Photoactivatable Reporter to Perform Multiplexed and Temporally Controlled Measurements of Kinase and Protease Activity in Single Cells.

Peptide bioreporters were developed to perform multiplexed measurements of the activation of epidermal growth factor receptor kinase (EGFR), Akt kinase (Akt/protein kinase B), and proteases/peptidases in single cells. The performance characteristics of the three reporters were assessed by measuring the reporter's proteolytic stability, kinetic constants for EGFR and Akt, and dephosphorylation rate. The reporter displaying optimal performance was composed of 6-carboxyfluorescein (6-FAM) on the peptide N-terminus, an Akt substrate sequence employing a threonine phosphorylation site for Akt, followed by a tri-D arginine linker, and finally an EGFR substrate sequence bearing a phosphatase-resistant 7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (L-htc) residue as the EGFR phosphorylation site. Importantly, use of a single electrophoretic condition separated the mono- and diphosphorylated products as well as proteolytic forms permitting the quantitation of multiple enzyme activities simultaneously using a single reporter. Because the Akt and EGFR substrates were linked, a known ratio (EGFR/Akt) of the reporter was loaded into cells. A photoactivatable version of the reporter was synthesized by adding two 4,5-dimethoxy-2-nitrobenzyl (DMNB) moieties to mask the EGFR and Akt phosphorylation sites. The DMNB moieties were readily photocleaved following exposure to 360 nm light, unmasking the phosphorylation sites on the reporter. The new photoactivatable reporter permitted multiplexed measurements of kinase signaling and proteolytic degradation in single cells in a temporally controlled manner. This work will facilitate the development of a new generation of multiplexed activity-based reporters capable of light-initiated measurement of enzymatic activity in single cells.

On Command Drug Delivery via Cell-Conveyed Phototherapeutics.

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.

Taking phototherapeutics from concept to clinical launch.

More than four decades have passed since the first example of a light-activated (caged) compound was described. In the intervening years, a large number of light-responsive derivatives have been reported, several of which have found utility under a variety of in vitro conditions using cells and tissues. Light-triggered bioactivity furnishes spatial and temporal control, and offers the possibility of precision dosing and orthogonal communication with different biomolecules. These inherent attributes of light have been advocated as advantageous for the delivery and/or activation of drugs at diseased sites for a variety of indications. However, the tissue penetrance of light is profoundly wavelength-dependent. Only recently have phototherapeutics that are photoresponsive in the optical window of tissue (600-900 nm) been described. This Review highlights these recent discoveries, along with their limitations and clinical opportunities. In addition, we describe preliminary in vivo studies of prospective phototherapeutics, with an emphasis on the path that remains to be navigated in order to translate light-activated drugs into clinically useful therapeutics. Finally, the unique attributes of phototherapeutics is highlighted by discussing several potential disease applications.

Light-Controlled Release of Therapeutic Proteins from Red Blood Cells.

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.

Compartmentalized cAMP Generation by Engineered Photoactivated Adenylyl Cyclases.

Because small-molecule activators of adenylyl cyclases (AC) affect ACs cell-wide, it is challenging to explore the signaling consequences of AC activity emanating from specific intracellular compartments. We explored this issue using a series of engineered, optogenetic, spatially restricted, photoactivable adenylyl cyclases (PACs) positioned at the plasma membrane (PM), the outer mitochondrial membrane (OMM), and the nucleus (Nu). The biochemical consequences of brief photostimulation of PAC is primarily limited to the intracellular site occupied by the PAC. By contrast, sustained photostimulation results in distal cAMP signaling. Prolonged cAMP generation at the OMM profoundly stimulates nuclear protein kinase (PKA) activity. We have found that phosphodiesterases 3 (OMM and PM) and 4 (PM) modulate proximal (local) cAMP-triggered activity, whereas phosphodiesterase 4 regulates distal cAMP activity as well as the migration of PKA's catalytic subunit into the nucleus.

Design and Application of Sensors for Chemical Cytometry.

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.