Visualizing Photodynamic Therapy in Transgenic Zebrafish Using Organic Nanoparticles with Aggregation-Induced Emission

Photodynamic therapy (PDT) employs accumulation of photosensitizers (PSs) in malignant tumor tissue followed by the light-induced generation of cytotoxic reactive oxygen species to kill the tumor cells. The success of PDT depends on optimal PS dosage that is matched with the ideal power of light. This in turn depends on PS accumulation in target tissue and light administration time and period. As theranostic nanomedicine is driven by multifunctional therapeutics that aim to achieve targeted tissue delivery and image-guided therapy, fluorescent PS nanoparticle (NP) accumulation in target tissues can be ascertained through fluorescence imaging to optimize the light dose and administration parameters. In this regard, zebrafish larvae provide a unique transparent in vivo platform to monitor fluorescent PS bio-distribution and their therapeutic efficiency. Using fluorescent PS NPs with unique aggregation-induced emission characteristics, we demonstrate for the first time the real-time visualization of polymeric NP accumulation in tumor tissue and, more importantly, the best time to conduct PDT using transgenic zebrafish larvae with inducible liver hyperplasia as an example. Electronic supplementary material The online version of this article (10.1007/s40820-018-0214-4) contains supplementary material, which is available to authorized users.


Characterization
UV-Vis spectra were recorded on a Shimadzu UV-1700 spectrometer. Photoluminescence (PL) spectra were recorded on an Edinburgh FS5 spectrofluorometer. Average particle size and size distribution were measured by Zetasizer Nano ZS (Malvern Instruments Ltd, UK) at room temperature. The sample morphology was studied by transmission electron microscopy (TEM, JEM-2010F, JEOL, Japan).

Cell Culture
Human liver cancer cell-line Hep G2, was provided by American Type Culture Collection (ATCC). The cells were cultured in DMEM medium containing 100 μg mL -1 streptomycin, 10% heat-inactivated FBS, 100 U mL -1 penicillin, and maintained in a humidified incubator with 5% CO2 at 37 °C.

Confocal Imaging
The cells were cultured in 6-well plates with glass slides and precultured overnight. Then the medium was replaced with fresh one and incubated with the PPDCT nanoparticles (30 µg mL -1 ). The cells were fixed, mounted and imaged by confocal laser scanning microscope (CLSM, Zeiss LSM 800, Jena, Germany). For zebrafish imaging, the zebrafish larvae were immobilized by placing them in 1% low melting agarose in glass bottom dishes. Using an upright confocal microscope (CLSM, Zeiss LSM 800, Jena, Germany), the zebrafish larvae were imaged using 10X and 40X lens.

Flow Cytometry
In order to confirm cellular internalization, precultured cells in 6-well plates were incubated with PPDCT NPs (30 µg mL -1 ) for 24 h. The cells were then trypsinized and fixed in suspension using 70% ethanol. The cells were then subjected to flow cytometric fluorescence measurement using 405 nm laser (Beckman Coulter CyAn ADP).

Cytotoxicity Studies
The metabolic activity of the cells was assessed by MTT assays. After incubation of the cells in DMEM medium overnight, the medium was removed, washed with PBS and the cells were incubated with PPDCT nanoparticles at different concentrations for 24 h and subjected to white light for different intervals. The cells were further washed with 1× PBS before the addition of 100 μL of MTT solution (0.5 mg mL -1 ) into each well. After 3 h incubation, the MTT solution was removed and DMSO (100 μL) was added into each well. The absorbance of MTT at 570 nm was studied by microplate reader (Genios Tecan). The cells without any treatment were used as control.

Photodynamic Therapy in Zebrafish
For zebrafish photodynamic therapy, the zebrafish larvae were immobilized by placing them in 1% low melting agarose in glass bottom dishes. Specific delivery of white light of 0.15 W cm -2 to the liver was enabled by placing mounted zebrafish larvae behind an opaque sheet with a slit that exposed the zebrafish liver. Using an upright confocal microscope (CLSM, Zeiss LSM 800, Jena, Germany), the zebrafish larvae were imaged using 10× and 40× lens. Confocal λex = 488 nm, Green fluorescent protein λem = 509 nm, PPDCT λem = 660 nm.

Zebrafish Line
Four to five pairs of zebrafish were placed in crossing tanks for spawning overnight. Embryos were settled to the bottom of the tank, and were collected using a sieve and transferred to petri dishes for embryo culture. They were screened, incubated at 27 °C , 0.4% CO2 and grown in egg water (10% NaCl; 1.63% MgSO4· 7H2O; 0.4% CaCl2; 0.3% KCl). At 22 h post fertilization, 1-phenyl 2-thiourea (PTU) was added to prevent melanin formation to yield optically transparent fish. EGFP:kras V12 transgenic zebrafish larvae were incubated with 5 µM of mifepristone in egg water with PTU. The embryos were imaged using confocal imaging S 3 / S 4 (Carl Zeiss LSM 800).

Microinjection
Local in vivo injection of the NPs intravenously was done using a nitrogen gas injector. The needle used for injection was made by pulling glass capillary tubes (O.D. 1.0 mm, I.D. 0.75 mm) in a needle puller which pulls the glass tubes into fine needles (20 µm). The NPs were filled into the needles which were loaded into the nitrogen gas injector. The injector was operated in continuous mode for the retro-orbital delivery of the NPs. The fishes were mounted in a mixture of 1% low melting agarose and 5% methyl cellulose for the injection and confocal imaging.

Statistical Analysis
Quantitative data were expressed as the mean ± standard deviation (SD). Statistical evaluations were made by comparing the area under the therapeutic trend curves in Fig. 5 by Student's t-test. P value < 0.05 was considered statistically significant.