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Photodynamic therapy (PDT) leads to cancer remission via the production of cytotoxic species under photosensitizer (PS) irradiation. However, concomitant damage and dark toxicity can both hinder its use. With this in mind, we have implemented a versatile peptide‐based platform of bioorthogonally activatable BODIPY‐tetrazine PSs. Confocal microscopy and phototoxicity studies demonstrated that the incorporation of the PS, as a bifunctional module, into a peptide enabled spatial and conditional control of singlet oxygen (1O2) generation. Comparing subcellular distribution, PS confined in the cytoplasmic membrane achieved the highest toxicities (IC50=0.096±0.003 μm) after activation and without apparent dark toxicity. Our tunable approach will inspire novel probes towards smart PDT., Modular peptide‐based platform for organelle‐confinement of the photodynamic effect. We report a general approach capable of directing the bioorthogonal activation of tetrazine‐BODIPY photosensitizers to specific subcellular organelles.

A persisting obstacle in human immunology is that blood-derived leukocytes are notoriously difficult to manipulate at the RNA level. Therefore, our knowledge about immune-regulatory RNA-networks is largely based on tumour cell-line and rodent knockout models, which do not fully mimic human leukocyte biology. Here, we exploit straightforward cell penetrating peptide (CPP) chemistry to enable efficient loss-of-function phenotyping of regulatory RNAs in primary human blood-derived cells. The classical CPP octaarginine (R8) enabled antisense peptide-nucleic-acid (PNA) oligomer delivery into nearly 100% of human blood-derived macrophages without apparent cytotoxicity even up to micromolar concentrations. In a proof-of-principle experiment, we successfully de-repressed the global microRNA-155 regulome in primary human macrophages using a PNA-R8 oligomer, which phenocopies a CRISPR-Cas9 induced gene knockout. Interestingly, although it is often believed that fairly high concentrations (μM) are needed to achieve antisense activity, our PNA-R8 was effective at 200 nM. RNA-seq characterized microRNA-155 as a broad-acting riboregulator, feedback restraining a late myeloid differentiation-induced pro-inflammatory network, comprising MyD88-signalling and ubiquitin-proteasome components. Our results highlight the important role of the microRNA machinery in fine-control of blood-derived human phagocyte immunity and open the door for further studies on regulatory RNAs in difficult-to-transfect primary human immune cells.

We report the use of bioorthogonal reactions as an original strategy in photodynamic therapy to achieve conditional phototoxicity and specific subcellular localization simultaneously. Our novel halogenated BODIPY-tetrazine probes only become efficient photosensitizers (ΦΔ ≈0.50) through an intracellular inverse-electron-demand Diels-Alder reaction with a suitable dienophile. Ab initio computations reveal an activation-dependent change in decay channels that controls 1 O2 generation. Our bioorthogonal approach also enables spatial control. As a proof-of-concept, we demonstrate the feasibility of the selective activation of our dormant photosensitizer in cellular nuclei, causing cancer cell death upon irradiation. Thus, our dual biorthogonal, activatable photosensitizers open new venues to combat current limitations of photodynamic therapy.