Name: in vitro and in vivo evaluation of photocontrolled biologically active compounds - potential drug candidates for cancer photopharmacology
Uploaded: 2023-09-29T13:10:00
Duration: 13 min 17 s
Description: Watch this Scientific Journal Video about In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology at JoVE.com

The authors acknowledge EU funding by the H2020-MSCA-RISE program through PELICO (#690973) and ALISE (#101007256) projects. This work was supported by the DFG-GRK 2039 (SA, TS and ASU), the NACIP program of the Helmholtz Society (SA and ASU), and the VIP+ of the BMBF (OB and ASU). We acknowledge Dr. Serhii Koniev, Karlsruhe Institute of Technology, who has synthesized compound LMB002, purified it and kindly provided the compound for the study. The authors also thankful to Chupryna Maksym who filmed and compiled the video in Ukraine, and to all brave defenders of Ukraine that made the experimental work, writing, and filming this publication possible.

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IVK, OB, SA, and ASU are inventors on the issued patent family: "Peptidomimetics possessing photocontrolled biological activity" (WO2014127919 [A1], EP2958934 [B1], US9481712 [B2], UA113685 [C2]) licensed to Lumobiotics GmbH. IVK, OB, TS, and SA are founders and shareholders of Lumobiotics GmbH. IVK is a scientific advisor, HK, TM, IP, and PB are employees of Enamine LLC. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the publication apart from those disclosed.

Photocontrolled compounds are unprecedented in drug development; however, no methods have been established for their preclinical and clinical evaluation. The closest monotherapy analog, photodynamic therapy (PDT), is the treatment modality for clinical use adopted by many countries against cancer and is in the development for other indications19,20. Similar to photopharmacology, PDT is also based on the use of light to activate the bioactive substance (singlet oxygen). Therefore, some experimental methods used for preclinical and clinical studies in PDT can be adopted for photopharmacology. For example, light sources, light delivery approaches, and medical devices are well-developed and approved for PDT; they can be directly used for the evaluation of photocontrolled drugs. However, PDT and photopharmacology have many distinctions from one another4, which justifies the need to establish specific methods for the latter.
First, the non-activated substance in PDT (oxygen) is always present in living tissues at&#160;nontoxic concentrations. By contrast, non-activated photocontrolled biologically active compounds can have residual activity and unwanted toxicity. Therefore, ideal photopharmacology drugs should have minimized biological activity in their administered form and must be highly active in their light-generated form, the &#34;phototherapeutic window&#34;21 must be as large as possible. Finding the hit and performing hit-to-lead optimization requires the identification of suitable compounds and the screening of relatively large libraries, already at early stages of drug development. Here, we proposed an automated high-throughput confocal fluorescent microscopy to identify efficient photoswitching compounds.
The chosen method of cytotoxicity evaluation allows easy implementation of the most critical requirement - maintenance of the PSS or stability of the visible-light-sensitive photoisomer. This is because, upon its implementation, the light exposure is minimized. Hence, if selecting alternative methods, automated ones should be preferred. This approach is reliable and informative. The use of 3D cell cultures (spheroids) at this stage provides a holistic understanding of the cell&#39;s response to the treatment in a more realistic tissue-like microenvironment. In addition, valuable insights into the action mechanism of the compounds can be obtained using microscopy as the direct method. The confocal fluorescent microscopy with proper staining protocol allows for the visual assessment of the morphology of the cells and spheroids; important details on the cell death and changes inside the cells can also be detected.
Second, light application requires a careful choice of light dosage. In PDT, light overdose is extremely harmful to tissues22. Photopharmacological therapy can be advantageous under excessive light irradiation. The upper limit of the activated substance is defined by the administered dose of the non-activated substance and its pharmacokinetics. However, light dosage is still an issue in photopharmacology. Care should be taken to ensure that the irradiating power density and exposure time are not less than the requirement for the therapy. In principle, the generation of the activated substance can be monitored in vivo. However, for bioethics reasons, we proposed an experiment with a model tissue (fresh minced meat) mixed with the non-activated compound15. This experiment is simple and can be modified to use different light sources. It can also be adapted for the photophysical estimation of light dosage and the measurement of thermal influences. Here again, by using model tissues, the light exposure is possible to minimize, compared, for example, to the more accurate photoswitching efficiency determination in the in vivo conditions, an alternative that may always be interesting to consider.
Finally, the compounds that demonstrate superior characteristics in the in vitro toxicity screens and are efficiently photoswitching at least 1-1.5 cm deep in the model tissue can be selected for costly, laborious, and lengthy in vivo studies. In this protocol, we used the same cell line (LLC) as in the in vitro assessment to generate the allograft cancer model. The tumor growth dynamics, mortality, and metastasis count are the parameters most suitable for assessing anticancer efficacy. Compared with conventional chemotherapy, an additional factor is applied in the photopharmacological treatment - the light. Therefore, two control animal groups are needed: one that receives only the vehicle and the other that receives the vehicle and irradiation. This setup enables the evaluation of the impact of light on the measured parameters. In our experiment, the animals of the two experimental groups received the non-activated compound, and the tumors of the mice in one group were irradiated. The irradiation regime was identical for the control and treatment groups. Comparison with benchmark chemotherapy is not necessary at this stage because the main purpose of the experiment is to demonstrate the combined effect of light and compound application. The best-performing compounds exhibiting this effect can then be selected for further study on their in vivo toxicity and comparison with benchmarks for making important go-no-go decisions on their development. Technically, the in vivo experiment that we describe can be easily adapted to pharmacokinetic or pharmacodynamics studies, for example, of a compound that is already selected as the drug lead.

Photocontrolled biologically active compounds have emerged in recent decades as a promising component of safe chemotherapies for human diseases and to specifically eradicate malignant solid tumors1. These compounds contain reversibly photoisomerizable fragments (molecular photoswitches) and can toggle between inactive and active photoisomers upon irradiation with light of different wavelengths.
Compared with their non-photocontrollable analogs, photocontrolled drugs may be safer because they can be systemically introduced into the patient&#39;s body in less active and essentially nontoxic forms, and are then activated by light only where necessary, such as in tumors, ulcers, and wounds. Although multiple exciting demonstrations of such molecular drug prototypes can be found in recent academic papers2,3,4,5,6,7, the field of clinical photopharmacology - an application of approved drug/medical device/disease combinations - does not exist. Photopharmacology is yet in the drug discovery stage, and systematic preclinical studies are unknown.
We only very recently demonstrated the in vivo safety advantage for some photocontrolled anticancer peptides, namely, the analogs of the peptide antibiotic gramicidin S8. These photocontrolled derivatives contain a diarylethene (DAE) photoswitch, which undergoes reversible photoinduced transformations between the so-called red light-generated &#34;ring-open&#34; and UV-generated &#34;ring-closed&#34; photoforms (illustrated in Figure 1 for one of the derivatives, compound LMB002).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64902/64902fig01.jpg" /> 
Figure 1: Photocontrolled cytotoxic peptide LMB002 and its photoisomerization. The diarylethene fragment is shown in red. Abbreviation: DAE = diarylethene. <a href="https://www.jove.com/files/ftp_upload/64902/64902fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Finding hits and performing hit-to-lead optimization often require in vitro and in vivo screening of appropriate compound libraries9,10. Here, we demonstrate a methodology suitable for the systematic high-throughput screening of cytotoxicity of photocontrolled compounds. We also determine the photoisomerization efficiency, estimate the light dose in model tissues, and evaluate the in vivo efficacy of the best-performing candidates. The approach is compliant with bioethics and animal care considerations.
In this work, traditional preclinical methods are modified to avoid the noncontrolled photoisomerization of tested compounds. The overall goal of applying these modified methods herein is to develop a general strategy that is straightforward and fast and yields statistically significant data to reliably compare in vitro activities and rationalize the in vivo efficacy testing of photoswitchable compounds for lead identification and further development.
The strategy consists of three consecutive steps. The first step involves the determination of IC50 (apparent 50% cell viability) in serial dilutions for the active and inactive photoforms of selected photocontrolled biologically active compounds using two-dimensional (2D, monolayer) and three-dimensional (3D, spheroid) cell cultures and confocal high-throughput automated fluorescence microscopy. Phototherapeutic windows are compared with respect to the IC50 difference between the two photoforms, and the best-performing candidates are selected. There is no specific advantage in toxicity assessment by automated microscopy and other cytotoxicity screening platforms (assays)11; more complex cell-based tumor models12 could be easily implemented at this stage.
For the compounds selected in step 1, the second step is to realistically estimate their photoswitching efficiency inside the tissues as a function of depth from the irradiated tissue surface by quantifying the photoswitching efficiency of the less active photoforms in a tissue surrogate using UV-detected high-performance liquid chromatography (HPLC) of irradiated sample extracts. In vivo, photoswitching efficiency could be studied, but we propose to use a simple tissue surrogate - minced pork meat. We have tested the validity of this approach. We measured the conversion of our photoswitchable compounds in vivo on a mice cancer model and observed approximately the same photoconversion at a depth measured in previous experiments with mice8. Any suitable alternative artificial tissue13, 3D bioprinted tissue/organ14, biopsy materials, or another exempt animal material could be used. However, this setup is a good compromise as it is economical, fast, and ethical.
The third step is the determination of in vivo anticancer efficacy in a murine cancer model. The compounds demonstrating superior characteristics in the in vitro experiments and efficiently photoswitching at a depth of at least 1-1.5 cm in the model tissues are selected for this experiment.
This protocol can be applied to compounds possessing different types of photoswitches, provided their photoforms (or their photostationary states, PSS) are stable for a reasonable time (a few days or longer). For illustration, a previously described DAE-derived LMB002 is used15. The LMB002 photoforms are thermally stable and can be stored at &#8722;20 &#176;C for at least a year without substantial degradation. Lewis lung carcinoma (LLC) cells are chosen for this in vitro and in vivo demonstration, but no restrictions are imposed on the cell type. LLC cells are adherent, readily culturable in 3D, and used to generate tumoroids (as described in the reference16). In vivo LLC cells are used to model metastatic processes and can readily generate solid tumors in immunocompetent mice after subcutaneous injection. This in vivo methodology can be universally applied to other cancer models17,18. The detailed implementation of this strategy is described below.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agilent 1100 Series capillary LC system</td>
			<td>ALSI-Chrom (Agilent distributor)</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>ATCC CRL-1642, LL/2 (LLC1) Lewis lung carcinoma cell line</td>
			<td>ECACC</td>
			<td>90020104</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6NCrl mice, female, inbred</td>
			<td>Charles River</td>
			<td>Strain code: 027</td>
			<td></td>
		</tr>
		<tr>
			<td>CelCulture, CO2 incubator</td>
			<td>Esco Micro</td>
			<td>CCL-170B</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel Basement membrane matrix</td>
			<td>Merck</td>
			<td>CLS354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning, 384- well spheroid microplates</td>
			<td>Merck</td>
			<td>CLS3830</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Merck</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco, DPBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>21600044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gramicidin S</td>
			<td>Lumobiotics</td>
			<td></td>
			<td>Custom synthesis</td>
		</tr>
		<tr>
			<td>HyClone, DMEM/high glucose</td>
			<td>Cytiva</td>
			<td>SH30003.04</td>
			<td></td>
		</tr>
		<tr>
			<td>IN Cell Analyzer 6500HS, imaging system</td>
			<td>Cytiva</td>
			<td>29240358</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen, Calcein AM</td>
			<td>Thermo Fisher Scientific</td>
			<td>C1430</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane anesthesia machine</td>
			<td>ASA</td>
			<td>S/N ASA 1305</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine, 200 mM solution</td>
			<td>Merck</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>LIKA-surgeon, diode surgery laser</td>
			<td>Fotonika plus</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>LMB002</td>
			<td>Lumobiotics</td>
			<td></td>
			<td>Custom synthesis</td>
		</tr>
		<tr>
			<td>Penicillin&#8211;Streptomycin, solution stabilized</td>
			<td>Merck</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>PhenoPlate, 96-well plates</td>
			<td>PerkinElmer</td>
			<td>6055302</td>
			<td></td>
		</tr>
		<tr>
			<td>Photometer PCE-LED 20</td>
			<td>PCE Instruments</td>
			<td>PCE-LED 20</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific, Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific, Propidium iodide</td>
			<td>Thermo Fisher Scientific</td>
			<td>J66764-MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4% solution</td>
			<td>Merck</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#8211;EDTA, 10 x solution</td>
			<td>Merck</td>
			<td>T4174</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraCruz Cell culture flasks with vented caps, 75 cm2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-200263</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraCruz, bottle top filters, PES, 0.22 &#956;m</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-360882</td>
			<td></td>
		</tr>
		<tr>
			<td>Vydac 218TP, C18 HPLC column (4.6 mm &#215; 250 mm, 5 &#181;m)</td>
			<td>Altmann Analytik (Avantor distributor)</td>
			<td>GR5103827</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro and in vivo evaluation of photocontrolled biologically active compounds - potential drug candidates for cancer photopharmacology

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic chemotherapy due to their precise spatiotemporal activation by directing a benign, non-ionizable light to a specific location within the patient&#39;s body. This paper presents a set of methods to evaluate the in vitro potency and ex vivo efficiency of the photoactivation of photocontrolled, biologically active compounds as well as the in vivo efficacy at early stages of drug development. The methodology is applied to anticancer cytotoxic peptides, namely, the diarylethene-containing analogs of a known antibiotic, gramicidin S. The experiments are performed using 2D (adherent cells) and 3D (spheroids) cell cultures of a cancer cell line (Lewis lung carcinoma, LLC), live tissue surrogates (pork meat mince), and an allograft cancer model (subcutaneous LLC) in immunocompetent mice. The selection of the most effective compounds and estimation of realistic phototherapeutic windows are performed via automated fluorescence microscopy. The photoactivation efficiency at varying illumination regimens is determined at different depths in a model tissue, and the optimal light dosage is applied in the final therapeutic in vivo experiment.

In this work, 2D and 3D cell experiments were conducted to determine the IC50 for &#34;ring-closed&#34; and &#34;ring-open&#34; forms of LMB002 (see Figure 1) at different incubation times. These values were compared with those obtained for the prototype peptide, gramicidin S (used as a positive control). A typical set of images of the incubation in 2D-grown LLC culture after staining is shown in Figure 5. Co-staining with Hoechst 33342 (blue) and propidium iodide (red) resulting in different shades of purple in a bigger fraction of cells in the case of treatment with &#34;ring-open&#34; form in comparison to &#34;ring-closed&#34; indicates a noticeable difference in cytotoxicity between two forms that can easily be quantified. The demonstrated example of a successful experiment is based on the data collected using the 96-well plate format, where the peptide variants at varying concentrations were added, as shown in Figure 2. Similar data can be acquired with 384-well and high-density plates. However, since per well volumes are reduced, technical and systematic errors and, as a result, the accuracy of the IC50 determination will decrease with increasing the well density.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64902/64902fig05.jpg" /> 
Figure 5: Representative images from the cytotoxicity assay in the monolayer-grown LLC. Cells were stained with Hoechst 33342 (blue) and propidium iodide (red). The times shown: 10 min, 60 min, 24 h, and 72 h are incubation times with compounds. Scale bars = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64902/64902fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The photoconversion of LMB002 by laser light irradiation in a model tissue - fresh pork mince - was determined using a sample composed of minced meat mixed with LMB002 &#34;ring-closed&#34; (inactive) form dissolved in PBS and measuring the conversion of this inactive form to the LMB002 &#34;ring-open&#34; (activated) form in the direction of radiation propagation. The sample was placed in a syringe and irradiated from one side with a flat beam of laser radiation for the exposure time of ~10 min (usually used in in vivo experiments), as shown in Figure 3. After the exposure, the sample cylinder was divided into parts by pressing the syringe piston and cutting the slices of the same height with a scalpel. The concentration of LMB002 &#34;ring-open&#34; in the extracts from the slices was determined using RP HPLC.
Figure 6&#160;illustrates the dose-effect curves Figure 6A-D obtained from data analysis. To identify the percentage of dead cells with nuclear co-staining of Hoechst 33342 and PI dyes, we used a built-in classifier tool that sets numerical thresholds at selected measured parameters to split all the cell counts into several categories. For example, when the red channel (propidium iodide) signal in the control was at the threshold (approximately 110-130 units), the cells could be classified as PI-positive, considered as dead, or PI-negative, considered as unaffected by the compounds. For LMB002, sigmoidal dependences of the percentage of propidium iodide-positive cells on the compound concentration can be seen. From these data, the IC50 values can be determined.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64902/64902fig06.jpg" /> 
Figure 6: Analysis of cytotoxicity in 2D culture. Sigmoid fits as were obtained in the LLC culture for (A) 10 min, (B) 60 min, (C) 24 h, and (D) 72 h-time intervals taken for incubation with compounds. Fitting allows for the accurate determination of IC50 values (not shown). Error bars are SEM. Abbreviations: LLC = Lewis lung carcinoma; PI = propidium iodide. <a href="https://www.jove.com/files/ftp_upload/64902/64902fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Considering the obtained IC50 values, we can conclude that the toxicity of all three compounds increased with the incubation time. Our experiment revealed that &#34;ring-open&#34; form of LMB002 is about one dilution step less toxic than the prototype peptide, gramicidin S. Whereas the &#34;ring-closed&#34; form demonstrates three to four dilution steps lower toxicity, which increases with incubation time. The difference between the two dilution steps is not affected by the increase in incubation time and can be used numerically as an experimentally determined phototherapeutic window6 for comparison with other compounds in a potential library screening. The IC50 value for gramicidin S was set as the reference point to correct experimental errors or differential outputs in biological replicates.
The 3D cell experiments produced the same type of raw data - the single cell-resolved one-per-well 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). By using 384-well plates, increasing the number of technical replicates, excluding redundant co-incubation time points, and changing the dilution fold, we were able to directly compare several compounds in a single test run (using single plate) as illustrated in the plate map in Figure 7.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64902/64902fig07.jpg" /> 
Figure 7: Plate map for the 3D culture experiment with two compounds. Color codes for the compounds and control are indicated. Numbers in wells are concentrations in &#181;M. 10 min, 24 h, and 72 h are incubation times. <a href="https://www.jove.com/files/ftp_upload/64902/64902fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 8&#160;displays the images of selected technical replicates of LLC spheroids grown at a density of 1 spheroid/well in the presence of tested compounds and control spheroids captured after staining.
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64902/64902fig08.jpg" /> 
Figure 8: Representative images from 3D culture cytotoxicity assay. Images show 48-h-old LLC spheroids stained with Hoechst 33342 (blue), calcein AM (green), and propidium iodide (red) after 10 min, 24 h, and 72 h co-incubation with both LMB002 photoforms and gramicidin S. Scale bars = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64902/64902fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Using the instrument software, the dose-effect curves, like those in the 2D experiment, were obtained from the z-stacked piles of images (Figure 9A). In addition, compact and nondeformed spheroids in the 3D cultures could be characterized by the whole-spheroid diameter (Figure 9B). It was also noted that the overall spheroid diameter varies with compound concentration.
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/64902/64902fig09.jpg" /> 
Figure 9: Cytotoxicity evaluation with 3D cultures. (A) Concentration-dependent cytotoxicity fitting curves and (B) concentration-dependent spheroid&#39;s diameter plots obtained in the 3D cultures of LLC co-incubated with gramicidin S for 10 min, 24 h, and 72 h and captured before staining. Error bars are SEM. <a href="https://www.jove.com/files/ftp_upload/64902/64902fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The experiment for Step 2 allows for the determination of LMB002 concentrations in both photoforms by using UV-detected high-performance liquid chromatography. The efficiency of photoconversion in model tissues was easily assessed and quantified using this setup (Figure 3). The data were obtained from the quantitative analysis of the chromatograms of the sample extracts. In these test experiments, LMB002 chromatograms were detected spectroscopically at 270 nm and 570 nm. At 270 nm, many additional signals were observed and attributed to the compounds co-extracted from the model tissue (verified from the control extract without the compound). Both photoforms were sufficiently different in retention times and absorbance. However, the LMB002 &#34;ring-open&#34; signal was baseline-separated from these background signals (see a representative chromatogram in Figure 10A). Therefore, this signal can be integrated without problems. At 570 nm, the chromatograms contained only LMB002 &#34;ring-closed&#34; form signal (Figure 10B). Here, we performed concentration determination using RP HPLC. Nevertheless, even higher accuracy and lower detection limits could be achieved using LC/MS as the analytical method.
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/64902/64902fig10.jpg" /> 
Figure 10: Representative chromatograms of LMB002 extracted from model tissues. (A) Sample at 2 mm from the irradiated surface, recorded at 270 nm (LMB002 &#34;ring-open&#34; form is integrated); (B) sample at 38 mm from the irradiated surface, recorded at 570 nm (the peak of LMB002 &#34;ring-closed&#34; is integrated). Retention time values (indicated) additionally confirmed the compound&#39;s identity. <a href="https://www.jove.com/files/ftp_upload/64902/64902fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The data obtained after integrating the corresponding signals of all the collected samples were used to build the concentration-depth graphs, as shown in Figure 11. On the basis of these graphs, the efficiency of photoconversion at different depths of the model tissue was easily assessed. It confirms that our red light source induces the &#34;ring-closed&#34; LMB002 photoconversion at a depth of up to 1 c, in the tissue surrogate, minced meat (at approximately 103 mW/cm2).
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/64902/64902fig11.jpg" /> 
Figure 11: Photoconversion efficiency evaluation. Concentration (A, mg/kg) of LMB002 &#34;ring-closed&#34; (non-activated, blue dots) and &#34;ring-open&#34; forms (activated, orange dots) at different distances from the irradiated surface of the model tissue (L, mm). <a href="https://www.jove.com/files/ftp_upload/64902/64902fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The results of the in vivo experiment - Step 3 of our methodology performed according to the schedule presented in Figure 4 - were represented by graphs showing tumor growth as a function of time (Figure 12) and Kaplan-Meier survival curves (Figure 13).
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/64902/64902fig12.jpg" /> 
Figure 12: Tumor growth dynamics in animals. Animals treated with LMB002 compared to the vehicle-treated animals (subcutaneous LLC allograft model in C57BL/6NCrl mice, compound dose 7 mg/kg, IV, 2 h 40 min incubation, then irradiation at 650 nm, 100 mW/cm2, 20 min). <a href="https://www.jove.com/files/ftp_upload/64902/64902fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/64902/64902fig13.jpg" /> 
Figure 13: Mortality curves for animals. Animals treated with LMB002 compared to the vehicle-treated animals (subcutaneous LLC allograft model in C57BL/6NCrl mice, compound dose 7 mg/kg, IV, 2 h 40 min incubation, then irradiation at 650 nm, 100 mW/cm2, 20 min). <a href="https://www.jove.com/files/ftp_upload/64902/64902fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

This protocol presents a set of experiments adopted for the evaluation of photoswitchable anticancer peptides, that can be used in the preclinical screening of such compounds. This includes cytotoxicity assessment in 2D and 3D cell cultures, the evaluation of ex vivo (model tissue) photoisomerization efficiency, and in vivo efficacy.

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic ...

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