Visualizing Photodynamic Therapy in Transgenic Zebrafish Using Organic Nanoparticles with Aggregation-Induced Emission
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KeywordsNanomedicine Photodynamic therapy Transgenic zebrafish Aggregation-induced emission Organic nanoparticles
The key novelty of this work is the creation of an in vivo model that can be used to effectively visualize image-guided photodynamic therapy. This allows fast screening of the performance of photosensitizers and their formulations.
Transparent zebrafish larvae provide a visual understanding of bio-distribution of nanoparticles, thereby enabling smarter formulation strategies.
Photodynamic therapy (PDT) is a noninvasive triggered therapeutic modality, which involves the use of photosensitizer (PS) molecules capable of generating reactive oxygen species (ROS) upon light excitation for treatment of malignant and non-malignant tumors. Over the last three decades, several PSs have been developed and some of them have been successfully used to treat different kinds of cancers [1, 2]. PDT can also be combined with chemotherapy for synergistic therapeutic effect  and to achieve triggered drug release from vesicles . Typically, upon light illumination, the formed PS triplet state releases energy to convert ambient oxygen molecules to reactive oxygen species (ROS) such as 1O2, H2O2, O2−· and ·OH. The generated ROS can cause an increase in oxidative stress in cells. At elevated concentrations, ROS oxidize lipids, proteins and DNA, which leads to damage in various cell organelles and eventually cell death. Cancer cells operate at a sensitive oxidative threshold making them vulnerable to further increase in ROS . Therefore, directly causing ROS to exceed the threshold concentration that is toxic to cells can kill cancer cells more easily as compared to that for normal cells . Currently, PDT application potential is limited by low light penetration through tissue and poorly characterized tumor PS uptake. While the former can be addressed by chemiluminescence-induced ROS production [7, 8], so far there is no straightforward tool to characterize or visualize the PS uptake. However, the success of PDT is strongly dependent on PS versus light dosage, trigger time, PS distribution and oxygen concentration in the tumor microenvironment [9, 10]. Several clinical studies have characterized the PS dose and corresponding light dose for effective PDT in cancers of various organs [11, 12]. The oxygen concentration variation in the process of PDT has been modeled, simulated and validated in tumor spheroid models [13, 14]. Although the importance of quantifying PS accumulation has been realized [15, 16], a sensitive in vivo model to do the same has not been established. Real-time quantitative visualization of PS concentration in target tissue enables concentration-dependent trigger of the PS, thereby allowing unbiased evaluation of various PS molecules.
Theranostic cancer medicine aims at developing drug composites that can be successfully tracked in vivo post-delivery. A combination of therapeutic and imaging modalities for PDT enables real-time tracking, cancer characterization, targeted delivery, triggered drug release and pharmacokinetic profiling. Cancer imaging involves use of various modalities such as ultrasound imaging, computed tomography, magnetic resonance imaging, fluorescence imaging and nuclear imaging. Although various imaging modalities may be applied, high spatial resolution, low-cost and real-time display of fluorescence imaging provides a unique advantage [17, 18]. As most fluorescent drugs and dyes are small hydrophobic molecules, their selective accumulation in tumor tissue is contingent upon the circulation time. In order to impart stealth properties to drugs, their aggregates can be encapsulated into polymeric nanoparticles (NPs) with PEG polymer decorated on the surface, which provide a long circulation time leading to enhanced permeation and retention (EPR) in the tumor tissue . Using fluorescent PSs encapsulated in polymeric NPs can enable real-time tracking of their fate, their selective tumor localization and consecutive PDT.
To continuously track the NP tissue concentration, we need strongly fluorescent, photostable and efficient PS NPs. Different from traditional PSs, which show quenched fluorescence and reduced photosensitizing capabilities in aggregate state, recently some PSs with aggregation-induced emission (AIE) characteristics have been developed to show bright fluorescence and strong capabilities in ROS production as NPs [20, 21]. AIE molecules generally possess rotor-like structures. They are almost non-emissive in molecularly dissolved state due to the free intramolecular motions which consume the excited-state energy. Upon aggregation, the restriction of molecular motion is able to activate the radiative decay channel to yield fluorescence. Although mice are considered as the gold standard for pharmacokinetic and pharmacodynamic studies, real-time quantitative tracking and direct visualization of NP delivery, their bio-distribution for optimized PDT in mouse models is cumbersome, inefficient and invasive. In order to study the NP organ distribution, the mice need to be killed and imaged since fluorescence imaging typically has a depth resolution of 3–5 mm . In this regard, the transparent zebrafish larva model has shown to be effective for such studies [23, 24]. Zebrafish produces optically transparent embryos which are used by biologists to study development, genetics, environmental toxicology, pharmacology and cancer. Zebrafish genome is 70% homologous with the human genome, making it an attractive vertebrate model with high scalability . In this study, we use the inducible transgenic zebrafish line which expresses the oncogene (EGFP:krasV12) under a liver-specific promoter that develops liver hyperplasia when subjected to drug mifepristone (RU-486) . Continuous exposure to mifepristone at the adult stage can cause the progression of hyperplasia to a mix of hepatoblastoma, carcinoma, malignant ascites and metastasis. We, however, induce hyperplasia in transparent larval stage that enables visualization of PS NP distribution over time in the liver tissue followed by selective light treatment for initiating PDT. Owing to the presence of EGFP, the fluorescence from the hyperplastic liver can be used to monitor the change in liver tumor size. Upon introduction of the PS NPs into systemic circulation (retro-orbital injection), their progressive accumulation in liver tumors can be monitored, which facilitates treatment parameters optimization of PDT. In this paper, we demonstrate how the zebrafish liver tumor model enables optimized precise photodynamic therapy using AIE PS NPs as an example.
3 Results and Discussion
The ROS production of the NPs was characterized by measuring the absorbance decay of indicator ABDA (9,10-anthracenediyl-bis(methylene)dimalonic acid) due to its reaction with 1O2 [31, 32] generated by the PS NPs in aqueous media under 0.15 W cm−2 white light (400–700 nm) excitation. As shown in Fig. 1d, under white light irradiation, the presence of PPDCT NPs at a fixed PS concentration can lead to gradual decrease in absorbance of ABDA (64 µM) in aqueous media, and the 1O2 generation of the AIE PS NPs is evaluated by the relative degradation of ABDA. Within 1 min, 15.8 nmol of ABDA could be degraded by 5 μM PPDCT NPs (based on molecules), which is significantly enhanced relative to Ce6 (5 μM) for which 12.2 nmol ABDA could be degraded under the same condition. This proves that PPDCT NPs could generate singlet oxygen in aqueous media with a relatively high efficiency.
Confocal microscopy was used to image fli1:EGFP larvae (7 dpf), post-intravenous delivery. The PPDCT NPs accumulated passively in two regions—the caudal hematopoietic tissue (CHT) and the liver. The CHT is an equivalent for the bone marrow in zebrafish larvae and possesses most of the innate immune cells that can phagocytose PPDCT NPs. Progressive decrease in fluorescent labeling in CHT is used as an independent indicator of NPs biodegradation. The larvae were tracked up to 4 days post-injection. NPs in systemic circulation are depicted by yellow fluorescent signal where green fluorescent EGFP-labeled vessels co-localized with circulating red fluorescent PPDCT NPs. As shown in Fig. 3b, the red fluorescent NPs were initially in circulation within the vessels labeled with EGFP. As time progressed, the particles extravasated and penetrated into the developing liver. Exit from circulation is confirmed by distinct red fluorescent NPs that are away from neighboring GFP-positive vessels.
The liver blood vessel fenestrations and low blood flow rate  allowed blood carrying PPDCT NPs to interact with the hepatic cells. PPDCT NPs were recognized by hepatocytes as foreign materials and phagocytosed by scavenger receptors. Progressive NPs uptake in the liver was observed from 24 h post-injection (hpi). At 72 hpi, an absence in overlap of the EGFP and PPDCT NPs fluorescence indicated that almost all the NPs had extravasated. Progressive breakdown of internalized NPs followed from 96 h, which is suggested by the decrease in red fluorescence detected in CHT. A similar approach of tracking PPDCT NPs uptake was applied to the liver-tumor-bearing larvae (Fig. 3c). Seven dpf larvae were injected with the fluorescent PPDCT NPs and tracked over 4 days. In the hyperplastic liver tumor, successful uptake of red fluorescent PPDCT NPs is indicated by its detection in EGFP-positive liver cancer cells. Hence, confocal imaging using two different emission detection channels for EGFP emission at 509 nm and PPDCT emission at 660 nm identified co-localization of NPs in liver cancer cells, an unbiased in vivo assessment of uptake efficiency. Successful NPs internalization by liver cancer cells results in visualization of yellow fluorescence in the overlay image (Fig. 3d). The PPDCT red fluorescent intensity in the liver tumor was computed as a percentage of the EGFP intensity using ImageJ, to ascertain the concentration of the PPDCT NPs in the tumor tissue (Fig. S2). At 24 hpi, since most NPs were in circulation, the red fluorescence as a percentage of EGFP fluorescence in the liver was low. It gradually increases as the liver filters out the PPDCT NPs with time. Since the liver was hyper-proliferative and responsible for hepato-biliary excretion and degradation of the NPs, the uptake of PPDCT NPs from the single intravenous delivery would change significantly with time. At 96 hpi, the total concentration of PPDCT in continuously dividing liver cancer cells became suboptimal, such that there was less co-localization of red fluorescent PPDCT in EGFP-positive liver cancer cells (Fig. S2). Therefore, 48 hpi or 2 days post-injection (dpi) was chosen as the earliest trigger time after analyzing the uptake profile (Fig. 3d). We considered this period as the ideal time to conduct PDT testing in injected zebrafish to trigger an optimal therapeutic response because of the maximized cellular uptake of the PPDCT NPs.
In this study, we developed theranostic polymer-encapsulated NPs to carry out PDT. PPDCT was encapsulated in a polymeric shell, thereby imparting it with stealth properties. These NPs were shown to have effective PDT properties in vitro. Upon intravenous delivery, the PPDCT NPs passively accumulated in the hyperplastic liver of transgenic (EGFP:krasV12) zebrafish larvae. The PPDCT NP bio-distribution was profiled for normal (fli:EGFP) and liver-tumor-bearing larvae. Fluorescence-guided tissue accumulation data in zebrafish suggest the optimal time to conduct PDT. Effective duration of white light illumination was adjusted based on anticancer effect in treated zebrafish. This study demonstrates the importance of correlation between the tumor NP uptake, light dose and trigger time. There is a sweet spot for initializing triggered therapy which may vary based on the choice of nano-delivery system and the photosensitizer dose. Our research demonstrates how transparent zebrafish larvae can be used to study the effect of multiple light doses for PDT. Better in vivo understanding of therapeutic response will facilitate development of efficient triggered combination therapy like PDT-enhanced chemotherapy.
We thank Dr. Deepan Balakrishnan for assisting in the development of the MATLAB code for unbiased voxel quantification. The authors acknowledge the financial support from National Research Foundation Investigatorship (R279-000-444-281) and National University of Singapore (R279-000-482-133).
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