Best Measurement
This section introduces a novel measurement method designed to evaluate the interaction between liposomes containing PURE system and living cancer cells. We provide comprehensive information to ensure the accessibility and reproducibility of this method for other iGEM teams. This includes a precise description of the protocol to be followed and a demonstration of its utility for the years ahead. Additionally, we outline the necessary controls that should be conducted to validate the measurements.
In the previous sections, we have presented our efforts to (1) functionalize the liposome membrane with targeting molecules, (2) culture cancer cells in the laboratory, (3) regulate cell-free gene expression using an oncometabolite, and (4) encapsulate a prodrug for therapeutic activity (Figure 1).
In this Best Measurement, we took an integrative approach and combined all working modules into one liposomal platform targeting tumor cells. Once the three modules are combined, we will have to assess their individual functionality, i.e, are the liposomes targeting the cancer cells, are they able to sense them, do they produce the drugs and kill cancer cells? However, the complexity of this set-up is prone to other questions, such as the stability of liposomes in the cellular medium, the means to detect liposomes and their interaction with living cells, the efficacy of the therapeutic response, etc.
From our experience, we feel that liposomes will become more and more utilized in the future. The combination of liposomes and synthetic biology as envisioned here provides a new chassis with unforeseen applications for future iGEMers. Therefore, the methodologies described below and the obtained results will be of great interest to the synthetic biology community.
Figure 1: Explanatory diagram of our Best Measurement method.
Our integrated liposome platform contains both anti-HER2 nanobodies (anti-HER2 nb) and folate ligands for specific anchoring to cancer cells, the PURE system components, purified DhdR proteins and Tegafur, respectively at 1.5 µM and 1 mM final concentration, and the DNA template encoding sfGFP under an 2-HG inducible promoter. This last component was chosen to assess the response of liposomes to cancer cells since we failed to express the 5-FU producing enzyme in PURE system (see Module 3: Anticancer drug in situ production).
Protocol:
Many protocols exist to form liposomes. Our goal was to use a method that (i) is compatible with virtually all natural or synthetic lipids for functionalization purposes, (ii) enables PURE system reactions, and (iii) does not require sophisticated equipment like microfluidic. We chose a technique for lipid film rehydration that makes use of lipid-coated beads as a support of the lipid film. Dried lipid-coated beads can be stored and easily distributed across laboratories. Lipid-coated beads were prepared by mixing lipids listed in the In-liposome gfp expression protocol with beads in a round-bottom flask. The solution was subjected to rotary evaporation as shown in Video 1.
Video 1: Rotary evaporation.
A 20 μL PUREfrex2.1 reaction solution was assembled as described in the PURE system protocol and added to the lipid-coated beads for swelling at 4°C. After freeze/thaw cycles, the liposome suspension was handled carefully to prevent liposome breakage.
Two conditions were tested for exposing liposomes to cancer cells. In the first protocol, liposomes were incubated at 37°C for gene expression prior to their functionalization with anti-HER2-nb and injection on top of cancer cells (sample 1). In a second protocol, liposomes pre-coated with anti-HER2-nb were injected in the growth medium on top of Caco-2 cells, where they have been incubated for in situ gene expression (sample 2). This latter protocol more closely mimics the in vivo conditions for drug delivery.
Detailed protocols for Caco-2 cell culture, liposome preparation and reagents mixing are available on our Protocol page. Fluorescence microscopy was used to image living cells, liposomes, and sfGFP expression.
1. Place the sample in an environment controlled imaging chamber set to 37°C during imaging for longer-term cell viability.
2. Find a field of view with living cells (about 50% confluence) using brightfield imaging.
3. Use the fluorescence channel of the lipid dye to localize the liposomes.
4. Open the images or film in a .czi format on ImageJ2 (v2.14.0). Check “Use Virtual Stack”.
5. Split the channels using “Image” → “Color” → “Split Channels”.
6. Adjust the contrast and brightness for each channel using “Image” → “Adjust” → “Brightness/contrast”.
7. Add color using “LUT” → choose the color needed.
8. If necessary, superimpose the channels using “Image” → “Color” → “Merge Channels” and indicate the color corresponding to the channel.
9. Download the image in a .tif format and the video in a gif. format.
Identification of liposomes
Liposomes are generally not visible in brightfield as a simple membrane is not thick enough to induce phase contrast (Fig. 2a). They can be localized using a fluorescent membrane dye as shown in Figure 2b.
Figure 2: Fluorescence microscopy images of anti-HER2-nb- and folate-decorated liposomes on top of Caco-2 cells (sample 1). a) Imaging of Caco-2 cells in brightfield. b) Imaging of liposomes localized with a fluorescent membrane dye.
However, liposomes with a multilamellar structure can be visible in brightfield (Fig. 3a). Multilamellar membranes are thicker and therefore induce a more pronounced phase contrast. To confirm its liposomal nature, it is recommended to acquire fluorescence images using a fluorescent membrane dye (Fig. 3b), as extracellular vesicles or blebs may display similar features.
Figure 3: Fluorescence microscopy images of anti-HER2-nb- and folate-decorated liposomes on top of Caco-2 cells (sample 2). a) Imaging of Caco-2 cells in brightfield. b) Imaging of a multilamellar liposome localized with a fluorescent membrane dye.
Anchoring of liposomes onto Caco-2 cancer cells
Now we could unambiguously detect liposomes, we assessed their capacity to anchor to tumor cells. We reasoned that free liposomes in solution undergo brownian diffusion, whereas attached liposomes are expected to be immobile or slowly diffusing. To discriminate between these two diffusion states, we acquired movies of our samples. Video 2 displays one freely diffusing liposome and one immobile liposome on cancerous cells. Follow-up experiments and statistical analysis with control samples without anti-HER2 nb and folate on the liposome surface will be necessary to ascertain binding specificity. Anyway, this result is very promising as it strongly suggests the possibility to anchor advanced liposomes on tumor cells, which has not been realized so far.
Video 2: film of an anchored liposome and a freely diffusing liposome on Caco-2 cells (sample 1).
In-liposome sfGFP expression
The most challenging assay of this whole project is the biosensing of cancer cells by our liposomes. Since the 2-HG biosensor module is our most advanced system (see Module 2: Biosensing systems), we used DhdR purified protein, the DNA template encoding sfGFP under the 2-HG inducible promoter and Tegafur at 1 mM (see Module 3: Anticancer drug in situ production). Assuming full repression of the sfGFP gene by DhdR under basal conditions (without 2-HG), observation of sfGFP fluorescence can be seen as the manifestation of transcription activation by 2-HG molecules produced by cancer cells. In sample 1, two liposomes were localized using the red fluorescent membrane dye in the field of view shown in Fig. 4b. Very interestingly, one liposome exhibited sfGFP signal (Fig. 4c).
Figure 4: Fluorescence microscopy images of anti-HER2-nb- and folate-decorated liposomes on top of Caco-2 cells (sample 1). a) Imaging of Caco-2 cells in brightfield. b) Imaging of liposomes localized with a fluorescent membrane dye. c) Imaging of liposomes in the GFP channel.
Similar results were obtained with another sample as shown in Figure 5.
Figure 5: Fluorescence microscopy images of anti-HER2-nb- and folate-decorated liposomes on top of Caco-2 cells (sample 2). a) Imaging of Caco-2 cells in brightfield. b) Imaging of a liposome localized with a fluorescent membrane dye. c) Imaging of the same liposome in the GFP channel.
No difference was observed between liposomes pre-incubated at 37°C and those incubated directly on cancer cells.
It is important to note that autofluorescent debris or cells may appear in the GFP channel. However, they are usually not visible in the red fluorescence channel, which allowed us to identify liposomes. Furthermore, when tracking the liposome displayed in Fig. 5, we observed a fast photobleaching of the fluorescence intensity in the GFP channel, which is expected for sfGFP but not for autofluorescent objects (Fig. 6). Altogether, we established several criteria that allowed us to unambiguously identify GFP-expressing liposomes in samples of cultured living cells.
Figure 6: Imaging of photobleaching.
These results are very exciting because they demonstrate that genetically engineered liposomes exhibit activity in cell-compatible conditions. Although several controls remain to be performed to strengthen these findings (e.g., using cells not producing 2-HG or HER2-negative, and liposomes without DNA template) the observed production of sfGFP in some liposomes is an achievement, especially when considering the suboptimal conditions (presence of a partly inhibiting Tegafur concentration, for instance).
Our approach involves a reporter gene such as sfGFP to measure the interaction between PURE system-containing liposomes and living cancer cells. The expression of GFP is conditional to the interaction of the gene regulator 'DhdR' encapsulated within the liposomes with the oncometabolite 2-HG produced by cancer cells. The results have demonstrated GFP expression in liposomes alongside living cell cancer. However, further complementary experiments are required to confirm that the intensity of the measured GFP depends on the concentration of 2-HG. Additionally, our results have also shown the stability of functionalized liposomes in a cellular medium for at least 6 hours. These findings contribute to the emerging utilization of functionalized liposomes for targeting cellular biomarkers. In our study, we functionalized our liposomes with nanobodies and folate to enhance their specificity toward HER2-positive cancer cells. To broaden the scope of applications, we could also imagine decorating liposomes with nanobodies or ligands to target other disease biomarkers.
Despite the frame of the iGEM competition with limited time, competences and manpower, we are proud to report on the feasibility of the method and the wide perspectives offered by what we nick-named “second-generation” or “intelligent” liposomes. This work combining cutting-edge acellular system and synthetic biology is indeed very promising for any situation, where a genetic response is necessary but where GMOs are ill-suited, as it is mostly the case with therapeutic finalities. The fact that the liposomal system seems functional in a semi-complex environment like in culture medium with mammalian cells is a definitive step forward to its use in vivo.
Once we have succeeded in producing the enzyme responsible for drug synthesis in place of sfGFP, we should obtain the ultimate liposomal system that is able to anchor, detect, produce 5-FU, and kill the cancer cells in its vicinity. We are therefore very close to our initial objective to produce toxic compounds where and when it is needed, hence offering a new strategy for a better cancer treatment less prone to side-effects.
While these second-generation liposomes are paving the way to a plethora of applications, they are clearly in their infancy, with plenty of development and optimization opportunities. Anyway, we showed here that using genetically engineered liposomes as a chassis for systems biology applications is not so foolish and we invite future iGEMers to build upon CALIPSO and bring this new technology even further.
In this scope, we did our best to provide ample details about how to set up such experiments, from liposome production, to their manipulation and the subsequent measurements. We envision plenty of exciting research directions, such as high-throughput liposome screening, the discovery of new nanobodies capable of targeting tumors, the production of any organic molecule in a spatially and timely controlled way... the choice is yours!
