Tetraphenylethylene-Based Photoluminescent Self-Assembled Nanoparticles: Preparation and Biological Evaluation

The conjugation of tetraphenylethylene (TPE) with podophyllotoxin, N-desacetylthiocolchicine, and cabazitaxel through a sebacic acid linker led to the formation of fluorescent nanoparticles. Dynamic light scattering (DLS) and photoluminescence spectroscopy were used for the identification and characterization of the fluorescent nanoparticles. The biological evaluation was determined in three human ovarian (KURAMOCHI, OVCAR3, OVSAHO) and three human breast (MCF7, SKBR 3, and MDA-MB231) cancer cell lines. In the case of cabazitaxel, the nanoparticles maintained the activity of the parent drug, at the low nanomolar range, while exhibiting high blue fluorescence. The internalization of the fluorescent NPs into cells was detected using immunofluorescence assay.


Nanoparticles assembly
In accordance with standard solvent evaporation protocols the conjugate (2.0 mg) was first dissolved in the appropriate organic solvent (0.5 mL, THF was used for conjugates 7 and 8, while EtOH for conjugate 6) in a vial while stirring at rt.The resulting solution was added dropwise to a round bottom flask containing MilliQ grade distilled water (1 mL) under magnetic stirring (500 rpm).The resulting suspension was stirred for 5 min, then the organic solvent was thoroughly evaporated under reduced pressure, obtaining pure NPs as an opalescent suspension (1 mL, 2 mg/mL).

Nanoparticles characterization
NPs were characterized by dynamic light scattering (DLS), using a 90 Plus Particle Size Analyzer from Brookhaven Instrument Corporation (Holtsville, NY, USA) operating at 15 mW of a solid-state laser (λ = 661 nm), using a 90-degree scattering angle.The ζ-potential was determined at 25 °C using a 90 Plus Particle Size Analyzer from Brookhaven Instrument Corporation (Holtsville, NY, USA) equipped with an AQ-809 electrode, operating at an applied voltage of 120 V.Ten independent measurements of 60 s duration were performed for each sample.Hydrodynamic diameters were calculated using Mie theory, considering the absolute viscosity and refractive index values of the medium to be 0.890 cP and 1.33, respectively.The same aqueous samples at a concentration of 0.2 mg/mL were used for ζ-potential measurement, without any change for the ionic strength (no addition of KCl).The ζ-potential was calculated from the electrophoretic mobility of nanoparticles, by using the Smoluchowski theory.

Fluorescence study
UV-vis absorption spectra were obtained on a Shimadzu UV-vis-NIR 3600 spectrophotometer in a 1 cm path length quartz cell.Photoluminescence quantum yields were measured with a C11347 Quantaurus-QY absolute photoluminescence quantum yield spectrometer (Hamamatsu Photonics), equipped with a 150W xenon lamp, an integrating sphere, and a multi-channel detector.Emission spectra were obtained with an FLS 980 spectrofluorimeter (Edinburg Instrument Ltd.) under continuous excitation provided by a 450 W xenon arc lamp and corrected for detector sensitivity.
Photoluminescence experiments were carried out at room temperature in air equilibrated medium.
Absorption and emission measurements on nanoparticles were conducted after dilution of the suspension with MilliQ grade distilled water to reduce light scattering.Experiments in homogeneous solution were carried out in ethanol for conjugate 6 and in tetrahydrofuran for conjugates 7 and 8, at concentration between 2x10 -5 M and 4x10 -6 M, no fluorescence emission was detected.Absorption spectra of conjugates in diluted organic solution (left) and as dispersed nanoparticles in water (right).

Confocal Imaging of NPs
Ovarian cancer cell line, OVCAR-3, were cultured in 24-well plates on coverslips at 1x10 5 cells/well.Cytotoxicity screening with NCI-Sulforhodamine B assay.

Biological assays
OVCAR3, OVSAHO and KURAMOCHI high grade serous ovarian cancer cell lines and MDA-MB231, SK-BR-3 and MCF-7 breast cancer cell lines were inoculated into 96-well plates as 3000 cells/well.After 24h incubation, cells were treated with the compounds in increasing concentrations (0.078 µM -40 µM).Each and every drug treatment was performed in triplicate.DMSO was used as negative control.After 72h of incubation time, medium was discarded and plates were washed twice with 1xPBS.Then the cells were fixed with 10% (w/v) trichloroacetic acid (TCA) solution for 1h in dark at +4 °C.In order to remove TCA, cells were then washed with ddH2O about 4-5 times and left air-dry at room temperature.The plates were then stained using 0.4% sulforhodamine B (SRB) solution in 1% acetic acid and incubated in dark at room temperature for 10min.Finally, excess dye was discarded by washing off by 1% acetic acid 4-5 times until no dye comes out and left air dry at room temperature.Lastly, 10 mM cold TBS solution was used to solubilize the protein-bound SRB dye.Absorbance values were measured at 515nm with microplate reader.In order to calculate IC50 values, the recorded OD value for each well was normalized to the OD value of its corresponding DMSO control.
24 hours later, growth medium was changed with fresh medium containing the NPs of 6 and 8 conjugates at IC75 concentration or medium control for 24 and 48 hours.Cells were fixed with 4% Paraformaldehyde for 20 minutes.Fixed cells were permeabilized for 15 minutes at RT, dark with 0.1% TritonX-100 in 1X DPBS.The cells were washed once with 0.1% Tween-20 in 1x DPBS, and blocked with SuperBlock (ScyTek Laboratories) for 20 minutes at RT. Coverslips were placed on 17.5 µl of the DRAQ5 TM Fluorescent Probe Solution (10 µM working solution) (Thermo Fischer, #62254), which stained nuclei as red and incubated for 20 minutes at RT. 6 ul of Mounting Medium was placed on Thermo SuperFrost microscope slides (Thermo Fischer).The coverslips incubated S11 with DRAQ5 probe were immersed into washing buffer to remove excess antibody solution and then placed on mounting medium by sealing with nail polish to stabilize the coverslips and visualized by confocal microscope (Leica DMi8 /TCS SP8-DLS).The results obtained by n=2 independent experiments.