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Photodynamic quenched cathepsin activity based probes for cancer detection and macrophage targeted therapy.

Ben-Nun Y, Merquiol E, Brandis A, Turk B, Scherz A, Blum G - Theranostics (2015)

Bottom Line: Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics.Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties.Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS.

View Article: PubMed Central - PubMed

Affiliation: 1. The Institute of Drug Research, The School of Pharmacy, The Faculty of Medicine, Campus Ein Karem, The Hebrew University, Jerusalem, Israel.

ABSTRACT
Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics. We designed small molecule quenched activity-based probes (qABPs) that fluoresce upon activity-dependent covalent modification, yielding cell killing by Photodynamic Therapy (PDT). These novel molecules are highly selective theranostic probes that enable both detection and treatment of cancer with minimal side effects. Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties. Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS. YBN14 allowed rapid and selective non-invasive in vivo imaging of subcutaneous tumors and induced specific tumor macrophage apoptosis by light treatment, resulting in a substantial tumor shrinkage in an aggressive breast cancer mouse model. These results demonstrate for the first time that the PS-qABPs technology offers a functional theranostic tool, which can be applied to numerous tumor types and other inflammation-associated diseases.

No MeSH data available.


Related in: MedlinePlus

Imaging protease activity in live cells. Cultures of NIH-3T3 cells were either pretreated with protease inhibitor GB111-NH2, or with control DMSO for 1 hour and then labeled by addition of YBN7 or YBN8 (10 µM) to growth medium for 7 hours. (a) Cells were imaged before washing by an inverted fluorescent microscope equipped with a Cy5 filter, bright field images are on the right. (b) Cells were imaged after a 16-hour wash with growth medium and after addition of lysosomal marker LysoTracker. Red fluorescence marks protease activity (PS-ABP or PS-qABP labeling); yellow fluorescence - lysosomal compartments; orange color - overlap of red and yellow signals.
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Figure 2: Imaging protease activity in live cells. Cultures of NIH-3T3 cells were either pretreated with protease inhibitor GB111-NH2, or with control DMSO for 1 hour and then labeled by addition of YBN7 or YBN8 (10 µM) to growth medium for 7 hours. (a) Cells were imaged before washing by an inverted fluorescent microscope equipped with a Cy5 filter, bright field images are on the right. (b) Cells were imaged after a 16-hour wash with growth medium and after addition of lysosomal marker LysoTracker. Red fluorescence marks protease activity (PS-ABP or PS-qABP labeling); yellow fluorescence - lysosomal compartments; orange color - overlap of red and yellow signals.

Mentions: To verify the probe location and permeability in intact cells, we acquired fluorescence microscopic images of live NIH-3T3 cells treated with the probes. Images of PS-ABP YBN7 without washout show high accumulation and nonspecific staining of the entire cell as a result of free probe accumulation (Figure 2a). Such staining pattern of the non-quenched probe implied non-specific distribution throughout the cell without significant accumulation in any particular location. To confirm the probe specificity we next performed extensive washout of un-bound probe from cells, which resulted in punctate staining that overlapped with the signal of the lysosomal marker Lysotracker, and could be blocked by pretreatment of cells with a cathepsin inhibitor (Figure 2b). Similarly, imaging of cells that were incubated with the PS-qABP YBN8 also revealed a distinct punctate labeling pattern even without the washout step, as a consequence of efficient quenching of the probe. These results confirmed that the quenched YBN8 localized to the lysosomes where it interacted with active enzymes that removed the quencher. Moreover, the probe accumulated in cells in sufficient amounts to be detected by fluorescence microscopy.


Photodynamic quenched cathepsin activity based probes for cancer detection and macrophage targeted therapy.

Ben-Nun Y, Merquiol E, Brandis A, Turk B, Scherz A, Blum G - Theranostics (2015)

Imaging protease activity in live cells. Cultures of NIH-3T3 cells were either pretreated with protease inhibitor GB111-NH2, or with control DMSO for 1 hour and then labeled by addition of YBN7 or YBN8 (10 µM) to growth medium for 7 hours. (a) Cells were imaged before washing by an inverted fluorescent microscope equipped with a Cy5 filter, bright field images are on the right. (b) Cells were imaged after a 16-hour wash with growth medium and after addition of lysosomal marker LysoTracker. Red fluorescence marks protease activity (PS-ABP or PS-qABP labeling); yellow fluorescence - lysosomal compartments; orange color - overlap of red and yellow signals.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440442&req=5

Figure 2: Imaging protease activity in live cells. Cultures of NIH-3T3 cells were either pretreated with protease inhibitor GB111-NH2, or with control DMSO for 1 hour and then labeled by addition of YBN7 or YBN8 (10 µM) to growth medium for 7 hours. (a) Cells were imaged before washing by an inverted fluorescent microscope equipped with a Cy5 filter, bright field images are on the right. (b) Cells were imaged after a 16-hour wash with growth medium and after addition of lysosomal marker LysoTracker. Red fluorescence marks protease activity (PS-ABP or PS-qABP labeling); yellow fluorescence - lysosomal compartments; orange color - overlap of red and yellow signals.
Mentions: To verify the probe location and permeability in intact cells, we acquired fluorescence microscopic images of live NIH-3T3 cells treated with the probes. Images of PS-ABP YBN7 without washout show high accumulation and nonspecific staining of the entire cell as a result of free probe accumulation (Figure 2a). Such staining pattern of the non-quenched probe implied non-specific distribution throughout the cell without significant accumulation in any particular location. To confirm the probe specificity we next performed extensive washout of un-bound probe from cells, which resulted in punctate staining that overlapped with the signal of the lysosomal marker Lysotracker, and could be blocked by pretreatment of cells with a cathepsin inhibitor (Figure 2b). Similarly, imaging of cells that were incubated with the PS-qABP YBN8 also revealed a distinct punctate labeling pattern even without the washout step, as a consequence of efficient quenching of the probe. These results confirmed that the quenched YBN8 localized to the lysosomes where it interacted with active enzymes that removed the quencher. Moreover, the probe accumulated in cells in sufficient amounts to be detected by fluorescence microscopy.

Bottom Line: Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics.Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties.Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS.

View Article: PubMed Central - PubMed

Affiliation: 1. The Institute of Drug Research, The School of Pharmacy, The Faculty of Medicine, Campus Ein Karem, The Hebrew University, Jerusalem, Israel.

ABSTRACT
Elevated cathepsins levels and activities are found in several types of human cancer, making them valuable biomarkers for detection and targeting therapeutics. We designed small molecule quenched activity-based probes (qABPs) that fluoresce upon activity-dependent covalent modification, yielding cell killing by Photodynamic Therapy (PDT). These novel molecules are highly selective theranostic probes that enable both detection and treatment of cancer with minimal side effects. Our qABPs carry a photosensitizer (PS), which is activated by light, resulting in oxidative stress and subsequent cell ablation, and a quencher that when removed by active cathepsins allow the PS to fluoresce and demonstrate PD properties. Our most powerful and stable PS-qABP, YBN14, consists of a selective cathepsin recognition sequence, a QC-1 quencher and a new bacteriochlorin derivative as a PS. YBN14 allowed rapid and selective non-invasive in vivo imaging of subcutaneous tumors and induced specific tumor macrophage apoptosis by light treatment, resulting in a substantial tumor shrinkage in an aggressive breast cancer mouse model. These results demonstrate for the first time that the PS-qABPs technology offers a functional theranostic tool, which can be applied to numerous tumor types and other inflammation-associated diseases.

No MeSH data available.


Related in: MedlinePlus