No abstract

Herein, the use of red blood cells (RBCs) as carriers of cytoplasmically interned phototherapeutic agents is described. Photolysis promotes drug release from the RBC carrier thereby providing the means to target specific diseased sites. This strategy is realized with a vitamin B12‐taxane conjugate (B12‐TAX), in which the drug is linked to the vitamin via a photolabile CoC bond. The conjugate is introduced into mouse RBCs (mRBCs) via a pore‐forming/pore‐resealing procedure and is cytoplasmically retained due to the membrane impermeability of B12. Photolysis separates the taxane from the B12 cytoplasmic anchor, enabling the drug to exit the RBC carrier. A covalently appended Cy5 antenna sensitizes the conjugate (Cy5‐B12‐TAX) to far red light, thereby circumventing the intense light absorbing properties of hemoglobin (350–600 nm). Microscopy and imaging flow cytometry reveal that Cy5‐B12‐TAX‐loaded mRBCs act as drug carriers. Furthermore, intravital imaging of mice furnish a real time assessment of circulating phototherapeutic‐loaded mRBCs as well as evidence of the targeted photorelease of the taxane upon photolysis. Histopathology confirms that drug release occurs in a well resolved spatiotemporal fashion. Finally, acoustic angiography is employed to assess the consequences of taxane release at the tumor site in Nu/Nu‐tumor‐bearing mice.

The bulk cell population response to a stimulus, be it a growth factor or a cytotoxic agent, neglects the cell-to-cell variability that can serve as a friend or as a foe in human biology. Biochemical variations among closely related cells furnish the basis for the adaptability of the immune system but also act as the root cause of resistance to chemotherapy by tumors. Consequently, the ability to probe for the presence of key biochemical variables at the single-cell level is now recognized to be of significant biological and biomedical impact. Chemical cytometry has emerged as an ultrasensitive single-cell platform with the flexibility to measure an array of cellular components, ranging from metabolite concentrations to enzyme activities. We briefly review the various chemical cytometry strategies, including recent advances in reporter design, probe and metabolite separation, and detection instrumentation. We also describe strategies for improving intracellular delivery, biochemical specificity, metabolic stability, and detection sensitivity of probes. Recent applications of these strategies to small molecules, lipids, proteins, and other analytes are discussed. Finally, we assess the current scope and limitations of chemical cytometry and discuss areas for future development to meet the needs of single-cell research.

Protein therapeutics are a powerful class of drugs known for their selectivity and potency. However, the potential efficacy of these therapeutics is commonly offset by short circulatory half-lives and undesired action at otherwise healthy tissue. We describe herein a targeted protein delivery system that employs engineered red blood cells (RBCs) as carriers and light as the external trigger that promotes hemolysis and drug release. RBCs internally loaded with therapeutic proteins are readily surface modified with a dormant hemolytic peptide. The latter is activated via easily assigned wavelengths that extend into the optical window of tissue. We have demonstrated that photorelease transpires with spatiotemporal control and that the liberated proteins display the anticipated biological effects in vitro. Furthermore, we have confirmed targeted delivery of a clot-inducing enzyme in a mouse model. Finally, we anticipate that this strategy is not limited to RBC carriers but also should be applicable to nano-and microtransporters comprised of bilayer lipid membranes.

Highlights d Design of engineered organelle-targeted optogenetic adenylate cyclases d Assessment of organelle-based cAMP-induced protein kinase activity d Identification of phosphodiesterases that modulate cAMPsignaling activity