Conversion of a DNA-binding fluorophore (DAPI) to a photosensitizer via bromination retains high fluorescence and high affinity DNA binding but now produces light-induced reactive oxygen species directed towards DNA resulting in rapid cancer cell death.
Reactive oxygen species (e.g., singlet oxygen) are the primary cytotoxic agents used in the clinically approved technique photodynamic therapy (PDT). Although singlet oxygen has high potential to effectively kill tumor cells, its production via light excitation of a photosensitizer has been limited by the penetration depth and delivery of light in tissue. To produce singlet oxygen without light excitation, we describe the use of Schaap's chemiluminescent scaffold comprising an adamantylidene−dioxetane motif. Functionalizing this scaffold with a photosensitizer, Erythrosin B, resulted in spontaneous chemiluminescence resonance energy transfer (CRET) leading to the production of singlet oxygen. We show that this compound is cell permeable and that the singlet oxygen produced via CRET is remarkably efficient in killing cancer cells at low micromolar concentrations. Moreover, we demonstrate that protection of the phenol on the chemiluminescent scaffold with a nitroreductase-responsive trigger group allows for cancer-selective dark dynamic cell death. Here, we present the concept of dark dynamic therapy using a small cell-permeable molecule capable of producing the effects of PDT in cells, without light.
Human NAD(P)H: Quinone Oxidoreductase 1 (hNQO1) is an attractive enzyme for cancer therapeutics due to its significant overexpression in tumors compared to healthy tissues. Its unique catalytic mechanism involving the two‐electron reduction of quinone‐based compounds has made it a useful target to exploit in the design of hNQO1 fluorescent chemosensors and hNQO1‐activatable‐prodrugs. In this work, hNQO1 is exploited for an optical therapeutic. The probe uses the photosensitizer, phenalenone, which is initially quenched via photo‐induced electron transfer by the attached quinone. Native phenalenone is liberated in the presence of hNQO1 resulting in the production of cytotoxic singlet oxygen upon irradiation. hNQO1‐mediated activation in A549 lung cancer cells containing high levels of hNQO1 induces a dose‐dependent photo‐cytotoxic response after irradiation. In contrast, no photo‐cytotoxicity was observed in the normal lung cell line, MRC9. By targeting hNQO1, this scaffold can be used to enhance the cancer selectivity of photodynamic therapy.
Antimicrobial
photodynamic therapy (APDT) employs a photosensitizer,
light, and molecular oxygen to treat infectious diseases via oxidative
damage, with a low likelihood for the development of resistance. For
optimal APDT efficacy, photosensitizers with cationic charges that
can permeate bacteria cells and bind intracellular targets are desired
to not limit oxidative damage to the outer bacterial structure. Here
we report the application of brominated DAPI (Br-DAPI), a water-soluble,
DNA-binding photosensitizer for the eradication of both Gram-negative
and Gram-positive bacteria (as demonstrated on N99 Escherichia
coli and Bacillus subtilis, respectively).
We observe intracellular uptake of Br-DAPI, ROS-mediated bacterial
cell death via one- and two-photon excitation, and selective photocytotoxicity
of bacteria over mammalian cells. Photocytotoxicity of both N99 E. coli and B. subtilis occurred at submicromolar
concentrations (IC50 = 0.2–0.4 μM) and low
light doses (5 min irradiation times, 4.5 J cm–2 dose), making it superior to commonly employed APDT phenothiazinium
photosensitizers such as methylene blue. Given its high potency and
two-photon excitability, Br-DAPI is a promising novel photosensitizer
for in vivo APDT applications.
Carboxylesterase 2 (CES2) has crucial roles in both xenobiotic metabolism and formation of pathogenic states including cancer. Thus, it is highly critical to monitor intracellular CES2 activity in living cancer...
Photodynamic therapy (PDT) is a clinically approved cancer
treatment
that requires a photosensitizer (PS), light, and molecular oxygena
combination which produces reactive oxygen species (ROS) that can
induce cancer cell death. To enhance the efficacy of PDT, dual-targeted
strategies have been explored where two photosensitizers are administered
and localize to different subcellular organelles. To date, a single
small-molecule conjugate for dual-targeted PDT with light-controlled
nuclear localization has not been achieved. We designed a probe composed
of a DNA-binding PS (Br-DAPI) and a photosensitizing photocage (WinterGreen).
Illumination with 480 nm light removes WinterGreen from the conjugate
and produces singlet oxygen mainly in the cytosol, while Br-DAPI localizes
to nuclei, binds DNA, and produces ROS using one- or two-photon illumination.
We observe synergistic photocytotoxicity in MCF7 breast cancer cells,
and a reduction in size of three-dimensional (3D) tumor spheroids,
demonstrating that nuclear/cytosolic photosensitization using a single
agent can enhance PDT efficacy.
Metrics & MoreArticle Recommendations * sı Supporting Information N MR spectra of CL-E1a ( 1 H) and CL-E1 ( 1 H and 13 C) have been updated in the Supporting Information and their purity by HPLC reported. These spectra are now from samples used in assays described in the article and therefore are a better representation of the purity of the compounds employed. The corrections do not alter any findings or conclusions reported. An updated version of the Supporting Information is provided.
■ ASSOCIATED CONTENT
* sı Supporting InformationThe Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.2c00509.Synthetic procedures and compound characterization data (NMR, MS), in vitro characterization data (absorbance, fluorescence, CL spectral comparisons, CL half-life data, response to ROS sensors, and NTR-CL-E1 activation), and in cellulo data (fluorescence images showing response to ROS sensors and intracellular uptake of probes and cytotoxicity control data) (PDF)
Antimicrobial photodynamic therapy (APDT) employs a photosensitizer, light, and molecular oxygen to treat infectious diseases via oxidative damage, with a low likelihood for the development of resistance. For optimal APDT efficacy, photosensitizers with cationic charges that can permeate bacteria cells and bind intracellular targets are desired to not limit oxidative damage to the outer bacterial structure. Here we report the application of brominated DAPI (BrDAPI), a water-soluble, DNA-binding photosensitizer for eradication of both gram negative and gram positive bacteria (as demonstrated on N99 E. coli and B. subtilis, respectively). We observe intracellular uptake of BrDAPI, ROS mediated bacterial cell death via 1 and 2 photon excitation, and selective photocytotoxicity of bacteria over mammalian cells. Photocytotoxicity of both N99 E. coli and B. subtilis occurred at sub-micromolar concentrations (IC50 = 0.2 to 0.4 micromolar) and low light doses (5 minute irradiation times, 4.5 J cm-2 dose) making it superior to commonly employed APDT phenothiazinium photosensitizers such as methylene blue. Given its high potency and 2 photon excitability, BrDAPI is a promising novel photosensitizer for in vivo APDT applications.
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