Subject Number : S-16-NM-0039
Support Type : 機器利用
Proposal Title (English) : Developing new theories based on soft matter physics to describe and understand the mechanical properties of cells as they interact with nanoparticles
Username (English) :A. Andy Lam
Affiliation (English) :1) University of British Columbia, Canada-Japan Co-op Program

1. Summary

My internship project at NIMS was to study the effect of nanoparticles on the spreading of cellular aggregates. The work described here is a contribution to Dr. Grégory Beaune’s greater investigations into the effect of nanoparticles on the spreading of cellular aggregates. When a spherical aggregate is placed onto a fibronectin-coated surface, the aggregate would deform, spreading into a monolayer film over the substrate. The addition of particles would affect the rate of spreading, and this project aimed to measure that spreading, additionally observing for other effects on spreading. Originally, we expected to complete the full project by the end of my three-month internship, but due to setbacks, my objected shifted to completing as many experiments possible in the three-month time frame.

2. Experimental

I used modified mouse S180 expressing E-Cad GFP from Dr. Beaune’s previous work, and grew cellular aggregates through the hanging drop method. Experimental chambers were prepared using fibronectin-coated coverslips as the bottom, and were filled with DMEM medium, supplemented with FBS, pen.strep and glycomax, in the cell culture room at the NIMS Molecule & Materials Synthesis Platform at SENGEN site. The chambers were also laced with various concentrations of nanoparticles of different sizes and surface modifications, before the addition of aggregates into the chamber. The particles used in these experiments were 20 nm, 200 nm, 1000 nm, 10000 nm, and 20000 nm (diameter) carboxylated polystyrene. We also used 200 nm amineated polystyrene particles. Near the end of my term we also worked with 4500 nm carboxylated polystyrene particles too. The concentrations used were based on percentage of a monolayer area taken up by the particles, in addition to a volume based concentrations, φ, representing the volume of particles divided by the total volume of solution. Finally, the aggregate spreading was observed every 10 minutes over the course of 15 or more hours, using the Leica TIRF microscope in the bio-imaging area, also at the NIMS Molecule & Materials Synthesis Platform.
Using the image set data from the microscopes; the area of aggregate spreading film was manually measured using ImageJ, an image analysis tool. From measuring the area at various time points we could determine a certain spreading velocity for each condition. For smaller particles, which could not be visually observed, it was possible to use an automated MATLAB plugin used by other members in our lab group to measure spreading.

3. Results and Discussion

From the image analysis, we could see that the presence of particles acts to slow down the spreading of aggregates, with the spreading rate slowed in relation to the concentration of particles used. In addition, we observed that the size of particles appeared to slow the spreading by different methods. For the larger particles of 20000 nm and 10000 nm diameter, the particles prevented the spreading by simply blocking the path of spreading. Particles would be pushed away until they formed a wall that blocking further spreading. In the smaller particles, we observed a phenomenon where the particles became localized to the initial spreading cells.

FIGURE 1. Example aggregate spreading experiment. Cell aggregate spreading experiment with 200 nm carboxylated polystyrene nanoparticles at φ = 6.8e-5 (volume of particles divided by the total volume of solution). Images were taken with the Leica TIRF every 10 minutes for 15 hours total.
In addition to the spreading of cellular aggregates, we also observed the behavior of individual cells over long periods of times. This was interesting due to the fact, that the presence of nanoparticles was observed to cause the re-aggregation of the cells over long periods of times, also described as de-wetting. Experiments were set up in the same conditions, only with the addition of small volumes of confluent cells rather than aggregates.
In these experiments, we saw that there was indeed this aggregating behavior, as the cells, originally in a single layer, came together into small aggregates in the presence of nanoparticles from 20nm to 1000nm. This behavior was not observed in the absence of particles, nor in the presence of the large 20000 nm and 10000 nm particles. In addition, we also observed the concentration of the observable small nanoparticles (200 nm both carboxylate and amineated, 1000 nm) into the cells, as they aggregated. This leads us to believe that the small nanoparticles were either being internalized into the cells or that the particles adhered to the cell surfaces, but we were unable to confirm this using confocal microscopy.

FIGURE 2. Dewetting behaviour of cells in the presence of nanoparticles. Cells were dispersed according to a 10% area monolayer, over 20 nm carboxylated polystyrene nanoparticles. Left image taken at 0 h, shows no initial aggregation; right image taken at 39 h, shows formation of small aggregates.

FIGURE 3. Concentration of nanoparticles in the aggregates of dewetted cells. Cells in 10% monolayer film were dispersed onto polystyrene nanoparticles. Amineated 200 nm particles are initially dispersed, but show concentration of nanoparticles into the aggregating cells.

4. Others

I would like to acknowledge Dr. Grégory Beaune (NIMS-MANA), Prof. Winnik (The Université de Montréal, NIMS-MANA) and Prof. Brochard-Wyart (Institut Curie) who supervised the completion of this project. I would also like to acknowledge the staff of NIMS Molecule & Materials Platform, whom I have consulted for advice on the proper use and handle of equipment.

5. Publication/Presentation

The results described in this report will contribute to two papers, with one currently under writing process by Dr. Grégory Beaune, Prof. Winnik and Prof. Brochard-Wyart.

6. Patent
N/A