Team:UCL/Demonstrate
Demonstrate: Encapsulin drug delivery system
Expression, Purification and Assembly of Encapsulin+DARPin fusion system
The first step in demonstrating our work involved manufacturing our multicomponent drug delivery platform. This involved a lot of layers of cloning and experimentation. The first layer was to study the potential of expression and assembly of a T. maritima encapsulins with a surface-displayed DARPin929 targeting peptides. Thus, BBa_K3111501 composite BioBrick was made, which contained the T. maritima encapsulin coding sequence fused with DARPin929 coding sequence at its C terminus. It was cloned into the pSB1C3 plasmid and amplified in E. coli DH5a. After sequencing the plasmid, we proceeded with transformation in E. coli BL21 (DE3) and 50 mL scale-up culture for protein expression.
In order to observe whether Encapsulin-DARPin fusion protein was successfully expressed we analysed our cell pellet using SDS-PAGE. The pellet obtained from the 50 mL cultures was then resuspended in Tris Buffer Saline at an OD600 10. Once resuspended the sample was cell lysed using sonication. Following sonication, the sample were span to separate the soluble and insoluble fragments form the whole cell lysate. We proceeded on with purification of the soluble fragment using column chromatography containing a Strep Tactin resin.
The expected protein band was ca. at 51 kDa. As observed from Figure 1, a thick band at this size was observed majorly in the insoluble fragment indicating that even though expression was successful the fused construct has considerably reduced solubility compared to the individual parts. However, after purification we managed to elute some of the expressed protein from the soluble fragment observed in elution 2.
To observe assembly under non-reducing condition environment, we concentrated the purified sample from elution 2 and used it for Transmission Electron Microscopy and non-reducing PAGE gel.
Figure 2(a) shows the TEM image obtained. We could clearly observe assembled encapsulins at the expected size of 25 nm, concluding that although expression of the protein was mostly insoluble, the soluble fragment contained fully assembled encapsulins with surface displayed DARPin929. The native PAGE gel observed in Figure 2(b) let us confirm that the DARPin did not get spliced-off during assembly, as we observed a significant increase in band size from the TmEnc lane to the TmEnc_DARPin929.
Loading cytotoxic cargo
Once surface display was proven we moved on to cargo loading. To induce cytotoxic action, we proceeded with loading of the encapsulin system with a fluorescent photosensitiser protein, miniSOG. Under blue-light illumination, miniSOG is activated and generates reactive oxygen species which are damaging to cancer cells. To achieve cargo loading we used BioBrick cloning to construct the plasmid observed in Figure 3.
To enable cargo loading we utilised a short native C-terminal targeting peptide (1,2) that was previously described to allow foreign cargo loading. Thus, miniSOG was fused to the C-terminal targeting peptide downstream via a flexible linker. Once cloning was proven successful, we followed the same transformation and 50 mL shake flask culture protocol as mentioned previously to allow protein expression. Then we purified the soluble cell lysate and obtained a sample to run on an SDS-PAGE to investigate cargo loading.
In Lane 3 of Figure 3, we can see that bands representing both the Encapsulin-DARPin hybrid construct (~50 kDa) and photosensitiser miniSOG (~15 kDa) can be seen in this purified elution. As miniSOG does not possess a Strep-tag, we would not expect to see it in this fraction unless it was bound to the inside of an assembled T. maritima encapsulin. Fluorescence spectrophotometric analysis of the purified elution indicated fluorescence compared to non-loaded encapsulins as well, confirming that there's miniSOG present. Quantification of the loading can be found on BBa_K3111502 part page.
Mammalian cell experiments
Once the construction of the multicomponent drug delivery vehicle was proven successful through SDS-PAGE and fluorescent spectrophotometric analysis we proceeded with mammalian cell culture studies. The targeting peptide on the T. maritima encapsulin, namely DARPin929 binds specifically and at nanomolar affinities to HER2 (3,4) whose overexpression is associated with breast cancer and gastroesophageal cancer (5). For this project, we worked with SK-BR-3 breast adenocarcinoma cells which are overexpressing this receptor.
The cells were first incubated with the Encapsulin-DARPin929-miniSOG drug delivery construct for 30 min, then they were illuminated with white light at 40 lumens/cm2 for 30 mins in order to activate the cytotoxic effect of the encapsulated miniSOG at 37 °C and 20% oxygen. At the end of the experiment, the cells were visualised with EVOS FL microscope to observe uptake of the encapsulins. Following that, all the cell samples were stained using the Annexin V – Propidium Iodide staining kit for determination of potential cell death and percentage loss in viability using flow cytometry. Moreover, we wanted to examine the specificity of the cytotoxic effect thus we replicated the experimental set-up and conditions with Mesenchymal Stem Cells (MSCs), which do not possess the HER2 receptor. We also included two controls which comprised of the SK-BR-3 and MSCs alone.
From the confocal microscopy imaging in Figure 5, we could see that after 1-hour incubation green fluorescence from miniSOG was detected in both samples. Fluorescence was not expected for the MSCs, since from our previous experiments with DARPins we have not observed any unspecific binding. We hypothesise that the concentration of construct that we loaded was too high, thus non-specific passive uptake was initiated. However, since significantly more fluorescence was observed in SK-BR-3 cells, we can deem that the system is indeed selective.
The plots in Figure 6 are separated in 4 quadrants over which the cell population is distributed. The upper left quadrant indicated the percentage of necrotic cells, the upper right the percentage of late apoptotic cells, the lower left shows the percentage of viable cells and the lower right the percentage of early apoptotic cells. Cell controls in Figure 6 (a) and (d) helped to define the normal boundaries for their respective cell type
From Figure 6 (b) we could observe a shift in cell population majorly towards the lower right quadrant indicating early apoptosis of the cell before illumination compared to our control cell population in 6 (a). While this was not expected, we speculate that light falling on the well plates during sample processing could have induced miniSOG, thus resulting in a loss of cell viability. MSCs however presented only a minimal percentage of early apoptotic cells. After illumination, we observed a considerable shift towards the upper and lower right quadrants in Figure 6 (c), indicating a high rate of apoptosis with a total of around 80% reduction in cell viability. The loss of viability in MSC through early apoptosis observed in Figure 6 (d, e and f) could be associated to the greater sensibility of stem cells to environmental condition fluctuation (in this instance, strong illumination) or the handling of the cells required for imaging and staining.
Conclusion
To conclude, these experiments show that our drug delivery platform successfully targets HER2 overexpressing SK-BR-3 cells and kills them at a significantly higher degree than MSCs once illuminated with light, thus demonstrating double selectivity in terms of cytotoxicity. We believe this has proven the concept of Encapsulin-DARPin drug delivery platform, and it can be further adapted upon by using different encapsulins, DARPins and cargo.
References
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