Biological Functionalization of Conjugated Polymer Nanoparticles for

Jul 13, 2016 - Due to the long distance and wide diffusion range, MCF-7 tumor cells with CPNs/anti-EpCAM have no obvious change with or without white ...
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Biological Functionalization of Conjugated Polymer Nanoparticles for Targeted Imaging and Photodynamic Killing of Tumor Cells Liheng Feng,*,† Jiarong Zhu,† and Zhijun Wang*,‡ †

School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, P. R. China Department of Chemistry, Changzhi University, Changzhi 046011, P. R. China

‡

S Supporting Information *

ABSTRACT: Conjugated polymer nanoparticles composed of PFT/PS as a core and PEG-COOH on the surface were prepared by a reprecipitating method. The CPNs diaplay excellet properties such as good photostability, low cytotoxicity, and strong brightness, etc. The average diamater of CPNs is 30 nm with a spherical morphology. To realize specific imaging in different parts of tumor cells, the bare CPNs with the carboxyls on the surface were conjugated with antibody or peptide by a covalent mode. Studies display that CPNs modified with anti-EpCAM can recognize MCF-7 tumor cells and locate on the membrane, while CPNs conjugated with transcriptional activator protein (Tat) specifically locate in the cytoplasm of MCF-7 cells. On the basis of the ability of CPNs for producing reactive oxygen species (ROS) under light irradiation, photodynamic therapy for tumor cells was investigated. Due to the long distance and wide diffusion range, MCF-7 tumor cells with CPNs/anti-EpCAM have no obvious change with or without white light irradiation. However, CPNs/Tat exhibits higher killing ability for MCF-7 cells. Noticeably, multifunctional CPNs linked with anti-EpCAM and Tat simultaneously not only can specifically target MCF-7 tumor cells, but also may inhibit and kill these cells. This work develops a potential application platform for multifunctional CPNs in locating imaging, photodynamic therapy, and other aspects. KEYWORDS: conjugated polymer nanoparticles, preparation, functionalization, cell imaging, photodynamic therapy

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INTRODUCTION Recently, conjugated polymer nanoparticles (CPNs) are of great interest in chemistry and biology fields because of their combined features of nanoparticles and conjugated polymers (CPs).1−5 The fast development of CPNs in biological applications not only depends on their outstanding properties (such as low cytotoxicity, strong brightness, and excellent photostability), but also lies in their good water solubility and facile modification and purification steps.6−10 Currently, the applications of CPNs in biological fields mainly focus on fluorescence imaging and drug/gene delivery. As fluorescent imaging reagents, CPNs usually located in the cytoplasm by nonspecific cellular uptake method.11−16 Despite CPNs having the delivery capacity for drug and gene, the penetration efficiency and speed for tumor cells were low and slow.17,18 Additionally, modifying the CPNs with specific biomolecules to target different parts of tumor cells is crucial for developing further biological applications. Our and other groups’ research indicated CPNs conjugated with antibody could specifically recognize the tumor cells and locate on the membrane.19−21 In other words, the CPNs connected with antibodies may act as a high efficiency stain reagent for a cell membrane. Many works have verified that the peptides (such as arginine translocation protein) have a strong penetration ability for cells to speed the carrier transfer process.22−24 On the basis of the case, we © XXXX American Chemical Society

reasonably believe that CPNs modified with penetrating peptides will precisely and quickly locate in the cytoplasm. It is of interest for modifying CPNs with different functional biomolecules to achieve efficient staining cells in different positions. Previous research studies indicate that reactive oxygen species (ROS) that can inhibit and kill the bacteria and tumor cells are also produced by conjugated polymers (CPs) under light irradiation.25−27 The feature of CPs provides a new opportunity for their application in therapeutics. Photodynamic therapy (PDT) is a well-established treatment tool for cancers and other diseases by a noninvasive method.28−30 In a PDT process, the photosensitizer has a very important role: it first is activated by a given light beam, and then undergoes a series of processes, and finally generates ROS to kill cells in the neighborhood. Here, conjugated polymer also acts as a photosensitizer. To date, conjugated polymers (especially water-soluble conjugated polymers) have been widely applied as photosensitizers for inhibiting and killing the bacteria and cells by the PDT approach.31−35 Very recently, in this aspect, conjugated polymer nanoparticles have received overwhelming Received: June 3, 2016 Accepted: July 13, 2016

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DOI: 10.1021/acsami.6b06642 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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mL) at 0 °C. The system was put under ultrasonic conditions for 10 min. Lastly, through introducing nitrogen continually to the system, the vast tetrahydrofuran solvents were removed at room temperature, and the desired dispersion was achieved by concentrating the mixture to 10 mL. Biofunctionalization of the Bare CPNs. On the basis of the condensation reaction of carboxyl and amino by the EDCI-catalyzed, three types of bioconjugated CPNs were prepared by the following procedures. To the bare CPNs dispersion (2 mL with 50 μg/mL CPs and 20 μg/mL PS-PEG-COOH) were added polyethylene glycol (40 μL, MW 335 PEG, 012.5% w/v) and HEPES buffer (40 μL, 1.0 M). Then, 20 μL of EDCI solution (10.0 mg/mL) and 40 μL of Nhydroxysulfosuccinimide sodium salt (1.0 M) were introduced to the above mixture. The system was shaken for 30 min on an oscillator. Accordingly, antibody and Tat peptide were added to the mixture and unceasingly reacted for 3 h at room temperature. Sephadex HiTrap Desalting columns (GE) were used to remove small molecules. Using Micron centrifugal filter (100 000 NMWL, Millpore) eliminated the unreacted biomolecules. Thus, the biofunctionalization CPNs were obtained. Characterizations of CPNs. The SME imaging of CPNs was measured by scanning electron microscope (Hitachi S-4800). The sample was prepared with 10 μL of the CPNs dispersion on clean silicon slices and quickly frozen, and then the water was removed under freeze-drying. The sizes and zeta potentials of CPNs were performed with Nano ZS (ZEN3600) system. Agarose gel electrophoresis was used to verify the successful conjugations of CPNs with biomolecules. The measured and recorded imaging was performed with a DYY-6C electrophoresis system and Bio-Rad ChemiDoc XRS system. Assay for Photostability. A mercury lamp (450/70 nm excitation filter) with 100 W was used to irradiate the samples which were prepared by dropping the CPN dispersion onto a glass plate. The time of irradiation was 2 min. Fluorescence microscopy (Olympus 1X71) was used to record the fluorescence intensity change of CPNs. Cell Culture. The breast cancer cell line (MCF-7) was obtained from the cell culture center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). The culture medium was the Roswell park memorial institute (RPMI-1640) with 10% fetal bovine serum (FBS) at 37 °C and under 5% CO2 humidified atmosphere. Cell Imaging in Vitro. First, MCF-7 cells were seeded on a confocal dish and cultured at 37 °C. When the density of MCF-7 was up to 80%, 2.5 μg/mL concentrations of these CPNs (CPNs/ antibody, CPNs/Tat, and CPNs/antibody/Tat, respectively) were introduced to the medium. The cells were continuously cultured for 2 h under CO2 humidified atmosphere. Before the cells were fixed with 4% paraformaldehyde, PBS buffer was used to wash the cells twice. After 20 min, the paraformaldehyde solution was removed and washed for another two-washing steps by PBS buffer. The control groups were the bare CPNs at the same concentration. Assay of Cell Viability and Photodynamic Testing. Each well of 10000s of MCF-7 cells in 96-well tissue culture plates was first seeded, and then cultured in RPMI-1640 medium with 10% FBS for 24 h. Then, the bare CPNs and functionalized CPNs were, respectively, added into the cell media according to the given concentrations and further incubated at 37 °C for 3 h. Photodynamic killing measurements were carried under white light source at fluence rate of 10 mW cm−2 for 30 min. After removing the media, 100 μL/ well of MTT (1.0 mg/mL) was added these wells, respectively. Then, the cells were continuously cultured at 37 °C for 4 h, followed by abandoning the supernatant. Lastly, 150 μL of DMSO per well was added to the plates and shaken for 10 min. A microplate reader (BIOTEK Synergy HT) was used to record the absorbance values at 490 nm.

attention because of their low toxicity, good solubility, and facile delivery capacity for drugs, genes, and biomolecules, etc. In comparison to common organic small molecule photosensitizers, the advantages of CPNs for the ROS generation efficiency lie in the intrinsic capacity which comes from conjugated polymers and packaging capacity for dye photosensitizers. In order to improve the generation efficiency of ROS, dye photosensitizers encapsulated by CPNs for onephoton or two-photon photodynamic therapy were developed, and displayed good killing ability for tumor cells.36−39 However, a main deficiency for these works is no specific location and target role for tumor cells. Hence, how to target precisely and kill the tumor cells is imperative for promoting CPN applications in PDT. In this work, we provide yellow emission CPNs with the core of PFT/PS and the shell of PEG conjugation carboxyl groups with a reprecipitation method. Usually, the tumor cells are not easily stained by the CPNs with carboxyl groups because there are many negative charges on the surface of cells. To realize exact location imaging in the different parts of the cells, a strategy of modifying the CPNs with specific functional biomolecules is adopted. Here, we select a highly targeted antibody as the location group for tumor cell membrane and a facile transcriptional activator protein (Tat) as the cytoplasm location body. Noticeably, the CPNs simultaneously conjugated with antibody and Tat not only can target the tumor cells specifically, but also partially penetrate into the cytoplasm. On the basis of the capacity of producing reactive oxygen species (ROS) by white light irradiation, the multifunctional CPNs may efficiently target and inhibit tumor cells, and then kill the tumor cells under light irradiation, which will provide a strong support for photodynamic therapy of cancer.

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EXPERIMENTAL SECTION

Materials and Measurements. Di-tert-butyl ((2,7-bis(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-9H-fluorene-9,9-diyl)bis(hexane6,1-diyl))dicarbamate (monomer 1) was obtained as described in the previous literature.40,41 4,7-Dibromobenzothiadiazole (monomer 2) was provided by Aldrich Chemical Co. Polystyrene polymer PSPEGCOOH was obtained from Polymer Source of Canada. Polyfluorenethiazole (PFT) polymer was synthesized as described by previously reported work.21 Relevant chemicals were obtained from Aldrich. Common organic solvents were provided by Chemical Companies of Beijing. Fluorescence emission and UV−vis absorption spectra were determined by a JASCO V-550 spectrophotometer and a Hitachi F4500 fluorescence spectrophotometer with a xenon lamp as the excitation source, respectively. Bio-Rad Molecular Imager ChemiDoc XRS system was used to record the images of gel electrophoresis. With Quantity One software (Version 4.6.5), corresponding analysis data were provided. Scanning electron microscope (SEM) images were detected on a scanning electron microscope (Hitachi S-4800). Dynamic light scattering (DLS) and zeta potential (ζ) of the CPNs were given in a Nano ZS instrument. A microplate reader (BIO-TEK Synergy HT) with 490 nm wavelength was used to report the data from the MTT assay. The confocal laser scanning microscope (FV1000-IX81) was used to investigate the fluorescence imaging of CPNs. The range of white light is 400−800 nm, and the source can be obtained by a metal halogen lamp (MVL-210, Mejiro Genossen). A Millipore filtration system was applied to purify the experiment water. Preparation of Conjugated Polymer Nanoparticles. The bare CPNs (PFT/PS-PEG-COOH) with carboxyl groups were prepared according to our previously reported reprecipitation method. A mixed tetrahydrofuran solution (10 mL) with PFT (50 μg/mL) and PS-PEGCOOH (20 μg/mL) was obtained by combining the stock solutions of PFT (1.0 mg/mL), PS-PEG-COOH (2.0 mg/mL), and tetrahydrofuran. Then, the mixture was rapidly poured into the Milli-Q water (20

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RESULTS AND DISCUSSION Preparation and Biofunctionalization. The conjugated polymer (PFT) was synthesized by the Suzuki coupling

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DOI: 10.1021/acsami.6b06642 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Synthesis Route of PFT and the Preparation Processes of CPNs and Their Modification with Antibody and Peptide

Figure 1. (a) DLS distribution of CPNs; (b) scanning electron image of CPNs; (c) cell viability of MCF-7 cells; (d) photostabilities of CPNs and fluorescein under 450/70 nm excitation filter mercury lamp with 100 W power.

reprecipitation method was used to prepare conjugated polymer nanoparticles with carboxyl groups on the surface. On the basis of the hydrophobic interaction, the bare CPNs with PFT and PS-PEG-COOH were formed, which were

reaction of monomer 1 and monomer 2 under pd(dppf)2Cl2 catalysis (Scheme 1). The detailed synthetic procedures of PTF and monomers can be obtained by previously reported works.28,40,41 In order to realize biofunctionalization, a modified C

DOI: 10.1021/acsami.6b06642 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces composed of the core (PFT and PS) and shell (PEG-COOH). The biofunctionalization of CPNs with antibody (antiEpCAM) and transcriptional activator protein (Tat) was obtained by a condensation reaction between carboxyl and amino groups (Scheme 1). In the preparation process, NHSSO3Na as an activator for carboxyl on the surface of CPNs was employed to improve conjugation efficiency of biomolecules. In order to eliminate nonspecific absorption of CPNs and antibody or peptide, polyethylene glycol (PEG, 0.1−0.3 wt %) was introduced in these reaction media. In the work, we developed three types of biofunctionalization CPNs: CPNs/ antibody (anti-EpCAM), CPNs/Tat (peptide), and Tat/ CPNs/antibody. Properties. Fluorescence emission and UV−vis absorption techniques were used to investigate the photophysical properties of CPNs in water (Figure S1). The maximum absorption and emission peaks of CPNs are at 470 and 560 nm in water, respectively. The fluorescence quantum yield of CPNs is 0.29 in water medium, and the CPNs act as a yellow emitter. The modified CPNs exhibit similar photophysical properties with bare CPNs. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) were applied to investigate the size and morphology of these CPNs (Figure 1a,b). As shown from Figure 1, we can find that the CPNs display a mean diameter of ∼30 nm with a spherical morphology. The MTT method was used to assay the cytotoxicity of CPNs (Figure 1c). In the method, the cell viability lies on the conversion of 3-(4′,5′dimethylthiazol-2′-yl)-2,5-diphenyl-2H-tetrazolium hydrobromide (MTT) to formazan.42 MCF-7 cells can reach 80% viability in the presence of CPNs (2.5 μg/mL). As an optical probe, the photostability of CPNs was also studied and compared with a typical dye (fluorescein). The result indicates that the photostability of CPNs is much stronger than that of the fluorescein dye under irradiation for 120 s at 455 nm continuously. (Figure 1d). These results reveal that the CPNs are a desirable biological material for cell imaging and other applications because of their excellent characteristics. Characterization. Three covalently conjugated CPNs (CPNs/antibody, CPNs/Tat, and Tat/CPNs/antibody) were successfully prepared and verified by zeta potential (ζ) and electrophoresis measurements (Figure 2). As can be seen from Figure 2, the sizes of modified CPNs (≈ 55 nm) are larger than that of the bare CPNs (≈30 nm). Also, due to the covalent conjugation between amino moieties (in the antibody and peptide) and surface carboxyl units of CPNs, the zeta potential of bare CPNs possesses higher negative values than that of functionalized CPNs. Moreover, the bare CPNs show faster mobility in the gel medium than that of functionalized CPNs because of the effective conjugation effect between carboxyl and amino groups. In brief, the above results display that the antibody and peptide can successfully link with CPNs by the covalent mode of amide bonds. Targeted Location Imaging in Vitro. To date, reported research studies have indicated that conjugated polymers and bare CPNs as fluorescence reagents may be used for cell imaging in vitro and vivo. On the basis of nonspecific interactions (such as electrostatic interaction), polymers and CPNs (usually with positive charges) were first absorbed on the surface of cells. Then, they mainly located in the cytoplasm region via endocytosis. A note about the current cells imaging is that the specificity and specific region for cells are poor and scarce. Hence, we put forward a strategy to achieve specific location imaging for tumor cells. In the work, live human breast

Figure 2. Characterizations of CPNs before and after conjugation with antibody and peptide, respectively. Gel electrophoresis of CPNs modified with antibody and peptide was in a 0.7% agarose gel, respectively. In the figure, the PFT/PS-PEG-COOH is unmodified CPNs. The PFT/PS-PEG-COOH/Tat, PFT/PS-PEG-COOH/Tat/ Antibody, and PFT/PS-PEG-COOH/Antibody are covalently conjugated with Tat peptide, anti-EpCAM, and both together, respectively. Other bands are simply mixtures of CPNs with antibody or peptide, respectively.

cancer cells (MCF-7) as imaging objects were cultured with the bare CPNs and modified CPNs (CPNs/anti-EpCAm, CPNs/ Tat, and Tat/CPNs/anti-EpCAM) for 2 h at 37 °C. Fluorescence imaging of these CPNs for tumor cells was obtained by a confocal laser scanning microscope (Figure 3). The CPNs modified by carboxyl groups adhere differently to the cell membrane because of the repulsive interaction between surface negative charges of CPNs and cells. On the basis of this

Figure 3. Fluorescence imaging of MCF-7 cells with the bare CPNs, CPNs/antibody, CPNs/Tat, and CPNs/Tat/antibody, respectively. The control groups are MCF-7 and the bare CPNs. The excitation wavelength is 488 nm, and the collection range of the emission wavelength is from 500 to 620 nm. D

DOI: 10.1021/acsami.6b06642 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 4. (a) Viabilities of MCF-7 cells with various biofunctional CPNs without white light irradiation. (b) Viabilities of MCF-7 cells with various biofunctional CPNs with white light irradiation for 30 min at a fluence rate of 10 mW/cm2.

case, we observed the fluorescence imaging of bare CPNs with MCF-7 cells used as control groups (Figure 3). It is well-known that an antigen on the cell membrance and a primary antibody provide the ideal specific combination. Here, the EpCAM receptor on the MCF-7 cells membrance was used to bind the antibody as a target. As shown in Figure 3, the CPNs modified with anti-EpCAM not only can efficiently recognize the tumor cells (MCF-7), but may also specifically locate on the cell membrane. Additionally, in order to get fluorescence imaging of CPNs in the cytoplasm, the transcriptional activator protein (H-Lys-Lys-Lys-Arg-Lys-Val-Ala-Ala-Arg-OH (Tat)) was linked with bare CPNs. The CPNs conjugated with Tat peptide can be found in the cytoplsm of MCF-7 (Figure S2a). Thus, specific imaging in different parts of living tumor cells is realized by modifying bare CPNs with specific biomolecules. Noticeably, the CPNs modified by anti-EpCAM and Tat simultaneously display the targeting and penetrating biofunctions, which are positive and promising for photodynamics therapy (PDT) research. The cell imaging of the type CPNs on the surface and that for the inner part of the tumor cells have both distributions (Figure 3 and Figure S2b). Photodynamic Killing for Tumor Cells. Many studies indicate that conjugated polymers and conjugated polymer nanoparticles also may generate reactive oxygen species (ROS) by light irradiation.25−27 The ROS is capable of inactivating the neighboring proteins and killing the bacteria and tumor cells. Therefore, photodynamic therapy (PDT) has received wide attention as a treatment mode for cancer in recent years. Our previous work indicated that the CPNs modified by antibody had no obvious change for tumor cells before and after the light irradiation. In the work, we first investigated the capacity of generating reactive oxygen species for bare CPNs (PFT/PSPEG-COOH). The generation of ROS from the CPNs by the light irradiation was verified by adopting the 2,7-dichlorofluorescein diacetate (DCFH-DA).43 The detection principle is that 2,7-dichlorofluorescein diacetate (DCFH-DA) can be transformed to 2,7-dichlorofluorescin (DCFH) with the fluorescence enhancement in the presence of ROS. The fluorescence intensity of DCFH with CPNs was much higher than that of the DCFH without CPNs (control group) under the white light irradiation, which indicated the considerable generation of ROS (Figure S3). However, the viability of MCF7 cells incubated with CPNs/antibody has a slight decrease before and after the radiation. The reason may be faster diffusion and inactivation of ROS on the surface of tumor cells than that in the inner. Figure 4 gave the photodynamic killing results for MCF-7 tumor cells with and without white light irradiation. As shown in Figure 4a, these CPNs all have good

biocompatibility and low cytotoxicity. More than 80% cell viabilities of MCF-7 are maintained in 3.0 μg/mL concentration of these CPNs. Different from the CPNs/antibody, the viability of MCF-7 cells with introduced CPNs/Tat has an obvious reduction with the increase of CPNs/Tat concentrations, which reveals CPNs/Tat as a therapeutic agent can be applied in photodynamic killing for tumor cells. On the basis of the above cases, the multifunctional CPNs conjugated with Tat and antibody should have potential value for targeting and killing tumor cells because of their specific recognition and efficient damage roles simultaneously. As expected, CPNs with Tat and anti-EpCAM not only can specifically target MCF-7 tumor cells but also have a certain function for killing tumor cells (Figure 4b). The biggest advantage of the multifunctional CPNs lies in accurate positioning and effective killing for tumor cells.

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CONCLUSION In summary, the contribution of our work lies in providing an approach for specific location imaging and photodynamic killing for tumor cells based on a new class of biofunctional CPNs. The covalent conjugations based on amide bonds of CPNs with biomolecules can be verified by the experimental data. Experimental results indicate that the method is facile and efficient, and may be applied to CPNs modification not only by antibody and peptide, but also by drug or DNA. These CPNs have low cytotoxicity, good biocompatibility, and photostability. CPNs conjugated with antibody can exclusively locate on the membrane of MCF-7 tumor cells, while CPNs modified by Tat peptide may efficiently stain in the cytoplasm and reduce the viability of MCF-7 tumor cells under white light irradiation. In comparison with the above two types of CPNs, multifunctional CPNs linked with Tat and anti-EpCAM have the ability of precise targeting and photodynamic damage for tumor cells. This work develops a potential application platform for biofunctional CPNs in fluorescence imaging, photodynamic therapy, and other aspects in the biological field.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b06642. UV−vis absorption and fluorescence emission spectra of CPNs, and merged 3D fluorescent images of functionalized CPNs and MCF-7 cells with the generation of ROS for CPNs (PDF) E

DOI: 10.1021/acsami.6b06642 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (L.-H.F.). *E-mail: [email protected] (Z.-J.W.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work described in this paper was supported by the National Nature Science Foundation (No. 21571116 and 21371110), the Youth Science Foundation of Shanxi Province (No. 2014021006) and the Program for the Outstanding Innovative Teams of Higher Learning Institutions of Shanxi (2013). We also thank Prof. Libing Liu and Fengting Lv for assistance during the preparation of the manuscript.

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DOI: 10.1021/acsami.6b06642 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX