qiwei Tian, Qian Wang, Ke Xin Yao, Baiyang Teng, Jizhe Zhang, Shiping Yang, and Yu Han* Photothermal ablation (PTA) therapy, which enables selective killing of cancer cells by converting optical energy into heat, is a minimally invasive alternative to conventional approaches, such as surgery and chemotherapy, for therapeutic intervention at specific biological targets.[1] Given that near-infrared (NIR) lasers are preferred photosource due to their large penetration depth in biological tissues,[2] a prerequisite for the use of PTA is the development of biocompatible and efficient NIR (λ = 700–1100 nm) photothermal coupling agents. To improve the efficacy of therapies and to reduce side effects, ideal photothermal agents should be multifunctional with imaging capabilities, e.g., they should work in magnetic resonance imaging (MRI) for visualizing the location of the cancer or in thermal imaging for real-time monitoring of the treatment.[3] A general strategy to developing multifunctional photothermal agents is the integration of MRI agents (e.g., Fe3O4 nanoparticles) into various well-established NIR photothermal platforms, including Au nanostructures,[4] Cu2-xS nanoparticles,[5] and carbon-based materials.[6] Composite materials thus far obtained by this means have indeed exhibited multiple functions and are highly efficient in cancer therapy, but they have limitations. For instance, Au nanostructures drastically change their morphologies after a long period of laser irradiation, resulting in
Dr. Q. W. Tian, Dr. K. X. Yao, B. Y. Teng, J. Z. Zhang, Prof. Y. Han Advanced Membranes and Porous Materials Center Physical Science and Engineering Division King Abdullah University of Science and Technology Thuwal, 23955-6900, Saudi Arabia E-mail: [email protected] Dr. Q. Wang Department of Orthopedics Shanghai First People’s Hospital Affiliated with Shanghai Jiaotong University Shanghai, 200080, China Prof. S. P. Yang The Key Laboratory of Resource Chemistry of the Ministry of Education & Shanghai Key Laboratory of Rare Earth Functional Materials Shanghai Normal University Shanghai, 200234, China. DOI: 10.1002/smll.201302042 small 2014, 10, No. 6, 1063–1068
inconsistent absorption behaviors and conversion efficiencies;[7] copper sulfide and carbon-based nanomaterials cannot be easily metabolized from the body because they are nonbiodegradable.[7] In addition, the syntheses of these composite materials require multiple steps, which leads to high production costs, poor reproducibility, and inhomogeneity of the materials.[8] In comparison with conventional inorganic photothermal agents, organic-based photothermal agents have greater potential for in vivo applications because of their better biocompatibility and biodegradability.[9] Recently, polypyrrole (PPY) nanoparticles were reported to be a new type of lowcost, biocompatible photothermal agent, which demonstrated high photothermal conversion efficiency and good photostab ility.[7a,10] These attributes make PPY a desirable platform for preparing multifunctional PTA agents, but, up to now, little work has been done towards this end. On the other hand, owing to their excellent magnetic properties and low toxicity, iron oxides have been commercially used with the approval of the US Food and Drug Administration[11] as contrast agents for T2-weighted MRI that offers higher resolution and deeper tissue penetration as compared with other imaging technologies. It is interesting to note that Fe3+ ions are used as the oxidation agents for the preparation of PPY nanoparticles. Consequently, a large number of ferric (Fe3+) and ferrous ions (Fe2+) remain in the obtained PPY nanoparticles. In this study, we utilized these residual Fe ions as precursors to produce Fe3O4 crystals in situ on the surface of pre-synthesized PPY nanoparticles. The resulting PPY@Fe3O4 composite nanoparticles have integrated MRI imaging, infrared thermal imaging, and photothermal conversion functions, and they exhibit excellent therapeutic effectiveness against cancer, as demonstrated in both in vitro and in vivo studies. To the best of our knowledge, this is the first report on the synthesis of a PPY-based photothermal agent with dual-modal imaging functionalities. PPY@Fe3O4 nanoparticles were synthesized through a very simple process, as illustrated in Figure 1a. Specifically, pyrrole monomers were first added into an aqueous solution containing Fe3+ ions and polyvinyl alcohol (PVA) that were in the form of a PVA-Fe3+ complex.[12] Oxidative polymerization of pyrrole was then induced by Fe3+ ions, and the
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Q. Tian et al.
Figure 1. (a) Schematic illustration of the synthetic route to the PPY@ Fe3O4 composite nanoparticles. (b,c) TEM images of the as-synthesized PPY nanoparticles (b) and PPY@Fe3O4 nanoparticles (c). The insets show the TEM images of the corresponding individual particle. (d) The indexed powder X-ray diffraction pattern of the PPY@Fe3O4 nanoparticles, as referenced by the standard face-centered cubic Fe3O4 phase (JCPDS file number 65-3107).
power X-ray diffraction (Figure 1d) confirmed that the produced crystals were standard face-centered cubic Fe3O4 phases (JCPDS file number 65-3107). We used inductively coupled plasma optical emission spectrometry to determine that Fe3O4 accounted for 28% in weight in the composite. The aqueous dispersion of the PPY@Fe3O4 particles exhibited an intense and broad absorption band extending from the visible to the NIR region with the maximum at ≈900 nm (Figure 2a), which is characteristic of the bipolaron band transitions for PPY.[14] It is worth noting that as a result of higher dispersible ability of PPY in water by using PVA as the capping agent, the PPY@Fe3O4 nanoparticles are also readily dispersible in water, and the obtained suspension is very stable with little particle aggregation or precipitation observed (the inset of Figure 2a), which is beneficial to MRI application.[15] The absorption strength of the suspension linearly increased with the particle concentration (Figure S3), which further confirms the good dispersibility of PPY@Fe3O4 nanoparticles in water. Notably, the PPY@ Fe3O4 nanoparticles are also stable in phosphate buffer solution (PBS) (Figure S4), which is important for biomedical applications. The room-temperature magnetization curve (the M–H loop) of the PPY@Fe3O4 nanoparticles showed no magnetization or coercivity (Figure 2b), indicating superparamagnetic behavior. The lower saturated mass magnetization (22.78 emu/g) of PPY@Fe3O4 as compared with the value for Fe3O4 reported in the literature (≈70 emu/g)[4d] is due to the significant weight contribution (>70%) from the nonmagnetic PPY in the composite. The superparamagnetic nature of PPY@Fe3O4 nanoparticles allows them to be magnetically manipulated or to be used as T2 contrast agents in MRI. As shown in inset to Figure 2b, PPY@Fe3O4 nanoparticles can be collected from the suspension by a magnet, leaving the rest of the solution completely colorless. This result demonstrates the magnetic property of the composite particles and also reveals that all PPY nanoparticles had combined with Fe3O4 nanoparticles in our experiment. Figure 3a shows the proton transverse relaxation rates (1/T2) of the PPY@Fe3O4 aqueous solution as a function of the iron concentration, which were measured in a 0.5 T magnetic field with a spin-echo pulse sequence. The r2 value of PPY@Fe3O4 was calculated from
formed PPY particles were coated by PVA, which functioned as a capping agent to control the particle size as well as to make the particles highly hydrophilic.[7a,12] Meanwhile, the reduced Fe2+ ions along with the unreacted Fe3+ ions formed a complex with PVA on the surface of the PPY nanoparticles (Figure 1a). The as-prepared PPY nanoparticles were very uniform in size (≈50 nm in diameter), according to transmission electron microscopy (TEM) (Figure 1b) and dynamic light scattering (DLS) analysis (Figure S2). In the second step, an ammonia solution was added into the system with moderate heating at 70 °C to precipitate Fe3+ and Fe2+ ions, forming Fe3O4 nanocrystals in situ [13] on the PPY nanoparticles without any additional precursors. The successful conjugation of Fe3O4 with PPY was clearly verified by TEM. As shown in Figure 1c, each PPY nanoparticle combines several Fe3O4 crystals with sizes ranging from 5 to 15 nm. Accordingly, the overall particle Figure 2. (a) UV-vis-NIR spectrum of PPY@Fe O nanoparticles dispersed in water (80 ppm) 3 4 sizes increased to ≈70 nm, which is con- and the corresponding photograph of the stable colloidal suspension (inset). (b) Roomsistent with the DLS analysis (Figure S1). temperature magnetization curve of the PPY@Fe3O4 nanoparticles. The PPY@Fe3O4 High-resolution TEM (Figure S2) and nanoparticles can be collected from the water suspension by a magnet, as shown in the inset.
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Polypyrrole@Fe3O4 Nanoparticles for Dual-Modal Imaging and Photothermal Cancer Therapy
and efficiently convert the energy from an 808 nm laser into thermal energy. Following the method developed in the literature,[18] the photothermal conversion efficiency of the PPY@Fe3O4 nanoparticles at 808 nm was calculated to be 39.15% (Figure S5). It has been reported that PPY nanoparticles exhibit stable photothermal conversion efficiencies upon repeated NIR laser irradiation.[7a] Likewise, PPY@ Fe3O4 composite nanoparticles exhibit good photostability, as demonstrated by their very constant photothermal conversion behavior during eight cycles of on-and-off laser treatment (the laser was on for 10 min in each cycle) (Figure S6). The laser-induced temperature increase Figure 3. (a) The T2 relaxation rate of the PPY@Fe3O4 nanoparticles as a function of the Fe allows PPY@Fe3O4 nanoparticles to act concentration. (b) T2-weighted MRI photographs of the hydrophilic PPY@Fe3O4 nanoparticles dispersed in PBS with different Fe concentrations. (c and d) In vivo T2-weighted MR images as in vivo thermal imaging contrast agents. of the tumor-bearing mouse before and after intratumor injection of the PPY@Fe3O4 The spatial temperature distribution in the tumor-bearing mice was detected in realnanoparticles (the red circles point to the tumor sites). time using an infrared thermal imaging system after intratumoral injection of PBS the slope of this plot to be 290.91 mM−1.S−1, higher than that (control) or PPY@Fe3O4 nanoparticles containing PBS (treatof commercial MRI contrast agents (Feridex, 152 mM−1 S−1; ment). It was found that upon the laser irradiation, the tumor Resovist, 86 mM−1 S−1)[16] at the same magnetic field strength. region of the mice in the treatment group heated up very In T2-weighted MRI, a higher r2 relaxivity implies more neg- quickly with the temperature reaching 48.8 from 32.8 °C in ative enhancement.[17] As shown in Figure 3b, as the concen- 5 min (Figure 4c), and consequently a strong image contrast tration of iron is increased in water, the T2-weighted images exhibit increasingly darker contrast. This suggests that PPY@ Fe3O4 could be used as a T2-weighted MRI contrast agent. As a proof-of-concept experiment, we evaluated the in vivo contrast enhancing effect of PPY@Fe3O4 in a tumor-bearing nude mouse using a 0.5 T MRI system. Intratumoral injection of the PPY@Fe3O4 nanoparticles suspension in PBS was carried out at a dose of 0.16 mg Fe per Kg body weight. The T2-weighted images recorded before (Figure 3c) and 3 h after injection (Figure 3d) show that the background tissue is negatively enhanced with a marked T2 signal drop by 25% at the tumor site. Given the strong absorption of PPY@ Fe3O4 nanoparticles in the NIR region, we investigated their photothermal effect using an 808 nm laser with a power density of 0.25 W/cm2. After continuous irradiation for 5 min, the temperature of the aqueous dispersion of the PPY@Fe3O4 nanoparticles increased by ≈2.9–33.5 °C depending on the concentration of the nanoparticles, Figure 4. (a) Temperature profiles of pure water and aqueous dispersions of PPY@Fe3O4 while the same treatment resulted in a nanoparticles with different particle concentrations (10, 20, 40, 80, 160 ppm) as a function of irradiation time. (b) Infrared thermal images of the tumor-bearing mice injected with 100 μL negligible temperature increase (
Multifunctional Polypyrrole@Fe3O4 Nanoparticles for Dual-Modal Imaging and In Vivo Photothermal Cancer Therapy Qiwei Tian, Qian Wang, Ke Xin Yao, Baiyang Teng, Jizhe Zhang, Shiping Yang, and Yu Han* Photothermal ablation (PTA) therapy, which enables selective killing of cancer cells by converting optical energy into heat, is a minimally invasive alternative to conventional approaches, such as surgery and chemotherapy, for therapeutic intervention at specific biological targets.[1] Given that near-infrared (NIR) lasers are preferred photosource due to their large penetration depth in biological tissues,[2] a prerequisite for the use of PTA is the development of biocompatible and efficient NIR (λ = 700–1100 nm) photothermal coupling agents. To improve the efficacy of therapies and to reduce side effects, ideal photothermal agents should be multifunctional with imaging capabilities, e.g., they should work in magnetic resonance imaging (MRI) for visualizing the location of the cancer or in thermal imaging for real-time monitoring of the treatment.[3] A general strategy to developing multifunctional photothermal agents is the integration of MRI agents (e.g., Fe3O4 nanoparticles) into various well-established NIR photothermal platforms, including Au nanostructures,[4] Cu2-xS nanoparticles,[5] and carbon-based materials.[6] Composite materials thus far obtained by this means have indeed exhibited multiple functions and are highly efficient in cancer therapy, but they have limitations. For instance, Au nanostructures drastically change their morphologies after a long period of laser irradiation, resulting in
Dr. Q. W. Tian, Dr. K. X. Yao, B. Y. Teng, J. Z. Zhang, Prof. Y. Han Advanced Membranes and Porous Materials Center Physical Science and Engineering Division King Abdullah University of Science and Technology Thuwal, 23955-6900, Saudi Arabia E-mail: [email protected] Dr. Q. Wang Department of Orthopedics Shanghai First People’s Hospital Affiliated with Shanghai Jiaotong University Shanghai, 200080, China Prof. S. P. Yang The Key Laboratory of Resource Chemistry of the Ministry of Education & Shanghai Key Laboratory of Rare Earth Functional Materials Shanghai Normal University Shanghai, 200234, China. DOI: 10.1002/smll.201302042 small 2014, 10, No. 6, 1063–1068
inconsistent absorption behaviors and conversion efficiencies;[7] copper sulfide and carbon-based nanomaterials cannot be easily metabolized from the body because they are nonbiodegradable.[7] In addition, the syntheses of these composite materials require multiple steps, which leads to high production costs, poor reproducibility, and inhomogeneity of the materials.[8] In comparison with conventional inorganic photothermal agents, organic-based photothermal agents have greater potential for in vivo applications because of their better biocompatibility and biodegradability.[9] Recently, polypyrrole (PPY) nanoparticles were reported to be a new type of lowcost, biocompatible photothermal agent, which demonstrated high photothermal conversion efficiency and good photostab ility.[7a,10] These attributes make PPY a desirable platform for preparing multifunctional PTA agents, but, up to now, little work has been done towards this end. On the other hand, owing to their excellent magnetic properties and low toxicity, iron oxides have been commercially used with the approval of the US Food and Drug Administration[11] as contrast agents for T2-weighted MRI that offers higher resolution and deeper tissue penetration as compared with other imaging technologies. It is interesting to note that Fe3+ ions are used as the oxidation agents for the preparation of PPY nanoparticles. Consequently, a large number of ferric (Fe3+) and ferrous ions (Fe2+) remain in the obtained PPY nanoparticles. In this study, we utilized these residual Fe ions as precursors to produce Fe3O4 crystals in situ on the surface of pre-synthesized PPY nanoparticles. The resulting PPY@Fe3O4 composite nanoparticles have integrated MRI imaging, infrared thermal imaging, and photothermal conversion functions, and they exhibit excellent therapeutic effectiveness against cancer, as demonstrated in both in vitro and in vivo studies. To the best of our knowledge, this is the first report on the synthesis of a PPY-based photothermal agent with dual-modal imaging functionalities. PPY@Fe3O4 nanoparticles were synthesized through a very simple process, as illustrated in Figure 1a. Specifically, pyrrole monomers were first added into an aqueous solution containing Fe3+ ions and polyvinyl alcohol (PVA) that were in the form of a PVA-Fe3+ complex.[12] Oxidative polymerization of pyrrole was then induced by Fe3+ ions, and the
© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
1063
communications
Q. Tian et al.
Figure 1. (a) Schematic illustration of the synthetic route to the PPY@ Fe3O4 composite nanoparticles. (b,c) TEM images of the as-synthesized PPY nanoparticles (b) and PPY@Fe3O4 nanoparticles (c). The insets show the TEM images of the corresponding individual particle. (d) The indexed powder X-ray diffraction pattern of the PPY@Fe3O4 nanoparticles, as referenced by the standard face-centered cubic Fe3O4 phase (JCPDS file number 65-3107).
power X-ray diffraction (Figure 1d) confirmed that the produced crystals were standard face-centered cubic Fe3O4 phases (JCPDS file number 65-3107). We used inductively coupled plasma optical emission spectrometry to determine that Fe3O4 accounted for 28% in weight in the composite. The aqueous dispersion of the PPY@Fe3O4 particles exhibited an intense and broad absorption band extending from the visible to the NIR region with the maximum at ≈900 nm (Figure 2a), which is characteristic of the bipolaron band transitions for PPY.[14] It is worth noting that as a result of higher dispersible ability of PPY in water by using PVA as the capping agent, the PPY@Fe3O4 nanoparticles are also readily dispersible in water, and the obtained suspension is very stable with little particle aggregation or precipitation observed (the inset of Figure 2a), which is beneficial to MRI application.[15] The absorption strength of the suspension linearly increased with the particle concentration (Figure S3), which further confirms the good dispersibility of PPY@Fe3O4 nanoparticles in water. Notably, the PPY@ Fe3O4 nanoparticles are also stable in phosphate buffer solution (PBS) (Figure S4), which is important for biomedical applications. The room-temperature magnetization curve (the M–H loop) of the PPY@Fe3O4 nanoparticles showed no magnetization or coercivity (Figure 2b), indicating superparamagnetic behavior. The lower saturated mass magnetization (22.78 emu/g) of PPY@Fe3O4 as compared with the value for Fe3O4 reported in the literature (≈70 emu/g)[4d] is due to the significant weight contribution (>70%) from the nonmagnetic PPY in the composite. The superparamagnetic nature of PPY@Fe3O4 nanoparticles allows them to be magnetically manipulated or to be used as T2 contrast agents in MRI. As shown in inset to Figure 2b, PPY@Fe3O4 nanoparticles can be collected from the suspension by a magnet, leaving the rest of the solution completely colorless. This result demonstrates the magnetic property of the composite particles and also reveals that all PPY nanoparticles had combined with Fe3O4 nanoparticles in our experiment. Figure 3a shows the proton transverse relaxation rates (1/T2) of the PPY@Fe3O4 aqueous solution as a function of the iron concentration, which were measured in a 0.5 T magnetic field with a spin-echo pulse sequence. The r2 value of PPY@Fe3O4 was calculated from
formed PPY particles were coated by PVA, which functioned as a capping agent to control the particle size as well as to make the particles highly hydrophilic.[7a,12] Meanwhile, the reduced Fe2+ ions along with the unreacted Fe3+ ions formed a complex with PVA on the surface of the PPY nanoparticles (Figure 1a). The as-prepared PPY nanoparticles were very uniform in size (≈50 nm in diameter), according to transmission electron microscopy (TEM) (Figure 1b) and dynamic light scattering (DLS) analysis (Figure S2). In the second step, an ammonia solution was added into the system with moderate heating at 70 °C to precipitate Fe3+ and Fe2+ ions, forming Fe3O4 nanocrystals in situ [13] on the PPY nanoparticles without any additional precursors. The successful conjugation of Fe3O4 with PPY was clearly verified by TEM. As shown in Figure 1c, each PPY nanoparticle combines several Fe3O4 crystals with sizes ranging from 5 to 15 nm. Accordingly, the overall particle Figure 2. (a) UV-vis-NIR spectrum of PPY@Fe O nanoparticles dispersed in water (80 ppm) 3 4 sizes increased to ≈70 nm, which is con- and the corresponding photograph of the stable colloidal suspension (inset). (b) Roomsistent with the DLS analysis (Figure S1). temperature magnetization curve of the PPY@Fe3O4 nanoparticles. The PPY@Fe3O4 High-resolution TEM (Figure S2) and nanoparticles can be collected from the water suspension by a magnet, as shown in the inset.
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© 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2014, 10, No. 6, 1063–1068
Polypyrrole@Fe3O4 Nanoparticles for Dual-Modal Imaging and Photothermal Cancer Therapy
and efficiently convert the energy from an 808 nm laser into thermal energy. Following the method developed in the literature,[18] the photothermal conversion efficiency of the PPY@Fe3O4 nanoparticles at 808 nm was calculated to be 39.15% (Figure S5). It has been reported that PPY nanoparticles exhibit stable photothermal conversion efficiencies upon repeated NIR laser irradiation.[7a] Likewise, PPY@ Fe3O4 composite nanoparticles exhibit good photostability, as demonstrated by their very constant photothermal conversion behavior during eight cycles of on-and-off laser treatment (the laser was on for 10 min in each cycle) (Figure S6). The laser-induced temperature increase Figure 3. (a) The T2 relaxation rate of the PPY@Fe3O4 nanoparticles as a function of the Fe allows PPY@Fe3O4 nanoparticles to act concentration. (b) T2-weighted MRI photographs of the hydrophilic PPY@Fe3O4 nanoparticles dispersed in PBS with different Fe concentrations. (c and d) In vivo T2-weighted MR images as in vivo thermal imaging contrast agents. of the tumor-bearing mouse before and after intratumor injection of the PPY@Fe3O4 The spatial temperature distribution in the tumor-bearing mice was detected in realnanoparticles (the red circles point to the tumor sites). time using an infrared thermal imaging system after intratumoral injection of PBS the slope of this plot to be 290.91 mM−1.S−1, higher than that (control) or PPY@Fe3O4 nanoparticles containing PBS (treatof commercial MRI contrast agents (Feridex, 152 mM−1 S−1; ment). It was found that upon the laser irradiation, the tumor Resovist, 86 mM−1 S−1)[16] at the same magnetic field strength. region of the mice in the treatment group heated up very In T2-weighted MRI, a higher r2 relaxivity implies more neg- quickly with the temperature reaching 48.8 from 32.8 °C in ative enhancement.[17] As shown in Figure 3b, as the concen- 5 min (Figure 4c), and consequently a strong image contrast tration of iron is increased in water, the T2-weighted images exhibit increasingly darker contrast. This suggests that PPY@ Fe3O4 could be used as a T2-weighted MRI contrast agent. As a proof-of-concept experiment, we evaluated the in vivo contrast enhancing effect of PPY@Fe3O4 in a tumor-bearing nude mouse using a 0.5 T MRI system. Intratumoral injection of the PPY@Fe3O4 nanoparticles suspension in PBS was carried out at a dose of 0.16 mg Fe per Kg body weight. The T2-weighted images recorded before (Figure 3c) and 3 h after injection (Figure 3d) show that the background tissue is negatively enhanced with a marked T2 signal drop by 25% at the tumor site. Given the strong absorption of PPY@ Fe3O4 nanoparticles in the NIR region, we investigated their photothermal effect using an 808 nm laser with a power density of 0.25 W/cm2. After continuous irradiation for 5 min, the temperature of the aqueous dispersion of the PPY@Fe3O4 nanoparticles increased by ≈2.9–33.5 °C depending on the concentration of the nanoparticles, Figure 4. (a) Temperature profiles of pure water and aqueous dispersions of PPY@Fe3O4 while the same treatment resulted in a nanoparticles with different particle concentrations (10, 20, 40, 80, 160 ppm) as a function of irradiation time. (b) Infrared thermal images of the tumor-bearing mice injected with 100 μL negligible temperature increase (
