Regulating Near-Infrared Photodynamic Properties of Semiconducting Polymer Nanotheranostics for Optimized Cancer Therapy Houjuan Zhu,†,# Yuan Fang,‡,# Qingqing Miao,† Xiaoying Qi,§ Dan Ding,*,‡ Peng Chen,*,† and Kanyi Pu*,† †
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 637457, Singapore State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China § Singapore Institute of Manufacturing Technology (SIMTech), Agency for Science Technology and Research (A*STAR), 71 Nanyang Drive, Singapore 638075, Singapore ‡
S Supporting Information *
ABSTRACT: Development of optical nanotheranostics for the capability of photodynamic therapy (PDT) provides opportunities for advanced cancer therapy. However, most nanotheranostic systems fail to regulate their generation levels of reactive oxygen species (ROS) according to the disease microenvironment, which can potentially limit their therapeutic selectivity and increase the risk of damage to normal tissues. We herein report the development of hybrid semiconducting polymer nanoparticles (SPNs) with self-regulated near-infrared (NIR) photodynamic properties for optimized cancer therapy. The SPNs comprise a binary component nanostructure: a NIR-absorbing semiconducting polymer acts as the NIR fluorescent PDT agent, while nanoceria serves as the smart intraparticle regular to decrease and increase ROS generation at physiologically neutral and pathologically acidic environments, respectively. As compared with nondoped SPNs, the NIR fluorescence imaging ability of nanoceria-doped SPNs is similar due to the optically inactive nature of nanoceria; however, the self-regulated photodynamic properties of nanoceria-doped SPN not only result in dramatically reduced nonspecific damage to normal tissue under NIR laser irradiation but also lead to significantly enhanced photodynamic efficacy for cancer therapy in a murine mouse model. This study thus provides a simple yet effective hybrid approach to modulate the phototherapeutic performance of organic photosensitizers. KEYWORDS: polymer nanoparticles, nanomedicine, photodynamic therapy, cancer therapy, near-infrared light dots,6,7 up-conversion nanoparticles,8,9 and carbon nanomaterials10,11 have been used as light-harvesting energy donors to sensitize the ROS generation from photosensitizers so as to amplify PDT or redshift light wavelength into deep-tissuepenetrating near-infrared (NIR) regions. However, most photodynamic theranostic nanoparticles are unable to change their ROS generation levels according to the disease microenvironment,12 which potentially limits their therapeutic
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hototheranostics have emerged as one of the avantgarde strategies for diagnosis and treatment of cancer due to their potential advantages such as negligible drug resistance and low systemic toxicity.1,2 In particular, photodynamic therapy (PDT) that utilizes photosensitizers to produce reactive oxygen species (ROS) under light irradiation has been widely integrated with fluorescence imaging for cancer theranostics, probably owing to the convenience that light is the solely needed source to trigger both processes.3 As most photosensitizers such as porphyrin, phthalocyanines, and bacteriochlorin derivatives are highly hydrophobic, encapsulation of them with other fluorescence agents into nanoparticles is a typical approach toward photodynamic nanotheranostics.4,5 Moreover, other fluorescent nanoparticles including quantum © 2017 American Chemical Society
Received: May 19, 2017 Accepted: August 25, 2017 Published: August 25, 2017 8998
DOI: 10.1021/acsnano.7b03507 ACS Nano 2017, 11, 8998−9009
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Scheme 1. Schematic Illustrations of (a) the Synthesis of Nanoceria-Doped SPNs and the Self-Regulated Photodynamic Properties of SPNs at Physiologically Neutral and Pathologically Acidic Conditions and (b) the Comparison between SelfRegulated and Conventional PDT-Mediated by Nanoceria-Doped SPNs and Nondoped SPNs, Respectively
vacancies,32 making it a smart regulator to act as ROS scavenger and converter at neutral and acidic pH, respectively. Such a combination endows nanoceria-doped SPNs with the pHregulated photodynamic properties, which not only amplifies phototherapy in the acidic microenvironment of tumor but also potentially reduces the side effect to normal tissues. Note that although nanoceria has been used for biological applications, most studies are based on its antioxidant properties, and its pHswitchable oxidation states have not been exploited to regulate the ROS generation of photosensitizers under different pHs.32 In the following, the synthesis and characterization of nanoceria-doped SPNs are described first, followed by the investigation on the doping effect on their optical and physical properties. Then, the ROS scavenging and photodynamic properties of nanoceria-doped SPNs are examined at different pHs. Next, the in vitro PDT performance of nanoceria-doped SPNs is validated along with nondoped SPNs. Lastly, the proofof-concept cancer therapy application of nanoceria-doped SPNs is demonstrated in xenograft mouse model.
selectivity and brings in the risk of potential damage to normal tissues. Semiconducting polymer nanoparticles (SPNs) have emerged as a new class of optical agents for molecular imaging and phototherapy.13−18 As SPNs are transformed from organic semiconducting polymers (SPs), they avoid metal-ion related toxicity issues and intrinsically possess good biocompatibility.19 The excellent fluorescent properties of SPNs enabled fluorescence imaging applications such as cell tracking,20 targeted tumor imaging21,22 and ultrafast hemodynamic imaging,23 as well as chemiluminescence imaging of neuroinflammation24 and hepatotoxicity.25 Recently, we have revealed that SPNs often have high photothermal conversion efficiencies,26 permitting photoacoustic imaging and photothermal cancer therapy.15,18,27 In contrast, PDT applications of SPNs for cancer therapy have been rarely explored in vivo, and the reported studies are limited to the nanoparticles with excitation at visible region which have the shallow tissue penetration and limited PDT efficiencies.28,29 The principle of PDT pinpoints the requirement for generation of single oxygen (1O2) is that the triple energy of a photosensitizer is larger than the energy difference between O2 and 1O2 (0.98 eV).30 Because the triple energy of most SPs exceeds 0.98 eV, it is theoretically feasible to develop NIR light excitable SPNs for cancer PDT. In this study, we report a hybrid approach to regulate the NIR photodynamic properties of SPNs for optimized cancer therapy. The SPN-based theranostics comprises two key active components (Scheme 1): poly(cyclopentadithiophene-altbenzothiadiazole) (PCPDTBT) and cerium oxide nanoparticle (nanoceria). PCPDTBT has strong absorption in the NIR region, where the triple energy is ∼1.0 eV,31 and thus can serve as both NIR fluorescence agent and photosensitizer; while nanoceria can switch oxidation states between III and IV based on pH conditions due to the high density of surface oxygen
RESULTS AND DISCUSSION Synthesis and Characterization. Nanoprecipitation method was used to prepare nanoceria-doped SPNs. As this method relies on the hydrophobic interactions between the components, nanoceria was synthesized via the thermal decomposition of cerium precursors in organic solutions and coated with the hydrophobic oleylamine.33 An amphiphilic triblock copolymer (PEG-b-PPG-b-PEG) was used to coprecipitate with PCPDTBT and nanoceria to endow SPNs with good water solubility. Such a method had no negative effect on the redox-activity of nanoceria, because the mixed valence states (Ce3+ and Ce4+) were retained after nanoprecipitation (Figure S1b, Supporting Information). High-resolution transmission 8999
DOI: 10.1021/acsnano.7b03507 ACS Nano 2017, 11, 8998−9009
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Figure 1. In vitro characterization of SPNs. (a) Representative TEM images of nanoceria, SPN-0, and SPN-C23. (b) DLS of SPN-C23. (c) Hydrodynamic average diameters of SPNs with different doping amount of nanoceria. (d) UV−vis absorption and (e) fluorescence spectra excited at 630 nm of SPNs. [SPN] = 4 μg/mL in 1 × PBS (pH 7.4).
Figure 2. In vitro studies on the ROS scavenging capability of SPNs. Schematic illustration for the ROS detection using (a) H2DCFDA and (d) Amplex Red. (b) Representative fluorescence spectra of H2DCFDA after addition of O2•−, O2•−/SPN-0, or O2•−/SPN-C23. (c) O2•− scavenging efficiency of SPNs measured by the fluorescence of H2DCFDA. [SPN] = 10 μg/mL, [H2DCFDA] = 5 μM; 1 × PBS (pH 7.4). Excitation for fluorescence: 480 nm with a long pass filter of 500 nm. (e) Representative fluorescence spectra of Amplex Red containing HRP after addition of H2O2, H2O2/SPN-0 or H2O2/SPN-C23. (f) H2O2 scavenging efficiency of SPNs measured by the fluorescence of HRP/ Amplex Red. [SPN] = 10 μg/mL, [Amplex Red] = 1 μM; [HRP] = 5 μU/mL; 1 × PBS (pH 7.4). Excitation for fluorescence: 530 nm with a long pass filter of 550 nm.
and 904.6 eV) and Ce4+ (peaks at 882.1, 900.9, and 916.4 eV) (Figure S1b, Supporting Information). To optimize the scavenging ability of SPNs, SPNs with different doping amounts of nanoceria were prepared and termed as SPN-0, SPN-C9, SPN-C16, SPN-C23, SPN-C28, and SPN-C33, which had the weight percentages of nanoceria of 0, 9, 16, 23, 28, and 33 w/w%, respectively. The representative TEM images (Figure 1a) revealed the spherical morphology for both nondoped and doped SPNs. However, dynamic light scattering (DLS) showed that the average diameters of SPNs increased with increasing doping amounts of
electron microscopy (HRTEM) image showed the size of nanoceria was in the range from 2 to 5 nm (Figure 1a). The lattice fringes were indexed to the (111) plane of cubic fluorite structure of ceria oxide with a plane to plane distance of 0.3 nm, which was different from the 0.32 nm spacing in bulk ceria. Moreover, the representative X-ray diffraction patterns (XRD) of the solid nanocrystalline powders showed all peaks matched with the cubic fluorite structure (JCPDS card no. 34-0394) (Figure S1a, Supporting Information). X-ray photoelectron spectroscopy (XPS) analysis revealed the mixed valence states of nanoceria, showing the valence state of Ce3+ (peaks at 885.0 9000
DOI: 10.1021/acsnano.7b03507 ACS Nano 2017, 11, 8998−9009
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Figure 3. In vitro ROS production from SPNs under NIR laser irradiation at 808 nm. Plot of fluorescence change of (a) H2DCFDA, (d) SOSG, (g) HRP/Amplex Red incubated with SPN-0 or SPN-C23 in PBS (pH = 6.5 or 7.4) as a function of NIR irradiation time. (b) Fluorescence enhancement (F/F0) of H2DCFDA in the presence of nanoceria encapsulated by PEG-b-PPG-b-PEG, SPN-0, or SPN-C23 in 1 × PBS (pH = 6.5 or 7.4) under NIR laser irradiation for 5 min. [SPN] = 10 μg/mL, [H2DCFDA] = 5 μM; excitation for fluorescence: 480 nm with a long pass filter of 500 nm. (e) Fluorescence enhancement (F/F0) of SOSG in the presence of ICG, nanoceria encapsulated by PEG-b-PPG-b-PEG, SPN-0, or SPN-C23 in 1 × PBS (pH = 6.5 or 7.4) under NIR laser irradiation for 4 min. [SPN] = 10 μg/mL, [SOSG] = 0.5 μM; excitation for fluorescence: 490 nm with a long pass filter of 500 nm. (h) Fluorescence enhancement (F/F0) of HRP/Amplex Red in the presence of nanoceria encapsulated by PEG-b-PPG-b-PEG, SPN-0, or SPN-C23 in 1 × PBS (pH = 6.5 or 7.4) under NIR laser irradiation for 5 min. [SPN] = 25 μg/mL, [HRP] = 5 μU/mL; [Amplex Red] = 1 μM; excitation for fluorescence: 530 nm with a long pass filter of 550 nm. The schematic illustration for (c) total ROS, (f) singlet oxygen, (i) hydrogen peroxide production from SPN-0 or SPN-C23 under NIR laser irradiation measured by H2DCFDA, Amplex Red, or SOSG in 1 × PBS (pH = 6.5 and 7.4). An 808 nm NIR laser was used with the power density of 0.44 W/cm2.
and hydrogen peroxide (H2O2), respectively. H2DCFDA is a ROS dye that can be oxidized to form a highly fluorescent dye (DCF) by ROS, such as superoxide and singlet oxygen (Figure 2a). Similarly, Amplex Red in combination with horseradish peroxidase (HRP) can be oxidized by hydrogen peroxide to produce the red-fluorescent product (resorufin) (Figure 2d). The fluorescence of both indicators was monitored and quantified upon addition of O2•− or H2O2 into the solution of SPNs at the neutral pH (7.4) (Figure 2b,e). In the presence of nondoped SPN (SPN-0), addition of O2•− and H2O2, respectively, led to 20.5- and 11.9-fold fluorescence enhancement for H2DCFDA and Amplex Red (Figure 2b,e), which was similar to that without SPNs. In contrast, in the presence of nanoceria-doped SPN (SPN-C23), the fluorescence of H2DCFDA and Amplex Red only increased by 1.26- and 1.60-fold, respectively. This clearly indicated that the presence of nanoceria in SPNs scavenged ROS and inhibited the oxidation of fluorescence indicators. It has been widely reported that nanoceria with the size
