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Nanosized carriers engineered from red blood cells (RBCs) provide a means for delivering various cargos, including drugs, biologics, and imaging agents. We have engineered nanosized particles from RBCs, doped with the near-infrared (NIR) fluorochrome, indocyanine green (ICG). An important issue related to clinical translation of RBC-derived nanocarriers, including these NIR nanoparticles, is their stability postfabrication. Freezing may provide a method for long-term storage of these and other RBC-derived nanoparticles. Herein, we have investigated the physical and optical stability of these particles in response to a single freeze-thaw cycle. Nanoparticles were frozen to -20 °C, stored frozen for up to 8 weeks, and then thawed at room temperature. Our results show that the hydrodynamic diameter, zeta potential, optical density, and NIR fluorescence emission of these nanoparticles are retained following the freeze-thaw cycle. The ability of these nanoparticles in NIR fluorescence imaging of ovarian cancer cells, as well as their biodistribution in reticuloendothelial organs of healthy Swiss Webster mice after the freeze-thaw cycle is similar to that for freshly prepared nanoparticles. These results indicate that a single cycle of freezing the RBC-derived nanoparticles to -20 °C followed by thawing at room temperature is an effective method to retain the physical and optical characteristics of the nanoparticles, and their interactions with biological systems without the need for use of cryoprotectants.

Erythrocyte-derived particles activated by near-infrared (NIR) light present a platform for various phototheranostic applications. We have engineered such a platform with indocyanine green as the NIR-activated agent. A particular feature of these particles is that their diameters can be tuned from micro- to nanoscale, providing a potential capability for broad clinical utility ranging from vascular to cancer-related applications. An important issue related to clinical translation of these particles is their immunogenic effects. Herein, we have evaluated the early-induced innate immune response of these particles in healthy Swiss Webster mice following tail vein injection by measurements of specific cytokines in blood serum, the liver, and the spleen following euthanasia. In particular, we have investigated the effects of particle size and relative dose, time-dependent cytokine response for up to 6 h postinjection, functionalization of the nanosized particles with folate or Herceptin, and dual injections of the particles 1 week apart. Mean concentrations of interleukin (IL)-6, IL-10, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP)-1 in response to injection of microsized particles at the investigated relative doses were significantly lower than the corresponding mean concentrations induced by lipopolysaccharide (positive control) at 2 h. All investigated doses of the nanosized particles induced significantly higher concentrations of MCP-1 in the liver and the spleen as compared to phosphate buffer saline (PBS) (negative control) at 2 h. In response to micro- and nanosized particles at the highest investigated dose, there were significantly higher levels of TNF-α in blood serum at 2 and 6 h postinjection as compared to the levels associated with PBS treatment at these times. Whereas the mean concentration of TNF-α in the liver significantly increased between 2 and 6 h postinjection in response to the injection of the microsized particles, it was significantly reduced during this time interval in response to the injection of the nanosized particles. In general, functionalization of the nanosized particles was associated with a reduction of IL-6 and MCP-1 in blood serum, the liver, and the spleen, and TNF-α in blood serum. With the exception of IL-10 in the spleen in response to nanosized particles, the second injection of micro- or nanosized particles did not lead to significantly higher concentrations of other cytokines at the investigated dose as compared to a single injection.

Nanoparticles activated by near-infrared (NIR) excitation provide a capability for optical imaging and photodestruction of tumors. We have engineered optical nanoconstructs derived from erythrocytes, which are doped with the FDA-approved NIR dye, indocyanine green (ICG). We refer to these constructs as NIR erythrocyte-mimicking transducers (NETs). Herein, we investigate the phototheranostic capabilities of NETs for fluorescence imaging and photodestruction of SKBR3 breast cancer cells and subcutaneous xenograft tumors in mice. Our cellular studies demonstrate that NETs are internalized by these cancer cells and localized to their lysosomes. As evidenced by NIR fluorescence imaging and in vivo laser irradiation studies, NETs remain available within tumors at 24 h postintravenous injection. In response to continuous wave 808 nm laser irradiation at intensity of 680 mW/cm(2) for 10−15 min, NETs mediate the destruction of cancer cells and tumors in mice through synergistic photochemical and photothermal effects. We demonstrate that NETs are effective in mediating photoactivation of Caspase-3 to induce tumor apoptosis. Our results provide support for the effectiveness of NETs as theranostic agents for fluorescence imaging and photodestruction of tumors and their role in photoinduced apoptosis initiated by their localization to lysosomes.

Pyrene derivatives are among the most widely used organic fluorescent photoprobes. Many of them are photosensitizers for hole injection. Pyrenes, however, are mostly UV absorbers, limiting their utility for photonic applications. Nitration of pyrene shifts its absorption to the visible region. Conversely, nitration of pyrene that is already derivatized for covalent labeling, produces mixtures of isomers that are challenging to separate. We present a robust procedure for attaining isomerically pure nitropyrenes. NMR analysis provides unequivocal assignments of the regioisomers and of the structures of the disubstituted nitropyrenes. The added substituents negligibly affect the electronic properties of the nitropyrenes. Photoexcited nitropyrenes undergo efficient triplet formation, making them an attractive choice for triplet sensitizers and photooxidants. Hence, facile and reliable preparation of disubstituted nitropyrenes provides venues for exploring their electronic and photonic utility.

We have engineered hybrid nanostructures derived from erythrocytes, which can be doped with various near infrared (NIR) organic chromophores, including the FDA-approved indocyanine green (ICG). We refer to these vesicles as NIR erythrocyte-mimicking transducers (NETs), as they are capable of generating heat, reactive oxygen species (ROS) or emit fluorescence light. We present preliminary results that demonstrate the effectiveness of NETs for fluorescence imaging and photodynamic therapeutic destruction of breast cancer cells, upon photo-excitation using NIR light. These hybrid nanostructures present a promising platform with theranostic capability for future biomedical clinical applications.

Ischemic stroke occurs when a blood clot obstructs or narrows the arteries that supply blood to the brain. Currently, tissue plasminogen activator (tPA), a thrombolytic agent, is the only United States Food and Drug Administration (FDA)-approved pharmacologic treatment for ischemic stroke. Despite its effective usage, the major limitation of tPA that stems from its short half-life in plasma (≈5 min) is the potential for increased risk of hemorrhagic complications. To circumvent these limitations, herein, the first proof-of-principle demonstration of a theranostic nanoconstruct system derived from erythrocytes doped with the FDA-approved near-infrared (NIR) imaging agent, indocyanine green, and surface-functionalized with tPA is reported. Using a clot model, the dual functionality of these nanoconstructs in NIR fluorescence imaging and clot lysis is demonstrated. These biomimetic theranostic nanoconstructs may ultimately be effective in imaging and treatment of blood clots involved in ischemic stroke.

Constructs derived from mammalian cells are emerging as a new generation of nano-scale platforms for clinical imaging applications. Herein, we report successful engineering of hybrid nano-structures composed of erythrocyte-derived membranes doped with FDA-approved near infrared (NIR) chromophore, indocyanine green (ICG), and surface-functionalized with antibodies to achieve molecular targeting. We demonstrate that these constructs can be used for targeted imaging of cancer cells in vitro. These erythrocyte-derived optical nano-probes may provide a potential platform for clinical translation, and enable molecular imaging of cancer biomarkers.