Cellular imaging using emission-tuneable conjugated polymer nanoparticles

New materials that exhibit tuneable optical properties, notable emission across the visible spectrum, are of immense interest to biologists as they present a broad palette of colours from a single imaging agent that can be utilised in biological detection. Such a flexible system, when combined with the advantages of using conjugated polymer nanoparticles in cell imaging results in a widely useful medical diagnostic system. Here, we describe tuneable emission observed through oxidation of a conjugated polymer followed by the formation of nanoparticles and their subsequent use in cell imaging.


Instrumentation
Absorption spectra were measured using a Hitachi U-4100 UV-Visible-NIR spectrometer using a 1 cm path length quartz cuvette. Photoluminescence spectra were measured using a Horiba Fluoromax-4 spectrofluorometer. Particle size distributions and zeta potentials were obtained using a Malvern Zetasizer (utilising dynamic light scattering). Transmission electron microscopy images were acquired on a Hitachi 7100 at St George's University of London, with a filament electron source at 100 kV.
Image analysis was performed with ImageJ software. The quantum yields (QYs) were measured using the dye comparison method. Photoluminescence quantum yields (PLQYs) were estimated using:  Confocal images were acquired with an inverted confocal microscope (Leica TCS SP2) and an internal analogue photomultiplier tube detector, whose detection wavelength range for the fluorescence emission was set to 500-650 nm. The samples were excited with a solid-state CW laser (CNI Laser MBL-III-100mW) at a wavelength of 473 nm and a power of ~5 mW after passing through a neutral density filter wheel. A RSP 500 excitation beam splitter and a 63X 1.2 N.A. water-immersion objective were used to acquire the images. The line scan speed was set to 400 Hz, the image size to 512x512 pixels with a pixel size of 470x470 nm 2 and a pinhole of 2 Airy units. The transmitted light images were taken at the same time as the confocal fluorescence images.

Controlled oxidation of MEH-PPV polymer chains
10mg of MEH-PPV was added to 10 mL of THF (final concentration of 1 mg/mL) and left stirring overnight to ensure MEH-PPV was completely dissolved. At the same time, 10 mL of H 2 O 2 (30%) was added to a clear vial. 5 mL of this was added to a vial containing 10 mL of THF. A serial dilution was made, adding 5 mL of the previously diluted H 2 O 2 to another 5 mL of THF until a final dilution of 0.03% was made. After a period of time (approximately 1 week), the colour of the solution changed from red to yellow to green to blue, with the photoluminescence and absorption spectra changing also.
Typically, a yellow emitting polymer was achieved with 1-2% of H 2 O 2 , green between 0.1-0.5% H 2 O 2 , and blue between 0.03% and 0.3% H 2 O 2 . The emission remained consistent over a year period.

Encapsulation of oxidised MEH-PPV loaded into PSMA
The method was adapted from 8 . 1.5 mL of each oxidised MEH-PPV solutions (0.05 mg/mL) were added to 1.5 mL of THF containing 0.03 mg of PSMA. The solution was sonicated in a 35 kHz ultrasound bath at 7-9 °C, in 30 second bursts for 5 minutes to ensure all polymers were completely dissolved. The solution was then injected into 5 mL deionised water and sonicated for 10 minutes. The solution was then stirred continuously at 400 rpm, at room temperature, for 24 hours to evaporate off THF. Loss of water was compensated by readjustment to 5 mL. The nano-suspension (10 µg/mL of MEH-PPV or total solid of 0.015 mg/mL) was subsequently filtered through a 0.2 µm cellulose acetate Gilson syringe filter. The filtrate was stored at room temperature.

Encapsulation of oxidised MEH-PPV loaded into PSMA with superparamagnetic iron oxide nanoparticles
As above, 1.5 mL of each oxidised MEH-PPV stocks were added to 1.5 mL of THF containing 0.03 mg of PSMA with an additional 10 µL of magnetic nanoparticles (690 mg/mL). The solution was sonicated in a 35 kHz ultrasound bath at 7-9 °C, in 30 second bursts for 5 minutes to ensure all polymers were completely dissolved. The solution was then injected into 5 mL of deionised water and sonicated for 10 minutes. The solution was then stirred continuously at 400 rpm, at room temperature, for 24 hours to evaporate off THF. Loss of water was compensated by readjustment to 5 mL. The nanosuspension (10 µg/mL of MEH-PPV or total solid of 0.015 mg/mL) was collected by using a magnet and washed. The particles were re-suspended and stored at room temperature.

Encapsulation of oxidised MEH-PPV loaded into F127
The method was adapted from Bourke et al, 9 . 1 mL of each oxidised MEH-PPV solutions (0.05 mg/mL) were added to a vial containing 50 mg of F127. The solution was sonicated in a 35 kHz ultrasound bath at 7-9 °C, in 30 second bursts for 5 minutes to ensure all polymers were completely dissolved. The solution was then injected into 9 mL deionised water and sonicated for 10 minutes. The solution was then stirred continuously at 400 rpm, at room temperature, for 24 hours to evaporate off THF. Loss of water was compensated by readjustment to 5 mL. The nano-suspension (10 µg/mL of MEH-PPV or total solid of 10.01 mg/mL) was subsequently filtered through a 0.2 µm cellulose acetate Gilson syringe filter. The filtrate was stored at room temperature.

MEH-PPV-PSMA nanoparticles associated with cells
HeLa and HEK cells were cultured as separate adherent monolayers DMEM supplemented with 10% heat inactivated FBS. HeLa cells were cultured on a sterilised 8 square well microplate, and HEK cells were cultured on a sterilised 96 well microplate. Cell cultures were kept at physiological temperature (∼37°C), 5% CO 2 in a humidified incubator. The oxidised MEH-PPV nanoparticle suspension was serially diluted in DMEM to have a polymer concentration of (7.5 µm/mL). 100 µl of the oxidised MEH-PPV nanoparticle suspension was added to 200 μL of the aforementioned media (for the 8 well plate) and 20 µl of the oxidised MEH-PPV nanoparticle suspension was added to 100 µl of the aforementioned media (for the 96 well plate). These were incubated for 24 hours.
HeLa cells were fixed in a 4% formaldehyde solution for 15 minutes, and then washed with phosphatebuffered saline (pH 7.0) six times. All images were acquired on a Leica inverted confocal using a 63X 1.2 N.A. water-immersion objective, with 473 nm excitation. Emission from the oxidised MEH-PPV nanoparticles was collected between 500-650 nm.
For the cytotoxicity study with HEK cells, after the time intervals of 1 hour, 24 hours and 48 hours, 100 μL CellTiter-Glo® reagent was added and cells were left to lyse for 10 minutes before the luminescence was recorded. SI Table 1 showing the photoluminescence spectra of MEH-PPV in THF oxidised by different % of H 2 0 2 , and how this changes over 7 days. Abs is absorption, Em is emission and QY is Quantum Yield.