Module 3: Anticancer drug in situ production
In our project, we encapsulated Tegafur in PURE system-containing liposomes. Through the action of the enzyme purine/pyrimidine nucleoside phosphorylase (Ppnp), Tegafur is converted into the drug 5-Fluorouracile (5-FU). We studied by HPLC the stability of these two compounds in PURE system, the effect of Tegafur on PURE system activity, and the activity of Ppnp. Additionally, we examined the toxicity of 5-FU on cultured cancer cells (see Protocols page).
Characterization of 5-FU and Tegafur
We first set out to construct a calibration curve of Tegafur in water. In our conditions, the retention time of Tegafur was between 19 and 20 minutes (Fig. 1). The peak area was calculated for various known concentrations of Tegafur and a linear regression was computed, with equation: Area = 9.056 [Tegafur] and the concentration in micromolar.
Figure 1: Chromatogram of various concentrations of Tegafur detected by HPLC.
Figure 2: Calibration curve of Tegafur in a range of concentrations from 0 to 100 µM.
The retention time of 5-FU was around 4.3 minutes (Fig. 3). Following the same procedure as described for Tegafur, we generated a calibration curve of 5-FU in water (Fig. 4), with an equation: Area = 8.66 [5-FU] and the concentration in micromolar.
Figure 3: Chromatogram of various concentrations of 5-FU detected by HPLC.
Figure 4: Calibration curve of 5-FU in a range of concentrations from 0 to 153.8 µM.
We concluded that both 5-FU and Tegafur can be detected and quantified by HPLC. Moreover, unknown concentrations of Tegafur and 5-FU were determined from measured peak areas using the equations above.
We then assessed if it is possible to detect both compounds in the same solution. A mix of 5-FU and Tegafur was prepared in water at the respective final concentration of 7.69 µM and 5 µM. A first injection was done at time zero (t-0) and a second injection of the same mix three days later (t-3 days) during which the solution was incubated at 35°C. Figure 5 shows that both 5-FU and Tegafur can easily be detected from the same solution.
Figure 5: Chromatogram of the mix containing 5-FU and Tegafur in water on Day 0.
We used the calibration curves described previously to quantify their amounts. The concentration of each compound into the mix was in very good agreement with the theoretical values (Table 1).
Table 1: Comparison of theoretical versus estimated concentrations by HPLC on Day 0.
Next step was to verify if the compounds in solution are stable over time. Both 5-FU and Tegafur could be detected after 3 days of incubation (Fig. 6). The measured concentrations were in agreement with the initial ones (Table 2), indicating that the two compounds are stable in water for at least 3 days.
Figure 6: Chromatogram of the mix containing 5-FU and Tegafur in water on Day 3.
Table 2: Comparison of theoretical versus estimated concentrations by HPLC on Day 3.
Next, we asked whether 5-FU and Tegafur could be detected in PURE system. Indeed, the rich molecular composition of PURE system may hinder the detection of these two compounds. After measuring the chromatogram of PURE system alone, we added either Tegafur or 5-FU. For both molecules, we could identify a signature peak at similar retention times as in water (Figures 7 and 9). The peak amplitudes are relatively low compared to that of more concentrated components in PURE system.
Figure 7: Detection of Tegafur in PURE system by HPLC.
Figure 8: Identification of the Tegafur peak in PURE system after zooming in.
Figure 9: Detection of 5-FU in PURE system (PUREfrex) solution by HPLC.
Figure 10: Identification of the pic of 5-FU peak detected in PURE system after zooming in.
We then quantified the concentrations in PURE system of Tegafur and 5-FU using the calibration curves obtained in water. A very good agreement with the expected concentrations was found, validating our approach for quantitation also in PURE system background.
Table 3: Comparison between theoretical and measured concentrations of Tegafur and 5-FU in PURE system by HPLC.
Figure 11 shows the detection of both 5-FU and Tegafur signals after 3-day incubation in PURE system at 35°C.
Figure 11: Chromatogram of the PURE system solution containing 5-FU and Tegafur on after 3 days incubation. As a reference, the chromatogram of PURE system only is displayed in the bottom panel.
Table 4: comparison of theoretical concentrations to estimated concentrations by HPLC on Day 3:
The results confirm the good agreement between the expected and measured concentrations, even after three days of incubation in PURE system.
We then wonder if the PURE system efficiency could be impacted by the concentration of our prodrug. The sfGFP protein was produced using PURE system with three different concentrations of Tegafur (0, 125 and 1000 µM ; Fig.12).
Figure 12: Effect of high concentration of Tegafur on PURE system activity. GFP reporter gene was used.
We observed that Tegafur can impair the efficiency of the PURE system down to half its capacity with 1000 µM tegafur. This information will be crucial in determining the final concentration of encapsulated Tegafur in liposomes for the commercial formulation.
Characterization of Ppnp
We attempted to produce E. coli purine/pyrimidine nucleoside phosphorylase (PpnP) in two different PURE system kits with different protein folding properties (PUREfrex2.0 and PUREfrex2.1). Tegafur at a final concentration of 119 µM and the gene encoding PpnP under T7 polymerase control were added in each sample. Tegafur and 5-FU concentrations were analyzed by HPLC at time zero and after 12 hours incubation at 37°C (Table 5)
Table 5: Estimated concentrations of Tegafur and 5-FU at time zero (T-0h) and after 12 hours (T-12h) of incubation at 37°C under different conditions (as indicated above).
After 12 hours of incubation, we expected to observe the enzymatic conversion of Tegafur into 5-FU by Ppnp. Unfortunately, the peak of Tegafur did not decrease and no additional peak of 5-FU appeared. We can conclude either that the produced enzyme was not active hence Tegafur was not converted into 5-FU, or that the amount of synthesized enzyme was too low to lead to detectable changes in Tegafur and 5-FU concentrations. We recommend doing some sequence optimization of the ppnP gene to increase expression levels and to supplement the PURE reaction with chaperones to enhance protein folding.
To simulate the enzymatic conversion of Tegafur in 5-FU and validate our approach for detecting changing amounts of the two compounds, we mixed 5-FU and Tegafur at different ratios (0/1; 0.2/0.8 0.5/0.5; 0.8/0.2 and 1/0, v/v), from an equimolar stock concentration of 500 µM. The expected change of the measured levels of Tegafur and 5-FU was demonstrated qualitatively (Fig. 13) and quantitatively (Fig. 14), which validated our analytical method to detect the activity of the PpnP enzyme.
Figure 13: Offset chromatograms of various 5-FU/Tegafur ratios to simulate the enzymatic conversion of Tegafur into 5-FU, which leads to varying amounts of the two compounds.
Figure 14: Quantitative analysis of the data shown in Figure 13. Decreasing amounts of Tegafur are correlated with increasing amounts of 5-FU.
5-FU cytotoxicity against cancer cells
Toxicity of 5-FU on Caco2 cells was investigated using an MTT assay (See our protocol page). We evaluated cell viability through cleavage of the tetrazolium ring of MTT by dehydrogenases in active mitochondria.
Figure 15: MTT assay shows cell survival decrease as 5-FU concentration increases.
Survival rate decreased with 5-FU concentration, reaching 50% of mortality at 200 μM. This test validated the toxicity of the chosen anticancer drug. In future experiments, we propose to carry out the same test with the anti-cancer agent encapsulated in liposomes in order to examine the influence of its molecular diffusion across the membrane.
In summary, our toxicity results confirm the adverse effect of 5-FU on cancer cells. Further experiments should focus on the permeability of Tegafur and 5-FU across the liposome membrane to assess their lifetime in the vesicle interior and diffusion rate into the tumor environment.
This study has established HPLC as a suitable analytical method to quantitatively detect Tegafur and 5-FU in PURE system. The stability of Tegafur in PURE system was demonstrated for at least three days, which is compatible with the expected treatment duration. It is important to note that Tegafur can reduce the efficiency of PURE system at high concentrations, which should be taken into account when defining the optimal amount of Tegafur to be encapsulated in each liposome.
Unfortunately, we did not observe the conversion of Tegafur into 5-FU by the Ppnp enzyme. Our recommendations for future experiments include the sequence optimization of the ppnP gene to increase expression levels, as well as the addition of chaperones to facilitate protein folding in PURE system. Due to the short time, we have preferred to focus on the biosensing module as it was the most challenging and central part of our project.
Considering the promising results we obtained throughout the three different modules we still wanted to advance our project realization as far as possible. We decided to integrate the three modules—anchoring, biosensing, and expression of a reporter gene—into a single liposomal platform targeting tumor cells. The experimental results obtained with this new liposomal platform led us to design a novel measurement method for assessing the interaction between liposomes containing the PURE system and living cancer cells. To discover the measurement system and the assays we performed, please consult our Best measurement page.
