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Following cellular internalization of FRB-VC loaded MSNs (MSN-FRB-VC) via endocytosis,36 the MSN carrier with cargo proceeds through the endosomal system where a decrease in pH causes dissociation of His-tagged FRB-VC from the MSN. An endosomal release trigger (chloroquine shock) is then applied to prompt endosomal rupture and cytosolic release of FRB-VC. FRB-VC can then diffuse to the nucleus and interact with FKBP-VN which is primed with rapamycin at saturation levels. FKBP-rapamycin-FRB forms a ternary complex which approximates the Venus halves thus facilitating complementation and chromophore maturation. Successful protein delivery can then be observed in a rapid and non-amplifying manner through visualization and measurement of Venus fluorescence in the nucleus. The fluorescent protein requires reconstitution prior to chromophore formation and thus only protein that has either evaded or escaped the endosome system and remains functional is able to produce signal in the nucleus.
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Figure 6-1 Synthesis and characterization of MSNs for His-tagged protein controlled binding and release. (a) Surface modification series of un-functionalized MSNs (un-MSNs) to MSN-Ni. (b) STEM (left) and SEM (right) images of MSN-NTA, scale bar: 50 nm.
Characterization of un-MSN and MSN-NTA: (c) dynamic light scattering, (d) N2 sorption isotherms and (e) pore size distribution calculated via NLDFT mode.
We applied our protein delivery sensor in parallel with MTT assays to optimize MSN dosage for protein delivery and biocompatibility. As expected, the HeLa-FKBP-VN cell line exhibits no increased fluorescence in the Venus channel (Venus fluorescence was visualized in the GFP channel) (Figure 6-2).
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Figure 6-2 Live cell confocal imaging of MSN-mediated intracellular protein delivery in the cytosolic protein delivery detection system. Different MSN-FRB-VC concentrations were incubated with HeLa-FKBP-VN cells. Images were taken 20 h post endosomal release trigger (chloroquine shock). Scale bar: 10 m.
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Introduction of unloaded MSNs to the cells also did not increase Venus fluorescence (Figure 6-2, 6-3a, 6-3b). In contrast, application of MSN-FRB-VC complexes at all examined concentrations generated nuclear Venus signal in the cells (Figure 6-2). To quantify the gross Venus signal present in the cell populations we used microplate fluorometry. Whilst a significant increase in protein delivery is seen between the 50 µg/ml and 100 g/ml concentrations, the fluorescent signal saturated thereafter (Figure 6-3a).
When we quantified the percentage of Venus positive cells via flow cytometry we saw a similar pattern (Figure 6-3b). A significant increase in protein transfection percentage was seen between the 50 g/ml and 100 g/ml concentrations indicating some dose dependency, with higher concentrations having no further positive effect on uptake efficiency.
In parallel with protein delivery experiments, MTT assays were performed to evaluate cytotoxicity at different MSN concentrations. No significant difference in cell viability was seen at concentrations of 50 µg/ml and 100 µg/ml when compared to the no-MSN control population. However, concentrations of 150 µg/ml and 200 µg/ml were shown to be significantly more cytotoxic than the control. The cytotoxicity displayed at higher levels of MSN concentration may explain the plateau in the Venus signal we see in the protein delivery experiments, as damage to the cells may impair protein uptake. Further, it might explain the slight decrease in delivery efficiency measured with microplate fluorometry above 100 µg/ml. Through combination of the three data sets (Figure 6-3a-c) 100 µg/ml was determined to be an optimal MSN concentration due to its high protein transfection efficiency and low cytotoxicity.
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Figure 6-3 Protein delivery efficiency analyses and cytotoxicity assay. (a) Fluorescence readout from microplate reader. (b) FACS analysis. (c) MTT assay. The results shown in (a) were calculated based on six biological repeats, (b) and (c) were triplicated. Error bars represent SDs.
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Next we sought to observe protein delivery in a cell population over time. Using spinning disc microscopy, HeLa-FKBP-VN cells incubated with MSN-FRB-VC complexes (100 µg/ml) and rapamycin were imaged at time points prior to and following chloroquine shock (2 h after MSN-FRB-VCs incubation). Before the chloroquine shock, no released FRB-VC protein was detected (Figure 6-4a). However, following destabilization of the endosomes with the chloroquine shock, Venus fluorescence was visible within the nuclei of some cells (1%) within just 30 minutes. At a time point of 5.5 h after chloroquine shock, over 50% of the cells can be seen to have functional proteins released to the nuclei. At 9 h protein release had reached a plateau where approximately 85% of the cells exhibit fluorescent nuclei. A live cell image of protein release saturation (10 h post chloroquine shock) is shown in Figure 6-4c. A timelapse video further demonstrates sensor detection of intracellular protein release (Appendix Figure 6-7). An alteration of the above experiment was performed where rapamycin addition (250 nM) was delayed until 24 h after MSN-FRB-VC incubation (Figure 6-4b). We reasoned that at this point the protein was likely already released into 80% of the cells and therefore the timing of Venus fluorescence should represent sensor complex formation and maturation rather than protein release.
When comparing the onset of nuclear fluorescence within cells of a population between the rapamycin primed and rapamycin delayed experiments (Figure 6-4a-b) a far greater spread (50% timings of the two experiments) can be seen in the rapamycin primed experiment indicating a staggered release timing in the population.
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Figure 6-4 Live cell tracking of protein release. (a) HeLa-FKBP-VN cells were primed with rapamycin before imaging. MSN-FRB-VC was added to the cells and the first image was recorded (time point -2 h). After 2 h, particles were washed away using PBS and followed by a chloroquine shock (0.5 mM in the medium, RT, 5 min) (time point: 0 h). Cells were tracked using confocal microscopy (spinning disc) for 22 h. (b) MSN-FRB-VC were incubated with HeLa FKBP-VN cells for 2 h, and subsequently the endosomal protein release was triggered by chloroquine shock. 22 h after endosomal protein release trigger, rapamycin was added to the medium, and cells were tracked using confocal microscopy (spinning disc) for another 5 h. (c) Live cell confocal imaging of rapamycin-primed sample 10 h after chloroquine shock. Scale bar: 200 m.
However, when we compared the timing of fluorescence increase after onset within individual cells between the two conditions, we observed similar durations (Figure 6-5a-b).
We interpret these results to be consistent with the aforementioned model for MSN uptake and his-tag mediated protein release (Figure 6-1c). The delay in fluorescence onset within cells in the rapamycin primed experiment can be attributed to the time taken for the
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relevant endosomal rupture event to occur. (Further evidence that the delay may be due to endosomal rupture timing is the prolonged presence of enlarged endosomes after chloroquine shock (appendix Figure 6-8).
Figure 6-5 Live cell tracking of protein release in individual cells within a population.
Fluorescence intensity tracking from 30 cells in (a) rapamycin-primed sample and (b) rapamycin-delayed sample. (c) Mean fluorescence increasing time evaluated from (a) and (b). The central 70% of the release curves from both (a) and (b) were evaluated . Mean values are indicated within the box. Box range represents standard deviation and the whisker range displays the minimum and maximum values in the population. (d) Fluorescence intensity distribution among 625 cells in the rapamycin-primed sample recorded at 15 h post chloroquine shock.
The similarity between the experiments in single cell fluorescence increase once initiated suggests that we are, in all likelihood, observing roughly analogous processes (Figure 6-5c).
Therefore, it seems probable that the once protein release begins it proceeds in a rapid fashion and that in both experimental variations we are simply observing sensor complementation and maturation timing. This logically aligns with the proposed mechanism of delivery, as bulk release of already MSN-detached protein should occur
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when the endosomal barrier is breached, thus permitting prompt protein complementation between the corresponding halves.
Unlike sensors that rely upon transcription or amplification mechanisms, the ratio-metric nature of our sensor enables us to measure variations not only between but also within populations. For example, through image analysis of cell nuclei we can assess parameters such as the range of protein released within a population as well as means and standard deviations of protein released (Figure 6-5c). In consideration of the significance of cellular protein stoichiometry, this should be a useful tool for understanding and adapting nanocarriers for various tasks.