(299i) Wide-Range Size Tuning of Conjugated Polymer Nanoparticles for Multi-Color Bioimaging

1. Introduction

Fluorescent nanoparticles have attracted significant attention as strong tools for bioimaging and biosensing. The representative example is quantum dot (QD), which shows size-dependent optical property due to the quantum confinement effect. QDs exhibit high photo-stability and tunable emission wavelength, but on the other hand, their toxicity caused by heavy metal components has been a potential concern. Recently, conjugated polymer nanoparticles, which are called polymer dots (Pdots), have been investigated as a new class of fluorescent nanoparticles without toxic heavy metals [1]. Whereas QDs show photoluminescence only when their size is within the single-nm range, Pdots maintain their optical property with a size up to several hundred nanometers.

The physiological property of nanoparticles, including size, shape, and surface chemistry, substantially affect their pharmacokinetic behavior in the biological systems. Particularly, the correlation between the size of nanoparticles and their interaction with cells and tissues in our body has recently attracted considerable research interest. Considering physiological phenomena, such as hepatic filtration, tissue extravasation, and kidney excretion, the size is a key parameter in the biodistribution and blood half-life of nanoparticles. In addition to enhanced permeability and retention (EPR) effect, tumor affects immunological system in whole body, which may alter the clearance behavior of nanoparticles from the bloodstream and its size dependency.

In general, Pdots are synthesized via nanoprecipitation method using commercially available conjugated polymers, which have delocalized π-electron system. The nanoprecipitation method demonstrated Pdot synthesis with a reliable size, usually up to tens of nanometers. Afterwards, many literatures have been investigated Pdot synthesis using stabilizing agents such as polystyrene grafted with ethylene oxide as well as poly(styrene-co-maleic anhydride) (PSMA). These stabilizing agents contribute to the colloidal stability of larger Pdots as well as the enhancement of biocompatibility.

Herein, we propose a size tunable Pdot synthesis strategy for controlling its behavior in the biological systems [2]. In the nanoprecipitation method, PSMA, which is widely used as a stabilizing agent, is initially hydrolyzed into a hydrophilic maleic acid copolymer to stabilize the particle surface. We succeeded in the size tuning of Pdots within the range from 15 nm to 220 nm by controlling the hydrolysis rate of PSMA and particle growth rate, and then demonstrated the ability of size tunable Pdots as a biomedical tool.

2. Materials and method

Pdots were synthesized based on the nanoprecipitation method using three different conjugated polymers; poly(9,9-dioctylfluorene-alt-benzothiazole) (F8BT), poly[2-methoxy-5-(2’-ethylhyxyloxy)-1,4-phenylene vinylene] (MEH-PPV), and poly(9,9-di-n-octylfluorenyl-2,7-diyl) (PFO). Conjugated polymer and PSMA were separately dissolved into tetrahydrofuran (THF), and then the mixture of these two solutions was added to water or 10 mM acetate buffer under sonication and stirring. After Pdots were formed, the THF was removed by evaporation at room temperature, and then purified by ultracentrifugation. The size of synthesized Pdots was evaluated using transmission electron microscopy (TEM) and dynamic light scattering (DLS), and their optical property was characterized by UV-vis spectrometer and fluorescent spectrometer. The surface of Pdots was modified with poly(ethylene glycol) (PEG) in order to increase the biocompatibility. The conjugation of PEG onto Pdots was evaluated by measuring zeta potential and Fourier transform infrared spectroscopy (FT-IR). The performance of PEG modified Pdots with different sizes as a bioimaging probe was then examined using mouse fibroblast (NIH/3T3) cells. The Pdots internalization into cells was evaluated using confocal laser microscopy and flow cytometry.

3. Results and discussion

Firstly, size-tunable synthesis method of Pdots was developed using F8BT as a conjugated polymer. Conjugated polymer and PSMA were dissolved in THF, followed by addition to the water phase with varying pH and ionic strength to synthesize Pdots. The synthesized Pdots were of spherical morphology and unimodal size distribution for all conditions. The size of Pdots decreased from approximately 220 nm to 60 nm with increasing pH from 5.4 to 6.2 when synthesized using 10 mM acetate buffer with 80 mM NaCl as the water phase. The decrease in Pdot size would be attributed to the accelerated hydrolysis rate of maleic anhydride units of PSMA in the basic condition. The hydrodynamic size of Pdots determined by DLS was also consistent with the TEM observation, thereby confirming the successful tuning of Pdots size by controlling pH of the water phase. In addition to the size-tuning based on pH, the size of synthesized Pdots also could be controlled by changing the volume of deionized water as the water phase during nanoprecipitation. When the volume of water increased, the size of Pdots decreased from 30 nm to 15 nm. Therefore, we succeeded in tuning the size of Pdots within the range from 15 nm to 220 nm. It is also confirmed that this size-tuning strategy can be adapted to other conjugated polymers, MEH-PPV and PFO.

The optical property of Pdots is crucial for their performance as fluorescent probes for bioimaging. The adsorption peak of synthesized Pdots was slightly red-shifted with increasing particle size, which could be due to the extension of the delocalization of π-electron conjugation. Meanwhile, Pdots showed the same fluorescent emission spectra regardless of their size. Quantum yield also did not change significantly, regardless of the size. For investigating the performance of Pdots with different diameters as fluorescent probes, the surface of Pdots was modified with PEG to minimize non-specific interaction with molecules in the physiological environment such as proteins. The surface zeta potential of Pdots increased after the reaction, which indicated that the PEG modification successfully proceeded. The successful surface modification of Pdots was also suggested by their FT-IR spectroscopic spectra before and after PEGylation.

The performance of Pdots with different sizes as bioimaging probes was then examined using NIH/3T3 cells in vitro. PEGylated Pdots were incubated with cells for 4 hours, followed by the observation using confocal laser microscopy. Cellular uptake of Pdots was successfully observed in all sizes within 60 nm to 220 nm. Furthermore, Pdots were colocalized with endosomes and lysosomes stained with LysoTracker, which suggested their internalization into cells through endocytosis. To further investigate their cellular uptake behavior, the fluorescent intensity from different sizes of Pdots internalized into each cell was quantified using flow cytometry. Cells were incubated with the presence of PEGylated Pdots under the same condition described above, and then trypsinized cells were analyzed using a flow cytometer. It was found that the Pdots showed higher fluorescent intensity in each cell with their increasing size. Therefore, it was confirmed that our size-tuned Pdots can demonstrate the size effect of nanoparticles in the biological environment.

4. Conclusions

In this study, we demonstrated the size-tunable synthesis of Pdots based on a nanoprecipitation method by regulating the hydrolysis rate of PSMA as a stabilizing agent. We found that the size of Pdots was successfully controlled by pH of the water phase during nanoprecipitation because it determined the hydrolysis rate of maleic anhydride units of PSMA. Additionally, the size of Pdots also tuned within the range smaller than 30 nm by changing the volume of water phase to control the particle growth rate. Taken together, facile on-demand size tuning of Pdots between 15 nm to 220 nm was achieved via optimizing reaction conditions. These strategies work as long as acid anhydrides are used during nanoparticle formation, and are expected to be adapted not only to fluorescent conjugated polymers but also to other hydrophobic polymers. We also succeeded in evaluating functions of size tuned Pdots using cultured cells. It is expected that size tunable Pdots will significantly broaden the application of Pdots as fluorescent materials in the biomedical field.

5. References

[1] C. Wu and D. Chiu, Angew. Chem., Int. Ed. Engl., 2013, 52,3086–3109. [2] N. Nakamura, N. Tanaka and S. Ohta, RSC Adv., 2022, 12, 11606-11611.