An optogenetic Bax has been designed that facilitates light-induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light-responsive Bax results in the release of mitochondrial proteins, downstream caspase 3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C-terminus mitigates background levels (dark) of apoptosis due to Bax overexpression. The mechanism of optogenetic Bax-mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis-induced channel analogous to that of endogenous Bax.

Light-activatable drugs offer the promise of controlled release with exquisite temporal and spatial resolution. However, light sensitive pro-drugs are typically converted to their active forms using short wavelengths, which display poor tissue penetrance. We report herein erythrocyte-mediated assembly of long wavelength-sensitive phototherapeutics. The activating wavelength of the constructs is readily pre-assigned by using fluorophores with the desired λex. Drug release from the erythrocyte carrier was confirmed by standard analytical tools and by the expected biological consequences of the liberated drugs in cell culture: methotrexate, binding to intracellular dihydrofolate reductase; colchicine, inhibition of microtubule polymerization; dexamethasone, induced nuclear migration of the glucocorticoid receptor.

Accumulation of intravenously injected cytotoxic liposomes in the skin induces serious toxicity. We used single time point and longitudinal intravital microscopy to understand skin accumulation dynamics of non-PEGylated and PEGylated liposomes after systemic injection into mice. Non-PEGylated egg phosphatidylcholine (PC) liposomes showed short circulation half-life (1.3 h) and immediate aggregation in the blood, with some aggregates lodging in skin microvasculature soon after the injection. At 24 h, and more prominently at 48 h postinjection, liposomes appeared in dermal and subdermal cells. PEGylated egg PC liposomes showed long circulation half-life (22 h) and no aggregation in the blood. PEGylated liposomes started to accumulate in the skin microvasculature as soon as 5 min after the injection. Within 3 h postinjection, PEGylated liposomes accumulated in extravascular cells in the dermis and subdermis. Liposomes were present in the skin for at least 7 days postinjection. A regulatory approved PEGylated liposomal doxorubicin (LipoDox) and empty liposomes of the same composition as LipoDox showed similar skin distribution as PEGylated egg PC liposomes, suggesting that this phenomenon is relevant to liposomes of different lipid composition. Decorating liposomes with shorter PEGs (350 or 700) in addition to PEG 2000 did not decrease the deposition. Outside the capillaries, liposomes partially colocalized with CD45−, F4/80+ cells. The accumulation of liposomes was not due to prior neutrophil/platelet binding and transport across endothelium. Moreover, our studies have excluded a role of complement in the skin accumulation of liposomes. Further understanding of mechanisms of this important phenomenon can improve the safety of liposomal nanocarriers.

A strategy for the light-activated release of bioactive compounds (BODIPY, colchicine, paclitaxel, and methotrexate) from membrane-enclosed depots is described. We have found that membrane permeable bioagents can be rendered membrane impermeable via covalent attachment to cobalamin (Cbl) through a photo-cleavable linker. These Cbl-bioagent conjugates are imprisoned within lipid-enclosed compartments in the dark, as exemplified by their retention in the interior of erythrocytes. Subsequent illumination drives the secretion of the bioactive species from red blood cells. Photo-release is triggered by wavelengths in the red, far red and near IR, which can be pre-assigned by affixing a fluorophore with the desired excitation wavelength to the Cbl-bioagent conjugate. Pre-assigned wavelengths allow different biologically active compounds to be specifically and unambiguously photo-released from common carriers.