Photocontrolled, biologically active compounds contain reversibly photoisomerizable fragments, photoswitches in their molecules. Compounds are cyclic peptides modified by a diarylethene fragment that undergoes reversible light-induced electrocyclic transformations. They can photoswitch, that is, toggle between a UV-generated, biologically inactive form and a red light-generated, biologically active form.
It is believed that photoswitchable drugs are safer than non-photoswitchable drugs. This is because you can administer a nontoxic, deactivated form to the human body and locally activate it with light only where you need it, which is, for example, tumors, augers, or wounds. There are no standard methods or protocols for the preclinical or clinical development of such photopharmacology drugs, so here, we want to show you experiments which can be used for this early preclinical research.
Kateryna Horbatok and Tetiana Makhnii will demonstrate the procedures. The animal care and experimental procedures were approved by the Bioethics Commission of the Bienta Company. Begin by preparing the buffers using standard procedures.
Alternatively, use commercially available solutions. Prepare stock solutions of compounds. For each compound, weigh two batches of 5.12 milligrams of the compound in the ring-closed photoform into two 1.5-milliliter Eppendorf tubes, one with clear and one with black, nontransparent walls.
As a positive control, weigh 2.28 milligrams of the parent, not photocontrolled, peptide, in an extra tube. Add 100 microliters of pure DMSO to each sample, and vortex for 30 seconds. Photoisomerize the stock solution in the clear-walled tube by irradiating the solution with a 650-nanometer laser, light power density 6 watts per centimeter square, with vortexing to ensure mixing.
Continue until the color changes from dark purple to light brown. Protect from light with aluminum foil. Seed 5, 000 to 10, 000 cells per well.
We used circa 8, 000 Lewis lung carcinoma cells in 200 microliters of DMEM in the central 60 wells of a 96-well sterile plate with a clear bottom and black, nontransparent walls. Fill the remaining 36 wells with pure DMEM. Incubate the plates at 37 degrees Celsius in a 5%CO2 atmosphere overnight.
The next day, prepare serial dilutions of compounds and the positive control in polypropylene autoclaved clear plates. Start with the stocks in DMSO, and dilute with DMEM, but do not exceed 1%volume DMSO in the final highest concentration. To prevent uncontrolled photoisomerization of the studied compounds, switch off the lights in the sterile cabinet.
Transfer 100 microliters of the dilutions into 56 wells with 100 microliters of previously added DMEM to reach the required final concentration of the compounds in the wells, typically within the five to 150 micromolar range. Next, add 100 microliters of DMEM to four wells, which serve as a negative control. Cover the plates with aluminum foil or a plastic, nontransparent cover to prevent uncontrolled photoswitching.
Place the plates in a cell culture incubator at 37 degrees Celsius for the chosen incubation time. After the incubations, add 50 microliters of staining solution per well to the plates. Use five-micromolar Hoechst 33342 and one-micromolar propidium iodide as the final staining solutions for this experiment.
Incubate again at 37 degrees Celsius for 20 minutes. Perform automated fluorescence imaging using a 20X magnification objective lens. The laboratory operations for this part of the method are identical to those described for the 2D experiment:preparation of the cell culture, incubation with the tested compounds, and imaging.
However, in this case, the cells are prepared as compact, mature spheroids in a 384-well, ultra-low-adhesion, U-bottom plate with black, nontransparent walls. Using a plate of this size allows for two compounds to be compared in one experiment. In this experiment, we used in addition calcein AM as a third component of the multicolor staining solution.
Images obtained from both the 2D and 3D experiments are analyzed using the instruments'automated image analysis software. Cells costained with Hoechst and propidium iodide dyes are considered necrotically dead, and their fraction as a function of the concentration is used to calculate the IC50 value. Assemble the optical train for the sample irradiation.
It consists of an optical cable from the laser light source, a lens with a variable focal length, a syringe with a nontransparent cover, and a flat cut end. All subsequent operations must be performed in a darkened room with minimum workplace illumination. Prepare a model tissue sample loaded with the inactive photoformal compounds.
In a typical run, five grams of fresh minced pork meat is mechanically mixed with the PBS solution of the compound to reach the final concentration of 50 milligrams per kilogram. Fill the syringe with the loaded sample. Irradiate the sample in the optical train for the required exposure and light dosage time.
After the exposure, make four millimeter-thick slices of the sample by pushing the mass off the syringe with the piston and cutting it with a scalpel. Weigh and extract the compound with an acetonitrile, water, TFA mixture, 70%acetonitrile and 01%trifluoroacetic acid in a 1.5-milliliter Eppendorf tube. Centrifuge at 20XG for 30 minutes two times to remove the insoluble material and collect the supernatant.
Record UV-detected chromatograms at 570-nanometer detection of their enclosed form and 270-nanometer detection of the ring-open form. Use the nonirradiated and irradiated control samples prepared similarly to the stock solutions in the cell experiments to determine the specific retention times and calibrate the method. Repeat the experiment three times, and plot the normalized percent of each photoform on the plot, percentage versus distance from the irradiated tissue surface.
At day zero, inoculate the C57 black 6 mice adult females weighing approximately 20 grams each subcutaneously with a suspension of circa half a million Lewis lung carcinoma cells in approximately 100 microliters of DMEM and Matrigel mix in the right hind leg. This procedure is done under 5%isoflurane sedation. The animals are ready for the treatment on days five to eight, when the tumors are palpable.
All operations involving being tested should be performed under semi-dark conditions. This condition also holds when treating the animals after receiving the compound dose for a period of two days. Assemble randomly four groups of eight animals, and remove the fur from the tumor area.
The two control groups receive an intravenous injection of the vehicle. 100 microliters saline per 20-gram animal, and the animals in the two experimental groups received the tested compound in the inactive photoform in a solution of one milligram per milliliter saline. Administer the compound bolus into the tail vein at five milliliters per kilo.
Two hours, 45 minutes after the compound has been injected, irradiate the tumor area for 20 minutes under isoflurane anesthesia with a 650-nanometer laser, using a light power density of 100 milliwatts per square centimeter. Carefully observe the animals'conditions over the next 30 minutes. Observe the animals daily, and measure their weight and the dimensions of their tumors.
Measure the tumor volumes, and note the progression of the necrosis. Determine the survival rate using the standard procedure. The results of our protocol for step one in the case of 2D cell culture experiments can be presented as images, as illustrated here on a representative image for Lewis lung carcinoma cells incubated with LMB002 at different concentrations and for different periods of the incubation.
Costaining with Hoechst and propidium iodide allows for the visualization of cell nuclei in the blue channel, thereby giving a total cell count. Cells with compromised integrity of the plasma membrane were observed in the red channel. Assuming the latter to be necrotically dead, the apparent cytotoxicity of a compound under study can be quantified as a percentage of the propidium iodide-positive cells.
The results of the data quantification obtained through automated image analysis are shown here. For LMB002, sigmoidal dependencies of the percentage of propidium iodide-positive cells in the compound concentration can be seen. From these data, the IC50 values can be determined.
Our experiment revealed that the open form of LMB002 is about one dilution step less toxic than the prototype peptide, Gramicidin S, whereas the closed form demonstrates three to four dilution steps lower toxicity, which increases with incubation time. The 3D cell experiments produced the same type of raw data, the single-cell resolved one-per-wall spheroid images. The inclusion of calcein as a third staining dye enables the quantification of the fraction of metabolically active cells observed in the green channel.
The dose effect curves, like those in the 2D experiment, were obtained from the Z-stack piles of images. Panel A illustrates such curves, corroborating the 2D results. As shown in panel B, the overall spheroid diameter varies with compound concentration.
The experiment for step two allows for the determination of LMB002 concentrations in both photoforms by using UV-detected, high-performance liquid chromatography. Both photoforms were sufficiently different in retention times and absorbance, shown integrated on the panels A and B of the figure. The obtained data are summarized in the figure, confirming that our red light source induces the ring-closed LMB002 photoconversion at a depth of up to one centimeter in the tissue surrogate, minced meat at approximately 103 milliwatts per square centimeter.
The results of the in vivo experiment, step three of our methodology, were represented by graphs showing tumor growth as a function of time in Kaplan-Meier survival curves. The strategy represented here for evaluating photoswitchable drug candidates is practical and suitable for compounds with diarylethene-type photoswitches. Step one, cytotoxicity quantification can be used for the initial screening of the libraries of the photocontrolled compounds.
Step two, photoswitching efficiency evaluation is both easy to set up and ethical. It does not require living animals. Step three, in vivo photopharmacology models, the therapy can be applied to small animals.
