CPN™ Flow Cytometry Proof of Concept Report:
Conducted by The CPI on Behalf of Stream Bio Ltd
The CPI undertook a proof of concept study into the application of Conjugated Polymer Nanoparticles (CPNs) in flow cytometry. This work was carried out under the ESM Outreach ERDF programme which is part funded by the European Development Fund 2014-2020. Three batches of CPNs were
provided for the study, namely: 170nm CPN-Red, 100nm CPN-Red, 100nm CPN-Green. The CPNs were loaded in to CHO-K1 cells by adding them to the culture media and allowing the cells to take up them by endocytosis. The cells were then analysed by both flow cytometry and fluorescent microscopy. The study demonstrated that the CPNs were highly fluorescent and readily visible using both these methodologies.
As depicted in Fig 1, cells readily took up CPNs from their medium. The intracellular CPN were seen to be punctate and were concentrated in the perinuclear region. This is consistent with the CPN being contained in intracellular vesicles and trafficked from the cell surface to the lysosomes and Golgi apparatus. It is currently unclear how many CPN would be contained in a single vesicle, comparisons with viral or particle uptake indicate that each vesicle may contain a single CPN. This indicates that each puncta may represent individual CPNs. Observation of the fields of cells show a range of fluorescent signal correlating with extent of CPN uptake may individual cells. A small subset (~3%) of cells appear to be very intensely labelled suggesting an elevated uptake of CPNs, the reason for this
is currently unclear and may represent uptake during cell division or other cellular growth process. The field of view also shows bright extracellular fluorescent signals. These are CPNs clustered together either through aggregation or by adhering to adhesive clumps of extracellular material. Studies at differing time points and using alternative cell types may further elucidate the behaviour of CPN with cells and of particular interest is the duration which CPN labelled cells continue to be fluorescent; are intracellular CPN rapidly broken down / expelled from the cell or do they persist in the cell?
Fig 1: Fluorescent microscope image of CPN loaded into CHO cell by
endocytosis. CPN fluorescence is predominantly perinuclear
Flow Cytometry Analysis
Fig2: Cells loaded with CPNs were analysed using flow cytometry to determine if the fluorescent signal was sufficient to identified CPN loaded cells over non-loaded cells. The figure clearly shows that the CPN loaded cells, loaded with the highest CPN concentration (0.02mg/ml), gave a signal over 250 fold more intense that the blank control (1.05x10^6 RFU CPN loaded vs. 3.8x10^3 RFU Blank). Cell loaded with low concentrations of CPN gave lower fluorescent signals (4.6x10^5 RFU, 0.01mg/ml; 2.4x10^5 RFU, 0.005mg/ml). These data clearly show that CPNs are compatible with the flow cytometry technique and give high intensity fluorescent signals that are readily detected using the standard excitation and emission configuration of the flow cytometer. Cells from these preparations were also analysed for cell health using propidium iodide, cells loaded with CPN showed now differences in cell health compared to the unloaded controls.
Fig 2: Flow cytometry data showing CPN loaded into CHO cell by
endocytosis at 0.02, 0.01 and 0.005mg/ml.
There is a clearly distinction from unloaded blank cells.
Mean fluorescence: 0.02mg/ml = 1,046,997 RFU, 0.01mg/ml = 456,484 RFU,
0.005mg/ml = 240369 RFU, Blank = 38,252 RFU
CPNs alone were also analysed on the flow cytometer, Fig 3. It can be seen that there is a single, tight peak of fluorescence in the CPN sample, well separated from any background signal seen in the blank media only control. It is thought that this signal may represent individual CPNs, as there is a single peak, aggregate would be expected to produce multiple peaks or significantly unbranded the peak. There is little signal from front scatter or side scatter that correlates with the 100nm CPN which is consistent with their small size, however the larger 170nm CPN can be isolated in the side scatter signal. The ability to detect single CPN confirms the potential to use the CPN to quantify the presence of cellular proteins of interest and potentially detecting the presence of individual proteins. Interestingly, the individual CPN were of similar intensity to cells loaded with multiple of CPN which may indicate a quenching of the CPN fluorescence inside the cell. This may be due to limited penetration of exciting or emitting light through the cellular membranes or may indicate quenching interactions of cellular proteins or other intra-vesicular conditions (e.g. pH, ion concentration) with
Fig 3: Data showing CPN loaded into directly on to a flow cytometer
The CPN are clearly distinct from the Blank media without CPN.
Mean fluorescence: 0.02mg/ml = 156 RFU, 0.01mg/ml = 797,095 RFU
This study has confirmed that CPNs can be used in flow cytometry and that they are readily visible both inside the cell and as individual nanoparticles. This builds confidence that their utility in flow cytometry can be extended through their conjugation to target molecules such as antibodies to bind
specific cellular proteins. Furthermore, this data points to the CPNs’ potential to be used to quantify the presence of proteins of interest.