Conjugated Polymers
Biocompatible Conjugated Polymer Nanoparticles for Efficient Photothermal Tumor Therapy Junlong Geng, Chunyang Sun, Jie Liu, Lun-De Liao, Youyong Yuan, Nitish Thakor, Jun Wang,* and Bin Liu*
Conjugated polymers (CPs) with strong near-infrared (NIR) absorption and high heat conversion efficiency have emerged as a new generation of photothermal therapy (PTT) agents for cancer therapy. An efficient strategy to design NIR absorbing CPs with good water dispersibility is essential to achieve excellent therapeutic effect. In this work, poly[9,9-bis(4-(2-ethylhexyl)phenyl)fluorene-alt-co-6,7-bis(4-(hexyloxy) phenyl)-4,9-di(thiophen-2-yl)-thiadiazoloquinoxaline] (PFTTQ) is synthesized through the combination of donor–acceptor moieties by Suzuki polymerization. PFTTQ nanoparticles (NPs) are fabricated through a precipitation approach using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) as the encapsulation matrix. Due to the large NIR absorption coefficient (3.6 L g−1 cm−1), the temperature of PFTTQ NP suspension (0.5 mg/mL) could be rapidly increased to more than 50 °C upon continuous 808 nm laser irradiation (0.75 W/cm2) for 5 min. The PFTTQ NPs show good biocompatibility to both MDA-MB-231 cells and Hela cells at 400 µg/mL of NPs, while upon laser irradiation, effective cancer cell killing is observed at a NP concentration of 50 µg/mL. Moreover, PFTTQ NPs could efficiently ablate tumor in in vivo study using a Hela tumor mouse model. Considering the large amount of NIR absorbing CPs available, the general encapsulation strategy will enable the development of more efficient PTT agents for cancer or tumor therapy.
Mr. J. L. Geng, Dr. J. Liu, Dr. Y. Y. Yuan, Prof. B. Liu Department of Chemical and Biomolecular Engineering National University of Singapore Singapore 117585 E-mail: [email protected] Mr. C. Y. Sun, Prof. J. Wang School of Life Sciences, Hefei National Laboratory for Physical Sciences at the Microscale University of Science and Technology of China Hefei, Anhui 230027, PR China E-mail: [email protected] Dr. L. D. Liao, Prof. N. Thakor Singapore Institute for Neurotechnology (SINAPSE) National University of Singapore Singapore 117456, Singapore DOI: 10.1002/smll.201402092 small 2014, DOI: 10.1002/smll.201402092
1. Introduction Photothermal therapy (PTT), employing the conversion of absorbed light energy into thermal energy to ablate cancer cells, has emerged as an alternative therapeutic methodology for cancer therapy.[1] PTT involves the delivery of therapeutic reagents to targeted location, light irradiation and heat conversion to kill cancer cells or tumors. Among various light sources, the light in the near infrared (NIR) range (700–1000 nm) is highly desirable due to the high penetration depth to targeted tumors and the minimal skin and tissue absorption in this region to minimize the damage to surrounding tissues.[2] As a result, a variety of materials with strong absorbance in NIR region have been extensively explored for PTT applications. Currently, the most widely used PTT agents are inorganic nanoparticles (NPs) including
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metal nanomaterials[3] (e.g. gold nanorods, gold nanocages, gold nanoshells and gold nanostars), carbonous nanostructures[4] (e.g. carbon nanotubes and graphene) and copper sulfide NPs.[5] However, these inorganic nanomaterials could stay in the body for a long period of time,[6] which raises safety concerns with limited practical applications. More recently, organic PTT agents have attracted great research interest as a result of their good biocompatibility and potential biodegradability in biological system. The typical example of NIR organic dye based PTT agent is Indocyanine Green (ICG), which has been formulated into polymer NPs with efficient photothermal conversion.[7] In addition, porphysome organic NPs have also been utilized as PTT agents for targeted tumor therapy.[8] However, these organic molecules usually show poor photostabilities and low efficiency in converting light to heat since part of the absorbed energy decays through radiative pathway. As a result, it is highly desirable to develop organic materials with good photostability, large absorption coefficient, and high heat conversion efficiency for PTT applications. In this regard, conjugated polymers (CP) have attracted great research interest due to their large absorption coefficients and good photostabilities.[9] Conjugated polymers (CPs) are macromolecules characterized with π-conjugated backbones and facilely modified electrical and optical properties, which have been widely applied in optoelectronic devices and biomedical field.[10] The successful examples of CP based PTT agents include poly(3,4-ethylenedioxythiophene):poly(4polyaniline,[11] styrenesulfonate) (PEDOT:PSS)[12] and polypyrrole.[13] Due to the poor processability of these materials, unique and customized approaches, such as layer-by-layer self-assembly and in-situ polymerization have been developed for the preparation of PEDOT:PSS nanoparticles (NPs), and polypyrrole NPs, respectively. As the obtained NPs generally do not have surface functional groups, further surface coating steps are necessary for subsequent functionalization, which is tedious and not genetic to be explored to other organic-soluble and processable CPs. The recent burst development of CPs for solar cells has led to the generation of a large amount of NIR absorbing CPs with low fluorescence, and some of them are even commercially available.[14] These materials offer a unique opportunity for the development and screening of CP based PTT agents, if a simple strategy could be developed to transform them into biological media.[15] In our previous studies, we have shown that one efficient way to transfer organic CPs to aqueous media is through a precipitation approach, which involves the precipitation of CPs dissolved in an organic solvent (e.g. tetrahydrofuran) to aqueous media to yield CP NP suspension.[16] These NPs have shown good photostability and benign biocompatibility for biosensing and bioimaging applications. A prerequisite condition for the CP nanoparticle preparation is that the NIR absorbing CP is soluble in organic solvents. In this contribution, using a donor-acceptor poly[9,9-bis(4-(2-ethylhexyl)phenyl)fluorene-alt-co-6,7-bis(4-(hexyloxy)phenyl)-4,9-di(thiophen2-yl)thiadiazolo-quinoxaline] (PFTTQ) as an example,[17] we show that NIR absorbing CP NPs could be easily prepared
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to serve as an effective PTT agent. PFTTQ based NPs have been fabricated through a facile precipitation approach using 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) as the encapsulation matrix.[16a,16c,18] The size of PFTTQ loaded DSPE-PEG2000 NPs (PFTTQ NPs) could be finetuned by controlling the starting CP and matrix concentration, demonstrating the good processability of PFTTQ. Due to the high electron-deficiency of thiadiazoloquinoxaline sections in PFTTQ,[19] the obtained PFTTQ NPs show strong NIR absorbance and high non-radiative decay rates, benefiting the conversion of light energy to heat irradiation. The photothermal effect of PFTTQ NPs has been investigated by monitoring the solution temperature evolution upon continuous 808 nm laser irradiation. The temperature of 0.5 mg/mL PFTTQ NP solution increased rapidly from 22 °C to 52 °C upon the laser irradiation at a power density of 0.75 W/cm2 for 10 minutes. Both in vitro and in vivo studies reveal that PFTTQ NPs are an efficient PTT agent.
2. Results and Discussion PFTTQ was synthesized by Suzuki polymerization according to the synthetic route shown in Scheme S1 in the supporting information. The chemical structure of PFTTQ is shown in Scheme 1, which contains a highly electron-deficient thiadiazoloquinoxaline unit (2 in Scheme S1), and a highly electron-rich bis(4-(2-ethylhexyloxy)phenyl)fluorene unit (1 in Scheme S1). Due to the alternating donor-acceptor backbones, PFTTQ shows broad absorption from 700 to 850 nm in THF as shown in Figure S1. Gel permeation chromatography (GPC) measurement reveals that PFTTQ has a number-average molecular weight (Mn) of 12900 with a polydispersity index of 2.1. The PFTTQ NPs were synthesized through a modified precipitation approach using DSPE-PEG2000 as the matrix (Scheme 1B).[16a,18] A THF solution containing PFTTQ and DSPE-PEG2000 matrix was poured into water under sonication. The hydrophobic DSPE segments were liable to entangle with PFTTQ chains and the hydrophilic PEG chains should extend into aqueous phase under sonication. The NP suspension was obtained under stirring overnight. The NP suspension was further filtered through a 0.2 µm syringe filter to yield PFTTQ NPs. Figure 1A shows the UV-vis absorption spectrum of PFTTQ NP suspensions in water. The absorption peak at 425 nm arises from π–π* transition of the conjugated backbone, while the broad absorption band from 700 to 850 nm results from charge transfer between fluorene and thiadiazoloquinoxaline units. The absorption coefficient of PFTTQ NPs at 800 nm is calculated to be 3.6 L g−1 cm−1 based on NP concentration. It is worth noting that almost no obvious fluorescence from PFTTQ NPs is detectable upon excitation at 800 nm, indicating that most of the excited excitons return to the ground state via non-radiative decay to generate heat. PFTTQ NPs (prepared from 0.25 mg/mL PFTTQ at the feed) show an average size of ∼25 nm based on the fieldemission transmission electron microscopy (FE-TEM) image
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small 2014, DOI: 10.1002/smll.201402092
Biocompatible Conjugated Polymer Nanoparticles for Efficient Photothermal Tumor Therapy
evolution of PFTTQ NP suspensions at various concentrations was examined with an 808 nm NIR laser at a power density of 0.75 W/cm2. As shown in Figure 1C, the temperature of 0.5 mg/mL PFTTQ NPs increases from 22 °C to 52 °C upon laser irradiation for 10 minutes. On the contrary, the temperature of pure water only increases from 22 °C to 31 °C under the same condition. In addition, the temperature elevation increases with the increased PFTTQ NP concentration as shown in Figure 1C. To further evaluate the photothermal conversion efficiency of PFTTQ NPs, Au nanorods (NRs), a widely used photothermal agent, have also been synthesized according to the seed-mediated growth approach.[20] Au NRs show an extinction maximum at 805 nm as shown in Figure S4. Upon laser irradiation, the temperature elevation of PFTTQ NPs is similar to that of Au NRs (with the same absorbance) as shown in Figure S5, illustrating that PFTTQ NPs are efficient phtoScheme 1. (A) Chemical structures of PFTTQ and DSPE-PEG2000. (B) Schematic illustration of thermal therapeutic agents. Noteworthy is PFTTQ NP synthesis. that the absorbance of PFTTQ NPs only slightly decreases by less than 5% after 1 h continuous NIR laser irradiation at a as shown in Figure 1B. Laser light scattering (LLS) results power density of 1 W/cm2 (Figure 1A), demonstrating good reveal that the volume averaged effective hydrodynamic photostability of PFTTQ NPs. diameter of PFTTQ NPs is 35 ± 10 nm. No obvious size variThe biocompatibility of NPs has been evaluated using ation of PFTTQ NPs is observed in 10 days at room tem- both MDA-MB-231 and Hela cells. Both cells were subculperature by laser light scattering measurement (Figure S2), tured in 96-well plates. At a confluence of 60 000 cell/mL, the indicating their excellent colloidal stability. In addition, the cultured cells were then incubated with PFTTQ NP suspensize of PFTTQ NPs could be easily fine-tuned by varying sions at different concentrations. No obvious cytotoxicity was the PFTTQ concentration at the feed. Doubling the PFTTQ observed for both cell lines upon incubation with PFTTQ as well as the matrix concentrations in THF at the feed NPs for 24 h at concentrations lower than 200 µg/mL and the could yield larger size PFTTQ NPs (∼40 nm), as shown in cell viabilities remained more than 90%. Even at 400 µg/mL Figure S3. The capability to fine-tune the PFTTQ NP size is of PFTTQ NPs, the cell viabilities remain more than 82% for highly desirable for different applications. both cell lines, demonstrating the low cytotoxicity of PFTTQ We then investigated the photothermal effect NPs. To further explore the PTT efficiency of PFTTQ NPs, of PFTTQ NPs upon laser irradiation. The temperature the cells after incubation with NPs were treated with laser
Figure 1. (A) UV-vis spectra of PFTTQ NPs before and after 1 h laser irradiation at a power density of 1W/cm2. (B) FE-TEM image of PFTTQ NPs. (C) The temperature evolution of PFTTQ NPs with various NP concentrations under 808-nm laser irradiation at a power density of 0.75 W/cm2. small 2014, DOI: 10.1002/smll.201402092
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Figure 2. Cell viabilities of (A) MDA-MB-231 and (B) Hela cells at various PFTTQ NP concentrations with and without 808-nm laser treatment at a power density of 0.75 W/cm2 for 10 minutes. Confocal fluorescence images of Hela cells incubated with 200 µg/mL PFTTQ NPs (C) before and (D) after illumination with 808-nm laser at 0.75 W/cm2 for 10 minutes. Live and dead cells were co-stained by fluorescein diacetate (C) and propidium iodide (D), respectively.
irradiation and their viabilities are shown in Figure 2A and B. The cell viabilities decreased quickly after laser irradiation. More than 80% of cells were damaged upon incubation with PFTTQ NPs (200 µg/mL), followed by 808 nm NIR laser irradiation for 10 minutes (shadowed columns in Figures 2A and B). In addition, Hela cells incubated with PFTTQ NPs (200 µg/mL) before and after laser irradiation have been co-stained with fluorescein diacetate (green for live cells) and propidium iodide (red for dead cells), and imaged by confocal fluorescence microscope. Before laser irradiation, obvious green fluorescence is observed from cells as shown in Figure 2C. In contrast, strong red fluorescence is detected after laser treatment (Figure 2D), indicating that Hela cells incubated with PFTTQ NPs have been effectively killed by laser irradiation. These results demonstrate the good therapeutic effect of PFTTQ NPs upon laser irradiation in in vitro experiments. After examining the PTT effect of PFTTQ NPs under in vitro conditions, we further investigated the photothermal effect of PFTTQ NPs under the same NIR irradiation density in vivo using a Hela tumor mouse model. PFTTQ NPs (1 mg/mL, 40 µL) were intratumorally injected to female NOD/SCID mice bearing Hela tumors. The temperature evolution of tumor injected with PFTTQ NPs upon laser irradiation treatment was monitored in 10 minutes by an infrared
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camera. As control groups, the temperature changes of mice tumor after injection with PBS or PFTTQ NPs but without laser irradiation as well as those treated with laser irradiation only without NPs have also been investigated. Without NIR irradiation, the temperature of mice tumors remains at ∼27 °C for PBS and PFTTQ NP groups (Figure 3A and B). In addition, no obvious temperature elevation is observed for the mice treated with NIR irradiation only without NP injection. On the other hand, the tumor temperature upon treatment with both PFTTQ NPs and NIR laser irradiation increases rapidly from 27 °C to 47 °C within 3 minutes and remains at ∼45 °C for the next 7 minutes (Figure 3A and 3B). Thanks to the superior spatial control of NIR laser, the temperature is only elevated within the tumor sites, while the other parts are not affected (Figure 3A). Previous studies have shown that a temperature increase in the range of 15–20 °C in 5 minutes is sufficient to induce the irreversible tissue damage,[13b,13c] implying that the Hela tumors could be greatly inhibited under this condition. Inspired by the efficient temperature elevation of PFTTQ NPs in mice tumor, we conducted the tumor inhibition experiments in vivo to investigate the anticancer effect of PFTTQ NPs using NOD/SCID mice bearing Hela tumors as a model. Mice were randomly divided into four groups with five in each group. For PFTTQ NPs with NIR laser irradiation
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Biocompatible Conjugated Polymer Nanoparticles for Efficient Photothermal Tumor Therapy
Figure 3. (A) IR thermal images of Hela tumor-bearing mice after treatment with PBS or PFTTQ NPs (1 mg/mL) without laser irradiation, only NIR laser irradiation (808 nm, 0.75 W/cm2) without NPs and 1 mg/mL of PFTTQ NPs with laser irradiation (808 nm, 0.75 W/cm2) at different times. (B) Temperature evolution of mice treated with PBS solution or PFTTQ NPs without laser irradiation, only NIR laser irradiation without NPs and PFTTQ NPs with laser irradiation. (C) Relative tumor size evolution of mice after different treatments.
group (PFTTQ NPs + NIR group), one dose of PFTTQ NPs (1 mg/mL, 40 µL) was intratumorally injected to each mouse, followed by the 808 nm laser irradiation at a power density of 0.75 W/cm2 for 10 minutes. For the control groups, mice were only given sole NIR irradiation (0.75 W/cm2, 10 minutes) without PFTTQ NPs injection (NIR group), or mice were only injected with either PBS (PBS group) or PFTTQ NPs (1 mg/mL, 40 µL, PFTTQ NPs group) without NIR treatment. The tumor sizes in each group were measured with a capillary and recorded every other day. The tumors treated with PBS or PFTTQ NPs and sole NIR irradiation grow rapidly, and the relative tumor volume of the control groups increases by 7-fold after 12 days as compared to the initial tumor sizes as shown in Figure 3C. In contrast, the tumors in PFTTQ+NIR group disappear in 4 days after the treatment and there is also no recurrence of the tumor in this group. The comparison between PFTTQ NPs + NIR group and three control groups illustrates that the tumor inhibition is induced by PFTTQ NPs and NIR irradiation, indicating the distinctly enhanced tolerability and antitumor efficacy of such treatment. To better understand the therapeutic effect of PFTTQ NPs upon laser irradiation, the tumors after various treatments (PBS, PFTTQ, NIR and PFTTQ+NIR) have also been studied with H&E staining. After 2 days of small 2014, DOI: 10.1002/smll.201402092
post treatment, a typical mouse in each group was sacrificed and the tumors were collected for hematoxylin and eosin (H&E) analysis. As shown in Figure S6, H&E stained slices from tumors do not show obvious lesion in controlled groups (PBS, PFTTQ NPs and NIR). In comparison, the significant cell damage is observed in PFTTQ+NIR group, which indicates that the higher temperature induced by PTT could kill tumor cells at both cellular and animal level. Meanwhile, no abnormalities of the liver, lung or spleen were observed at autopsy, suggesting the low systemic toxicity to other organs of tumoral injection of PFTTQ NPs upon NIR irradiation. In addition, the weight of each mouse maintains a relative stable and normal level as shown in Figure S7, also demonstrating the NPs exhibited excellent biocompatible and low toxicity effect under in vivo condition.
3. Conclusion We have developed PFTTQ NPs with strong NIR absorption through a facile precipitation approach utilizing DSPEPEG2000 as the matrix. Thanks to the processible property of PFTTQ, the sizes of PFTTQ NPs could be fine-tuned by controlling the CP and matrix concentrations at the feed.
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The temperature of PFTTQ NP suspension (0.5 mg/mL) could be rapidly increased from 22 °C to 52 °C upon laser irradiation (808 nm, 0.75 W/cm2) for 5 minutes. Additionally, the local temperature of tumor injected with PFTTQ NPs increased to 45 °C within 5 minutes upon laser irradiation under the same condition. The NPs have been utilized as an effective PTT agent for cancer therapy both in vitro and in vivo. This strategy could be easily extended to other organic-soluble and processible CP materials with both large absorption coefficient and high non-radiative decay rates for PTT application. As the PFTTQ NPs have almost no fluorescence, only intratumoral injection of the NPs was studied. Further development of multi-functional CP NPs will lead to image-guided photothermal therapy, which will offer new opportunities to study the biodistribution and tumor accumulation of CP NPs, and help to evaluate their potential utility in clinical applications.
4. Experimental Section Materials: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) was purchased from Avanti Polar Lipids, Inc. 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT), penicillin-streptomycin solution, fetal bovine serum (FBS), trypsin-EDTA solution, methanol and tetrahydrofuran (THF), 4-iodophenol, 1-bromohexane, trimethylsilylaceylene, copper iodide, 2-(tributylstannyl)thiophene, N-bromosuccinimide (NBS), palladium(II) acetate (Pd(AcO)2), tricyclohexylphosphine (Cy3P) and tetraethylammonium hydroxide (Et4NOH) solution (35 wt% in H2O) were purchased from SigmaAldrich and used as received. Toluene used for Suzuki polycondensation was pretreated with sulfuric acid followed by distillation. All other chemical reagents were used as received. Compounds 1 and 2 were prepared according to previous reports. Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation, Breford, USA). MDA-MB-231 and Hela cell lines were provided by American Type Culture Collection. Characterization: Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 500 NMR spectrometer (500 MHz for 1H, referenced to TMS at δ = 0.00 ppm and 125 MHz for 13C, referenced to CDCl3 at 77.0 ppm). Average particle size and size distribution of the NPs were determined by laser light scattering (LLS) with particle size analyzer (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of 90° at room temperature. Field emission transmission electron microscopy (FE-TEM) studies were performed on a JEOL JEM-2010 electron microscope with an accelerating voltage of 200 kV. UV-vis spectra were collected on a Shimadzu UV-1700 spectrometer. Synthesis of PFTTQ: PFTTQ was synthesized according to Scheme S1 in the supporting information. 4,9-Bis(5bromothiophen-2-yl)-6,7-bis(4-(hexyloxy)phenyl)-[1,2,5] thiadiazolo[3,4-g]quinoxaline (1) and 2,7-bis(4,4,5,5-tetramethyl-1,3,3-dioxaboralan-2-yl)-9,9-bis(4-(2-ethylhexyloxy)phenyl) fluorine (2) have been synthesized according to the previous reports.[19b,21] A Schlenk tube was charged with 1 (100.0 mg, 0.116 mmol), 2 (95.8 mg, 0.116 mmol), palladium acetate (3 mg) and tricyclohexylphosphine (7 mg) in toluene (10 mL) before it was sealed with a rubber septum. The Schlenk tube was degassed
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with three freeze-pump-thaw cycles to remove air and moisture. Then, the mixture was heated to 80 °C followed by injection with an aqueous Et4NOH solution (20 wt%, 1.5 mL) to initiate the reaction. After 18 h, the reaction was stopped and cooled down to room temperature. The mixture was precipitated with methanol and the crude polymer was obtained by centrifugation. The crude polymer was subsequently dissolved in chloroform, washed with water 3 times, and dried over MgSO4. After solvent removal, the polymer (84 mg, yield: 57%) was obtained as a black-green solid by precipitation in methanol. 1H NMR (500 MHz, CDCl3, ppm) δ: 9.00 (br, 4 H), 7.81-7.14 (br, 14 H), 6.80 (br, 8 H), 3.98 (br, 4 H), 3.78 (br, 4 H), 1.83 (br, 4 H), 1.67 (br, 2 H), 1.50-1.26 (br, 28 H), 0.93-0.85 (br, 18 H). Synthesis of PFTTQ NPs: A THF solution (1 mL) containing DSPE-PEG2000 (0.375 mg), and PFTTQ (0.25 mg) was poured into water (10 mL). This was followed by sonicating the mixture for 2 minutes at 12 W output using a microtip probe sonicator (XL2000, Misonix Incorporated, NY). The mixture was then stirred at room temperature overnight to evaporate the organic solvent. The NP suspension was further purified with a 0.2 µm syringe filter to obtain PFTTQ NPs. To tune the size of PFTTQ NPs, 1 mL of THF solution containing 1 mg of PFTTQ and 1.5 mg of DSPE-PEG2000 was also utilized to synthesize PFTTQ NPs, yielding NPs with bigger sizes. Synthesis of Au NRs: Au NRs were synthesized by a seed-mediated growth approach following the previous reports.[20] Briefly, Au NP seeds were prepared by rapidly injecting NaBH4 (0.01 M, 0.6 mL) to an aqueous mixture of HAuCl4 (0.01 M, 0.25 mL) and cetyltrimethylammonium bromide (CTAB, 0.1 M, 9.75 mL). After stirring for 5 minutes, the obtained brownish solution was kept for 2 h at room temperature before being used as the seed. To grow Au NRs, 2 mL of HAuCl4 and 400 mL of AgNO3 were added into 40 mL of 0.1 M CTAB under gentle stirring. Subsequently, 0.8 mL of 1.0 M HCl was added, followed by the addition of 0.32 mL of 0.1 M ascorbic acid. Next, 8 µL of the seed solution was injected into the mixture and gently stirred for another 5 minutes. The reaction mixture was then left undisturbed overnight to yield the Au NRs. Cytotoxicity of PFTTQ NPs: MDA-MB-231 and Hela cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin streptomycin at 37 °C in a humidified environment containing 5% CO2. The metabolic activities of both cells were evaluated using methylthiazolyldiphenyltetrazolium (MTT) assays. MDA-MB-231 and Hela cells were seeded in 96-well plates (Costar, IL, USA) at density of 4 × 104 cells mL−1. After 24 h incubation, the medium was replaced by PFTTQ NP suspensions at different mass concentrations, and the cells were then incubated for another 24 h. After the designated time interval, the wells were washed twice with 1×PBS and 100 µL of freshly prepared MTT (0.5 mg mL−1) solution in culture medium was added into each well. For the PTT experiment, the cells were treated with an 808 nm laser at a power density of 0.75 W/cm2 for 10 minutes, followed further incubation with MTT solution. The MTT medium solution was carefully removed after 3 h incubation in an incubator. DMSO (100 µL) was then added into each well and the plate was gently shaken for 10 minutes at room temperature to dissolve all the precipitates formed. The absorbance of MTT at 570 nm was monitored by the microplate reader (Genios Tecan) after subtracting the absorbance of the corresponding control cells incubated with PFTTQ NPs at the same concentration but without the addition of MTT to eliminate the absorbance interference from
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small 2014, DOI: 10.1002/smll.201402092
Biocompatible Conjugated Polymer Nanoparticles for Efficient Photothermal Tumor Therapy
PFTTQ. Cell viability was expressed by the ratio of absolute absorbance of the cells incubated with NP suspensions to that of the cells incubated with culture medium only. Photostability of PFTTQ NPs: The photostability of PFTTQ NPs was investigated by monitoring their respective UV-vis absorbance changes after 808 nm continuous laser irradiation for one hour. After one hour, the UV-vis absorbance of the NP suspension was measured with the spectrometer to compare with that of fresh PFTTQ NP suspension. Tumor model and tumor suppression study: Female NOD/ SCID mice were obtained from Beijing HFK Bioscience Co., Ltd. and used at 6 weeks of age. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. The xenograft tumor model was generated by subcutaneously injection of 4 × 106 Hela cells suspended in 100 µL phosphate buffered saline (PBS, with 30% Matrigel, BD Bioscience) into the right shoulder of each female NOD/SCID mouse. When the tumor volume was approximately 60 mm3, the mice were randomly divided into 4 groups. For the PTT treatment, each mouse was intratumorally injected with 40 µL of 1 mg/mL PFTTQ NPs in PBS solution and the control group received the same volume of PBS. The mice were irradiated with 808 nm NIR laser at 0.75 W/cm2 for 10 minutes. The temperature of the tumor sites was recorded by IR 7320 thermal camera and analyzed by IR Flash Software (Infrared Cameras. Inc). The tumor sizes were monitored by measuring the perpendicular diameter every other day. The volume was calculated based on the following equation: tumor volume = 1/2 × length × width2. Moreover, the typical mice in each group were sacrificed on day 2 after treatments, and the tumors were excised for histology observations. The organs were fixed in 10% neutral buffered formalin, which were then processed routinely into paraffin, sliced at thickness of 4 µm, and stained with hematoxylin and eosin (H&E). The H&E-stained slices were imaged by optical microscopy, which were assessed by 3 independent pathologists. Statistical Analysis: Quantitative data were expressed as mean ± SD. Statistical comparisons were made by ANOVA analysis and Student’s t-test. P value < 0.05 was considered statistically significant.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements J. L. Geng and C. Y. Sun contributed equally to this work. We are grateful to the financial support from the Singapore National Research Foundation (R-279–000–323–281), the JCO IMRE/ 12–8P1103 and JCO IMRE/14-8P1110. Mr. J. L. Geng thanks the National University of Singapore for the research scholarship.
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Received: July 15, 2014 Published online:
small 2014, DOI: 10.1002/smll.201402092
Biocompatible Conjugated Polymer Nanoparticles for Efficient Photothermal Tumor Therapy Junlong Geng, Chunyang Sun, Jie Liu, Lun-De Liao, Youyong Yuan, Nitish Thakor, Jun Wang,* and Bin Liu*
Conjugated polymers (CPs) with strong near-infrared (NIR) absorption and high heat conversion efficiency have emerged as a new generation of photothermal therapy (PTT) agents for cancer therapy. An efficient strategy to design NIR absorbing CPs with good water dispersibility is essential to achieve excellent therapeutic effect. In this work, poly[9,9-bis(4-(2-ethylhexyl)phenyl)fluorene-alt-co-6,7-bis(4-(hexyloxy) phenyl)-4,9-di(thiophen-2-yl)-thiadiazoloquinoxaline] (PFTTQ) is synthesized through the combination of donor–acceptor moieties by Suzuki polymerization. PFTTQ nanoparticles (NPs) are fabricated through a precipitation approach using 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) as the encapsulation matrix. Due to the large NIR absorption coefficient (3.6 L g−1 cm−1), the temperature of PFTTQ NP suspension (0.5 mg/mL) could be rapidly increased to more than 50 °C upon continuous 808 nm laser irradiation (0.75 W/cm2) for 5 min. The PFTTQ NPs show good biocompatibility to both MDA-MB-231 cells and Hela cells at 400 µg/mL of NPs, while upon laser irradiation, effective cancer cell killing is observed at a NP concentration of 50 µg/mL. Moreover, PFTTQ NPs could efficiently ablate tumor in in vivo study using a Hela tumor mouse model. Considering the large amount of NIR absorbing CPs available, the general encapsulation strategy will enable the development of more efficient PTT agents for cancer or tumor therapy.
Mr. J. L. Geng, Dr. J. Liu, Dr. Y. Y. Yuan, Prof. B. Liu Department of Chemical and Biomolecular Engineering National University of Singapore Singapore 117585 E-mail: [email protected] Mr. C. Y. Sun, Prof. J. Wang School of Life Sciences, Hefei National Laboratory for Physical Sciences at the Microscale University of Science and Technology of China Hefei, Anhui 230027, PR China E-mail: [email protected] Dr. L. D. Liao, Prof. N. Thakor Singapore Institute for Neurotechnology (SINAPSE) National University of Singapore Singapore 117456, Singapore DOI: 10.1002/smll.201402092 small 2014, DOI: 10.1002/smll.201402092
1. Introduction Photothermal therapy (PTT), employing the conversion of absorbed light energy into thermal energy to ablate cancer cells, has emerged as an alternative therapeutic methodology for cancer therapy.[1] PTT involves the delivery of therapeutic reagents to targeted location, light irradiation and heat conversion to kill cancer cells or tumors. Among various light sources, the light in the near infrared (NIR) range (700–1000 nm) is highly desirable due to the high penetration depth to targeted tumors and the minimal skin and tissue absorption in this region to minimize the damage to surrounding tissues.[2] As a result, a variety of materials with strong absorbance in NIR region have been extensively explored for PTT applications. Currently, the most widely used PTT agents are inorganic nanoparticles (NPs) including
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metal nanomaterials[3] (e.g. gold nanorods, gold nanocages, gold nanoshells and gold nanostars), carbonous nanostructures[4] (e.g. carbon nanotubes and graphene) and copper sulfide NPs.[5] However, these inorganic nanomaterials could stay in the body for a long period of time,[6] which raises safety concerns with limited practical applications. More recently, organic PTT agents have attracted great research interest as a result of their good biocompatibility and potential biodegradability in biological system. The typical example of NIR organic dye based PTT agent is Indocyanine Green (ICG), which has been formulated into polymer NPs with efficient photothermal conversion.[7] In addition, porphysome organic NPs have also been utilized as PTT agents for targeted tumor therapy.[8] However, these organic molecules usually show poor photostabilities and low efficiency in converting light to heat since part of the absorbed energy decays through radiative pathway. As a result, it is highly desirable to develop organic materials with good photostability, large absorption coefficient, and high heat conversion efficiency for PTT applications. In this regard, conjugated polymers (CP) have attracted great research interest due to their large absorption coefficients and good photostabilities.[9] Conjugated polymers (CPs) are macromolecules characterized with π-conjugated backbones and facilely modified electrical and optical properties, which have been widely applied in optoelectronic devices and biomedical field.[10] The successful examples of CP based PTT agents include poly(3,4-ethylenedioxythiophene):poly(4polyaniline,[11] styrenesulfonate) (PEDOT:PSS)[12] and polypyrrole.[13] Due to the poor processability of these materials, unique and customized approaches, such as layer-by-layer self-assembly and in-situ polymerization have been developed for the preparation of PEDOT:PSS nanoparticles (NPs), and polypyrrole NPs, respectively. As the obtained NPs generally do not have surface functional groups, further surface coating steps are necessary for subsequent functionalization, which is tedious and not genetic to be explored to other organic-soluble and processable CPs. The recent burst development of CPs for solar cells has led to the generation of a large amount of NIR absorbing CPs with low fluorescence, and some of them are even commercially available.[14] These materials offer a unique opportunity for the development and screening of CP based PTT agents, if a simple strategy could be developed to transform them into biological media.[15] In our previous studies, we have shown that one efficient way to transfer organic CPs to aqueous media is through a precipitation approach, which involves the precipitation of CPs dissolved in an organic solvent (e.g. tetrahydrofuran) to aqueous media to yield CP NP suspension.[16] These NPs have shown good photostability and benign biocompatibility for biosensing and bioimaging applications. A prerequisite condition for the CP nanoparticle preparation is that the NIR absorbing CP is soluble in organic solvents. In this contribution, using a donor-acceptor poly[9,9-bis(4-(2-ethylhexyl)phenyl)fluorene-alt-co-6,7-bis(4-(hexyloxy)phenyl)-4,9-di(thiophen2-yl)thiadiazolo-quinoxaline] (PFTTQ) as an example,[17] we show that NIR absorbing CP NPs could be easily prepared
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to serve as an effective PTT agent. PFTTQ based NPs have been fabricated through a facile precipitation approach using 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) as the encapsulation matrix.[16a,16c,18] The size of PFTTQ loaded DSPE-PEG2000 NPs (PFTTQ NPs) could be finetuned by controlling the starting CP and matrix concentration, demonstrating the good processability of PFTTQ. Due to the high electron-deficiency of thiadiazoloquinoxaline sections in PFTTQ,[19] the obtained PFTTQ NPs show strong NIR absorbance and high non-radiative decay rates, benefiting the conversion of light energy to heat irradiation. The photothermal effect of PFTTQ NPs has been investigated by monitoring the solution temperature evolution upon continuous 808 nm laser irradiation. The temperature of 0.5 mg/mL PFTTQ NP solution increased rapidly from 22 °C to 52 °C upon the laser irradiation at a power density of 0.75 W/cm2 for 10 minutes. Both in vitro and in vivo studies reveal that PFTTQ NPs are an efficient PTT agent.
2. Results and Discussion PFTTQ was synthesized by Suzuki polymerization according to the synthetic route shown in Scheme S1 in the supporting information. The chemical structure of PFTTQ is shown in Scheme 1, which contains a highly electron-deficient thiadiazoloquinoxaline unit (2 in Scheme S1), and a highly electron-rich bis(4-(2-ethylhexyloxy)phenyl)fluorene unit (1 in Scheme S1). Due to the alternating donor-acceptor backbones, PFTTQ shows broad absorption from 700 to 850 nm in THF as shown in Figure S1. Gel permeation chromatography (GPC) measurement reveals that PFTTQ has a number-average molecular weight (Mn) of 12900 with a polydispersity index of 2.1. The PFTTQ NPs were synthesized through a modified precipitation approach using DSPE-PEG2000 as the matrix (Scheme 1B).[16a,18] A THF solution containing PFTTQ and DSPE-PEG2000 matrix was poured into water under sonication. The hydrophobic DSPE segments were liable to entangle with PFTTQ chains and the hydrophilic PEG chains should extend into aqueous phase under sonication. The NP suspension was obtained under stirring overnight. The NP suspension was further filtered through a 0.2 µm syringe filter to yield PFTTQ NPs. Figure 1A shows the UV-vis absorption spectrum of PFTTQ NP suspensions in water. The absorption peak at 425 nm arises from π–π* transition of the conjugated backbone, while the broad absorption band from 700 to 850 nm results from charge transfer between fluorene and thiadiazoloquinoxaline units. The absorption coefficient of PFTTQ NPs at 800 nm is calculated to be 3.6 L g−1 cm−1 based on NP concentration. It is worth noting that almost no obvious fluorescence from PFTTQ NPs is detectable upon excitation at 800 nm, indicating that most of the excited excitons return to the ground state via non-radiative decay to generate heat. PFTTQ NPs (prepared from 0.25 mg/mL PFTTQ at the feed) show an average size of ∼25 nm based on the fieldemission transmission electron microscopy (FE-TEM) image
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Biocompatible Conjugated Polymer Nanoparticles for Efficient Photothermal Tumor Therapy
evolution of PFTTQ NP suspensions at various concentrations was examined with an 808 nm NIR laser at a power density of 0.75 W/cm2. As shown in Figure 1C, the temperature of 0.5 mg/mL PFTTQ NPs increases from 22 °C to 52 °C upon laser irradiation for 10 minutes. On the contrary, the temperature of pure water only increases from 22 °C to 31 °C under the same condition. In addition, the temperature elevation increases with the increased PFTTQ NP concentration as shown in Figure 1C. To further evaluate the photothermal conversion efficiency of PFTTQ NPs, Au nanorods (NRs), a widely used photothermal agent, have also been synthesized according to the seed-mediated growth approach.[20] Au NRs show an extinction maximum at 805 nm as shown in Figure S4. Upon laser irradiation, the temperature elevation of PFTTQ NPs is similar to that of Au NRs (with the same absorbance) as shown in Figure S5, illustrating that PFTTQ NPs are efficient phtoScheme 1. (A) Chemical structures of PFTTQ and DSPE-PEG2000. (B) Schematic illustration of thermal therapeutic agents. Noteworthy is PFTTQ NP synthesis. that the absorbance of PFTTQ NPs only slightly decreases by less than 5% after 1 h continuous NIR laser irradiation at a as shown in Figure 1B. Laser light scattering (LLS) results power density of 1 W/cm2 (Figure 1A), demonstrating good reveal that the volume averaged effective hydrodynamic photostability of PFTTQ NPs. diameter of PFTTQ NPs is 35 ± 10 nm. No obvious size variThe biocompatibility of NPs has been evaluated using ation of PFTTQ NPs is observed in 10 days at room tem- both MDA-MB-231 and Hela cells. Both cells were subculperature by laser light scattering measurement (Figure S2), tured in 96-well plates. At a confluence of 60 000 cell/mL, the indicating their excellent colloidal stability. In addition, the cultured cells were then incubated with PFTTQ NP suspensize of PFTTQ NPs could be easily fine-tuned by varying sions at different concentrations. No obvious cytotoxicity was the PFTTQ concentration at the feed. Doubling the PFTTQ observed for both cell lines upon incubation with PFTTQ as well as the matrix concentrations in THF at the feed NPs for 24 h at concentrations lower than 200 µg/mL and the could yield larger size PFTTQ NPs (∼40 nm), as shown in cell viabilities remained more than 90%. Even at 400 µg/mL Figure S3. The capability to fine-tune the PFTTQ NP size is of PFTTQ NPs, the cell viabilities remain more than 82% for highly desirable for different applications. both cell lines, demonstrating the low cytotoxicity of PFTTQ We then investigated the photothermal effect NPs. To further explore the PTT efficiency of PFTTQ NPs, of PFTTQ NPs upon laser irradiation. The temperature the cells after incubation with NPs were treated with laser
Figure 1. (A) UV-vis spectra of PFTTQ NPs before and after 1 h laser irradiation at a power density of 1W/cm2. (B) FE-TEM image of PFTTQ NPs. (C) The temperature evolution of PFTTQ NPs with various NP concentrations under 808-nm laser irradiation at a power density of 0.75 W/cm2. small 2014, DOI: 10.1002/smll.201402092
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Figure 2. Cell viabilities of (A) MDA-MB-231 and (B) Hela cells at various PFTTQ NP concentrations with and without 808-nm laser treatment at a power density of 0.75 W/cm2 for 10 minutes. Confocal fluorescence images of Hela cells incubated with 200 µg/mL PFTTQ NPs (C) before and (D) after illumination with 808-nm laser at 0.75 W/cm2 for 10 minutes. Live and dead cells were co-stained by fluorescein diacetate (C) and propidium iodide (D), respectively.
irradiation and their viabilities are shown in Figure 2A and B. The cell viabilities decreased quickly after laser irradiation. More than 80% of cells were damaged upon incubation with PFTTQ NPs (200 µg/mL), followed by 808 nm NIR laser irradiation for 10 minutes (shadowed columns in Figures 2A and B). In addition, Hela cells incubated with PFTTQ NPs (200 µg/mL) before and after laser irradiation have been co-stained with fluorescein diacetate (green for live cells) and propidium iodide (red for dead cells), and imaged by confocal fluorescence microscope. Before laser irradiation, obvious green fluorescence is observed from cells as shown in Figure 2C. In contrast, strong red fluorescence is detected after laser treatment (Figure 2D), indicating that Hela cells incubated with PFTTQ NPs have been effectively killed by laser irradiation. These results demonstrate the good therapeutic effect of PFTTQ NPs upon laser irradiation in in vitro experiments. After examining the PTT effect of PFTTQ NPs under in vitro conditions, we further investigated the photothermal effect of PFTTQ NPs under the same NIR irradiation density in vivo using a Hela tumor mouse model. PFTTQ NPs (1 mg/mL, 40 µL) were intratumorally injected to female NOD/SCID mice bearing Hela tumors. The temperature evolution of tumor injected with PFTTQ NPs upon laser irradiation treatment was monitored in 10 minutes by an infrared
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camera. As control groups, the temperature changes of mice tumor after injection with PBS or PFTTQ NPs but without laser irradiation as well as those treated with laser irradiation only without NPs have also been investigated. Without NIR irradiation, the temperature of mice tumors remains at ∼27 °C for PBS and PFTTQ NP groups (Figure 3A and B). In addition, no obvious temperature elevation is observed for the mice treated with NIR irradiation only without NP injection. On the other hand, the tumor temperature upon treatment with both PFTTQ NPs and NIR laser irradiation increases rapidly from 27 °C to 47 °C within 3 minutes and remains at ∼45 °C for the next 7 minutes (Figure 3A and 3B). Thanks to the superior spatial control of NIR laser, the temperature is only elevated within the tumor sites, while the other parts are not affected (Figure 3A). Previous studies have shown that a temperature increase in the range of 15–20 °C in 5 minutes is sufficient to induce the irreversible tissue damage,[13b,13c] implying that the Hela tumors could be greatly inhibited under this condition. Inspired by the efficient temperature elevation of PFTTQ NPs in mice tumor, we conducted the tumor inhibition experiments in vivo to investigate the anticancer effect of PFTTQ NPs using NOD/SCID mice bearing Hela tumors as a model. Mice were randomly divided into four groups with five in each group. For PFTTQ NPs with NIR laser irradiation
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Biocompatible Conjugated Polymer Nanoparticles for Efficient Photothermal Tumor Therapy
Figure 3. (A) IR thermal images of Hela tumor-bearing mice after treatment with PBS or PFTTQ NPs (1 mg/mL) without laser irradiation, only NIR laser irradiation (808 nm, 0.75 W/cm2) without NPs and 1 mg/mL of PFTTQ NPs with laser irradiation (808 nm, 0.75 W/cm2) at different times. (B) Temperature evolution of mice treated with PBS solution or PFTTQ NPs without laser irradiation, only NIR laser irradiation without NPs and PFTTQ NPs with laser irradiation. (C) Relative tumor size evolution of mice after different treatments.
group (PFTTQ NPs + NIR group), one dose of PFTTQ NPs (1 mg/mL, 40 µL) was intratumorally injected to each mouse, followed by the 808 nm laser irradiation at a power density of 0.75 W/cm2 for 10 minutes. For the control groups, mice were only given sole NIR irradiation (0.75 W/cm2, 10 minutes) without PFTTQ NPs injection (NIR group), or mice were only injected with either PBS (PBS group) or PFTTQ NPs (1 mg/mL, 40 µL, PFTTQ NPs group) without NIR treatment. The tumor sizes in each group were measured with a capillary and recorded every other day. The tumors treated with PBS or PFTTQ NPs and sole NIR irradiation grow rapidly, and the relative tumor volume of the control groups increases by 7-fold after 12 days as compared to the initial tumor sizes as shown in Figure 3C. In contrast, the tumors in PFTTQ+NIR group disappear in 4 days after the treatment and there is also no recurrence of the tumor in this group. The comparison between PFTTQ NPs + NIR group and three control groups illustrates that the tumor inhibition is induced by PFTTQ NPs and NIR irradiation, indicating the distinctly enhanced tolerability and antitumor efficacy of such treatment. To better understand the therapeutic effect of PFTTQ NPs upon laser irradiation, the tumors after various treatments (PBS, PFTTQ, NIR and PFTTQ+NIR) have also been studied with H&E staining. After 2 days of small 2014, DOI: 10.1002/smll.201402092
post treatment, a typical mouse in each group was sacrificed and the tumors were collected for hematoxylin and eosin (H&E) analysis. As shown in Figure S6, H&E stained slices from tumors do not show obvious lesion in controlled groups (PBS, PFTTQ NPs and NIR). In comparison, the significant cell damage is observed in PFTTQ+NIR group, which indicates that the higher temperature induced by PTT could kill tumor cells at both cellular and animal level. Meanwhile, no abnormalities of the liver, lung or spleen were observed at autopsy, suggesting the low systemic toxicity to other organs of tumoral injection of PFTTQ NPs upon NIR irradiation. In addition, the weight of each mouse maintains a relative stable and normal level as shown in Figure S7, also demonstrating the NPs exhibited excellent biocompatible and low toxicity effect under in vivo condition.
3. Conclusion We have developed PFTTQ NPs with strong NIR absorption through a facile precipitation approach utilizing DSPEPEG2000 as the matrix. Thanks to the processible property of PFTTQ, the sizes of PFTTQ NPs could be fine-tuned by controlling the CP and matrix concentrations at the feed.
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The temperature of PFTTQ NP suspension (0.5 mg/mL) could be rapidly increased from 22 °C to 52 °C upon laser irradiation (808 nm, 0.75 W/cm2) for 5 minutes. Additionally, the local temperature of tumor injected with PFTTQ NPs increased to 45 °C within 5 minutes upon laser irradiation under the same condition. The NPs have been utilized as an effective PTT agent for cancer therapy both in vitro and in vivo. This strategy could be easily extended to other organic-soluble and processible CP materials with both large absorption coefficient and high non-radiative decay rates for PTT application. As the PFTTQ NPs have almost no fluorescence, only intratumoral injection of the NPs was studied. Further development of multi-functional CP NPs will lead to image-guided photothermal therapy, which will offer new opportunities to study the biodistribution and tumor accumulation of CP NPs, and help to evaluate their potential utility in clinical applications.
4. Experimental Section Materials: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) was purchased from Avanti Polar Lipids, Inc. 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT), penicillin-streptomycin solution, fetal bovine serum (FBS), trypsin-EDTA solution, methanol and tetrahydrofuran (THF), 4-iodophenol, 1-bromohexane, trimethylsilylaceylene, copper iodide, 2-(tributylstannyl)thiophene, N-bromosuccinimide (NBS), palladium(II) acetate (Pd(AcO)2), tricyclohexylphosphine (Cy3P) and tetraethylammonium hydroxide (Et4NOH) solution (35 wt% in H2O) were purchased from SigmaAldrich and used as received. Toluene used for Suzuki polycondensation was pretreated with sulfuric acid followed by distillation. All other chemical reagents were used as received. Compounds 1 and 2 were prepared according to previous reports. Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation, Breford, USA). MDA-MB-231 and Hela cell lines were provided by American Type Culture Collection. Characterization: Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 500 NMR spectrometer (500 MHz for 1H, referenced to TMS at δ = 0.00 ppm and 125 MHz for 13C, referenced to CDCl3 at 77.0 ppm). Average particle size and size distribution of the NPs were determined by laser light scattering (LLS) with particle size analyzer (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of 90° at room temperature. Field emission transmission electron microscopy (FE-TEM) studies were performed on a JEOL JEM-2010 electron microscope with an accelerating voltage of 200 kV. UV-vis spectra were collected on a Shimadzu UV-1700 spectrometer. Synthesis of PFTTQ: PFTTQ was synthesized according to Scheme S1 in the supporting information. 4,9-Bis(5bromothiophen-2-yl)-6,7-bis(4-(hexyloxy)phenyl)-[1,2,5] thiadiazolo[3,4-g]quinoxaline (1) and 2,7-bis(4,4,5,5-tetramethyl-1,3,3-dioxaboralan-2-yl)-9,9-bis(4-(2-ethylhexyloxy)phenyl) fluorine (2) have been synthesized according to the previous reports.[19b,21] A Schlenk tube was charged with 1 (100.0 mg, 0.116 mmol), 2 (95.8 mg, 0.116 mmol), palladium acetate (3 mg) and tricyclohexylphosphine (7 mg) in toluene (10 mL) before it was sealed with a rubber septum. The Schlenk tube was degassed
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with three freeze-pump-thaw cycles to remove air and moisture. Then, the mixture was heated to 80 °C followed by injection with an aqueous Et4NOH solution (20 wt%, 1.5 mL) to initiate the reaction. After 18 h, the reaction was stopped and cooled down to room temperature. The mixture was precipitated with methanol and the crude polymer was obtained by centrifugation. The crude polymer was subsequently dissolved in chloroform, washed with water 3 times, and dried over MgSO4. After solvent removal, the polymer (84 mg, yield: 57%) was obtained as a black-green solid by precipitation in methanol. 1H NMR (500 MHz, CDCl3, ppm) δ: 9.00 (br, 4 H), 7.81-7.14 (br, 14 H), 6.80 (br, 8 H), 3.98 (br, 4 H), 3.78 (br, 4 H), 1.83 (br, 4 H), 1.67 (br, 2 H), 1.50-1.26 (br, 28 H), 0.93-0.85 (br, 18 H). Synthesis of PFTTQ NPs: A THF solution (1 mL) containing DSPE-PEG2000 (0.375 mg), and PFTTQ (0.25 mg) was poured into water (10 mL). This was followed by sonicating the mixture for 2 minutes at 12 W output using a microtip probe sonicator (XL2000, Misonix Incorporated, NY). The mixture was then stirred at room temperature overnight to evaporate the organic solvent. The NP suspension was further purified with a 0.2 µm syringe filter to obtain PFTTQ NPs. To tune the size of PFTTQ NPs, 1 mL of THF solution containing 1 mg of PFTTQ and 1.5 mg of DSPE-PEG2000 was also utilized to synthesize PFTTQ NPs, yielding NPs with bigger sizes. Synthesis of Au NRs: Au NRs were synthesized by a seed-mediated growth approach following the previous reports.[20] Briefly, Au NP seeds were prepared by rapidly injecting NaBH4 (0.01 M, 0.6 mL) to an aqueous mixture of HAuCl4 (0.01 M, 0.25 mL) and cetyltrimethylammonium bromide (CTAB, 0.1 M, 9.75 mL). After stirring for 5 minutes, the obtained brownish solution was kept for 2 h at room temperature before being used as the seed. To grow Au NRs, 2 mL of HAuCl4 and 400 mL of AgNO3 were added into 40 mL of 0.1 M CTAB under gentle stirring. Subsequently, 0.8 mL of 1.0 M HCl was added, followed by the addition of 0.32 mL of 0.1 M ascorbic acid. Next, 8 µL of the seed solution was injected into the mixture and gently stirred for another 5 minutes. The reaction mixture was then left undisturbed overnight to yield the Au NRs. Cytotoxicity of PFTTQ NPs: MDA-MB-231 and Hela cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin streptomycin at 37 °C in a humidified environment containing 5% CO2. The metabolic activities of both cells were evaluated using methylthiazolyldiphenyltetrazolium (MTT) assays. MDA-MB-231 and Hela cells were seeded in 96-well plates (Costar, IL, USA) at density of 4 × 104 cells mL−1. After 24 h incubation, the medium was replaced by PFTTQ NP suspensions at different mass concentrations, and the cells were then incubated for another 24 h. After the designated time interval, the wells were washed twice with 1×PBS and 100 µL of freshly prepared MTT (0.5 mg mL−1) solution in culture medium was added into each well. For the PTT experiment, the cells were treated with an 808 nm laser at a power density of 0.75 W/cm2 for 10 minutes, followed further incubation with MTT solution. The MTT medium solution was carefully removed after 3 h incubation in an incubator. DMSO (100 µL) was then added into each well and the plate was gently shaken for 10 minutes at room temperature to dissolve all the precipitates formed. The absorbance of MTT at 570 nm was monitored by the microplate reader (Genios Tecan) after subtracting the absorbance of the corresponding control cells incubated with PFTTQ NPs at the same concentration but without the addition of MTT to eliminate the absorbance interference from
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
small 2014, DOI: 10.1002/smll.201402092
Biocompatible Conjugated Polymer Nanoparticles for Efficient Photothermal Tumor Therapy
PFTTQ. Cell viability was expressed by the ratio of absolute absorbance of the cells incubated with NP suspensions to that of the cells incubated with culture medium only. Photostability of PFTTQ NPs: The photostability of PFTTQ NPs was investigated by monitoring their respective UV-vis absorbance changes after 808 nm continuous laser irradiation for one hour. After one hour, the UV-vis absorbance of the NP suspension was measured with the spectrometer to compare with that of fresh PFTTQ NP suspension. Tumor model and tumor suppression study: Female NOD/ SCID mice were obtained from Beijing HFK Bioscience Co., Ltd. and used at 6 weeks of age. All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. The procedures were approved by the University of Science and Technology of China Animal Care and Use Committee. The xenograft tumor model was generated by subcutaneously injection of 4 × 106 Hela cells suspended in 100 µL phosphate buffered saline (PBS, with 30% Matrigel, BD Bioscience) into the right shoulder of each female NOD/SCID mouse. When the tumor volume was approximately 60 mm3, the mice were randomly divided into 4 groups. For the PTT treatment, each mouse was intratumorally injected with 40 µL of 1 mg/mL PFTTQ NPs in PBS solution and the control group received the same volume of PBS. The mice were irradiated with 808 nm NIR laser at 0.75 W/cm2 for 10 minutes. The temperature of the tumor sites was recorded by IR 7320 thermal camera and analyzed by IR Flash Software (Infrared Cameras. Inc). The tumor sizes were monitored by measuring the perpendicular diameter every other day. The volume was calculated based on the following equation: tumor volume = 1/2 × length × width2. Moreover, the typical mice in each group were sacrificed on day 2 after treatments, and the tumors were excised for histology observations. The organs were fixed in 10% neutral buffered formalin, which were then processed routinely into paraffin, sliced at thickness of 4 µm, and stained with hematoxylin and eosin (H&E). The H&E-stained slices were imaged by optical microscopy, which were assessed by 3 independent pathologists. Statistical Analysis: Quantitative data were expressed as mean ± SD. Statistical comparisons were made by ANOVA analysis and Student’s t-test. P value < 0.05 was considered statistically significant.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements J. L. Geng and C. Y. Sun contributed equally to this work. We are grateful to the financial support from the Singapore National Research Foundation (R-279–000–323–281), the JCO IMRE/ 12–8P1103 and JCO IMRE/14-8P1110. Mr. J. L. Geng thanks the National University of Singapore for the research scholarship.
small 2014, DOI: 10.1002/smll.201402092
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Received: July 15, 2014 Published online:
small 2014, DOI: 10.1002/smll.201402092
