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.

<ol>
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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>

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

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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2.0K Views.  Taras Shevchenko National University of Kyiv. 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.

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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.

Synthesis, Cellular Delivery and In vivo Application of Dendrimer-based pH Sensors

Fluorescence sensors are powerful tools in life science. Here we describe a methodology to synthesize and use dendrimer-based fluorescent sensors to measure pH in living cells and in vivo. The dendritic scaffold enhances the properties of conjugated fluorescent dyes leading to improved sensing properties.

The development of  fluorescent indicators represented a revolution for life sciences. Genetically  encoded and synthetic fluorophores with sensing ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The composition of heavy crude oils and shale oils varies substantially depending on the origin of the mixture. In particular they contain an increased amount of nitrogen containing compounds compared to the conventionally used sweet crude oils. As nitrogen compounds have an influence on the operation of thermal processes occurring in coker units and steam crackers, and as some species are considered as environmentally hazardous, a detailed analysis of the reactions involving nitrogen containing compounds under pyrolysis conditions provides valuable information. Therefore a novel method has been developed and validated with a feedstock containing a high nitrogen content, i.e., a shale oil. First, the feed was characterized offline by comprehensive two-dimensional gas chromatography (GC &#215; GC) coupled with a nitrogen chemiluminescence detector (NCD). In a second step the on-line analysis method was developed and tested on a steam cracking pilot plant by feeding pyridine dissolved in heptane. The former being a representative compound for one of the most abundant classes of compounds present in shale oil. The composition of the reactor effluent was determined via an in-house developed automated sampling system followed by immediate injection of the sample on a GC &#215; GC coupled with a time-of-flight mass spectrometer (TOF-MS), flame ionization detector (FID) and NCD. A novel method for quantitative analysis of nitrogen containing compounds using NCD and 2-chloropyridine as an internal standard has been developed and demonstrated.

A method combining comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detection has been developed and applied to on-line analysis of nitrogen containing compounds in a complex hydrocarbon matrix.

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The ...

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA &#8211; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance ...

Bio-inspired Polydopamine Surface Modification of Nanodiamonds and Its Reduction of Silver Nanoparticles

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a simple protocol for the multifunctional surface modification of NDs by using mussel-inspired polydopamine (PDA) coating. In addition, the functional layer of PDA on NDs could serve as a reducing agent to synthesize and stabilize metal nanoparticles. Dopamine (DA) can self-polymerize and spontaneously form PDA layers on ND surfaces if the NDs and dopamine are simply mixed together. The thickness of a PDA layer is controlled by varying the concentration of DA. A typical result shows that a thickness of ~5 to ~15 nm of the PDA layer can be reached by adding 50 to 100 &#181;g/mL of DA to 100 nm ND suspensions. Furthermore, the PDA-NDs are used as a substrate to reduce metal ions, such as Ag[(NH3)2]+, to silver nanoparticles (AgNPs). The sizes of the AgNPs rely on the initial concentrations of Ag[(NH3)2]+. Along with an increase in the concentration of Ag[(NH3)2]+, the number of NPs increases, as well as the diameters of the NPs. In summary, this study not only presents a facile method for modifying the surfaces of NDs with PDA, but also demonstrates the enhanced functionality of NDs by anchoring various species of interest (such as AgNPs) for advanced applications.

A facile protocol is presented to functionalize the surfaces of nanodiamonds with polydopamine.

Surface functionalization of nanodiamonds (NDs) is still challenging due to the diversity of functional groups on the ND surfaces. Here, we demonstrate a ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...