Membrane cholesterol enrichment and folic acid functionalization lead to increased accumulation of erythrocyte-derived optical nano-constructs within the ovarian intraperitoneal tumor implants in mice.

Cytoreductive surgery remains as the gold standard to treat ovarian cancer, but with limited efficacy since not all tumors can be intraoperatively visualized for resection. We have engineered erythrocyte-derived nano-constructs that encapsulate the near infrared (NIR) fluorophore, indocyanine green (ICG), as optical probes for NIR fluorescence imaging of ovarian tumors. Herein, we have enriched the membrane of these nano-constructs with cholesterol, and functionalized their surface with folic acid (FA) to target the folate receptor-α. Using a mouse model, we show that the average fraction of the injected dose per tumor mass for nano-constructs with both membrane cholesterol enrichment and FA functionalization was ~ sixfold higher than non-encapsulated ICG, ~ twofold higher than nano-constructs enriched with cholesterol alone, and 33 % higher than nano-constructs with only FA functionalization at 24-h post-injection. These results suggest that erythrocyte-derived nano-constructs containing both cholesterol and FA present a platform for improved fluorescence imaging of ovarian tumors.

Indocyanine Green (ICG) Fluorescence Is Dependent on Monomer with Planar and Twisted Structures and Inhibited by H-Aggregation.

The optical properties of indocyanine green (ICG) as a near-infrared (NIR) fluorescence dye depend on the nature of the solvent medium and the dye concentration. In the ICG absorption spectra of water, at high concentrations, there were absorption maxima at 700 nm, implying H-aggregates. With ICG dilution, the main absorption peak was at 780 nm, implying monomers. However, in ethanol, the absorption maximum was 780 nm, and the shapes of the absorption spectra were identical regardless of the ICG concentration, indicating that ICG in ethanol exists only as a monomer without H-aggregates. We found that emission was due to the monomer form and decreased with H-aggregate formation. In the fluorescence spectra, the 820 nm emission band was dominant at low concentrations, whereas at high concentrations, we found that the emission peaks were converted to 880 nm, suggesting a new form via the twisted intramolecular charge transfer (TICT) process of ICG. The NIR fluorescence intensity of ICG in ethanol was approximately 12- and 9-times brighter than in water in the NIR-I and -II regions, respectively. We propose an energy diagram of ICG to describe absorptive and emissive transitions through the ICG structures such as the monomer, H-aggregated, and TICT monomer forms.

Proteomes of Micro- and Nanosized Carriers Engineered from Red Blood Cells.

Red blood cell (RBC)-derived systems offer a potential platform for delivery of biomedical cargos. Although the importance of specific proteins associated with the biodistribution and pharmacokinetics of these particles has been recognized, it remains to be explored whether some of the key transmembrane and cytoskeletal proteins responsible for immune-modulatory effects and mechanical integrity of the particles are retained. Herein, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantitative tandem mass tag mass spectrometry in conjunction with bioinformatics analysis, we have examined the proteomes of micro- and nanosized erythrocyte ghosts doped with indocyanine green and compared them with those of RBCs. We identified a total of 884 proteins in each set of RBCs, micro-, and nanosized particles, of which 8 and 45 proteins were expressed at significantly different relative abundances when comparing micro-sized particles vs RBCs and nanosized particles vs RBCs, respectively. We found greater differences in relative abundances of some mechano-modulatory proteins, such as band 3 and protein 4.2, and immunomodulatory proteins like CD44, CD47, and CD55 in nanosized particles as compared to RBCs. Our findings highlight that the methods utilized in fabricating RBC-based systems can induce substantial effects on their proteomes. Mass spectrometry data are available at ProteomeXchange with the identifier PXD038780.

Erythrocyte-Derived Nanoparticles with Folate Functionalization for Near Infrared Pulsed Laser-Mediated Photo-Chemotherapy of Tumors.

Despite its common side effects and varying degrees of therapeutic success, chemotherapy remains the gold standard method for treatment of cancer. Towards developing a new therapeutic approach, we have engineered nanoparticles derived from erythrocytes that contain indocyanine green as a photo-activated agent that enables near infrared photothermal heating, and doxorubicin hydrochloride (DOX) as a chemotherapeutic drug. We hypothesize that milliseconds pulsed laser irradiation results in rapid heating and photo-triggered release of DOX, providing a dual photo-chemo therapeutic mechanism for tumor destruction. Additionally, the surface of the nanoparticles is functionalized with folate to target the folate receptor-α on tumor cells to further enhance the therapeutic efficacy. Using non-contract infrared radiometry and absorption spectroscopy, we have characterized the photothermal response and photostability of the nanoparticles to pulsed laser irradiation. Our in vitro studies show that these nanoparticles can mediate photo-chemo killing of SKOV3 ovarian cancer cells when activated by pulsed laser irradiation. We further demonstrate that this dual photo-chemo therapeutic approach is effective in reducing the volume of tumor implants in mice and elicits an apoptotic response. This treatment modality presents a promising approach in destruction of small tumor nodules.

Morphological Characteristics, Hemoglobin Content, and Membrane Mechanical Properties of Red Blood Cell Delivery Systems.

Red blood cell (RBC)-based systems are under extensive development as platforms for the delivery of various biomedical agents. While the importance of the membrane biochemical characteristics in relation to circulation kinetics of RBC delivery systems has been recognized, the membrane mechanical properties of such carriers have not been extensively studied. Using optical methods in conjunction with image analysis and mechanical modeling, we have quantified the morphological and membrane mechanical characteristics of RBC-derived microparticles containing the near-infrared cargo indocyanine green (ICG). We find that these particles have a significantly lower surface area, volume, and deformability as compared to normal RBCs. The residual hemoglobin has a spatially distorted distribution in the particles. The membrane bending modulus of the particles is about twofold higher as compared to normal RBCs and exhibits greater resistance to flow. The induced increase in the viscous characteristics of the membrane is dominant over the elastic and entropic effects of ICG. Our results suggest that changes to the membrane mechanical properties are a result of impaired membrane-cytoskeleton attachment in these particles. We provide a mechanistic explanation to suggest that the compromised membrane-cytoskeleton attachment and altered membrane compositional and structural asymmetry induce curvature changes to the membrane, resulting in mechanical remodeling of the membrane. These findings highlight the importance of membrane mechanical properties as an important criterion in the design and engineering of future generations of RBC-based delivery systems to achieve prolonged circulation.

Membrane Cholesterol Enrichment of Red Blood Cell-Derived Microparticles Results in Prolonged Circulation.

Particles fabricated from red blood cells (RBCs) can serve as vehicles for delivery of various biomedical cargos. Flipping of phosphatidylserine (PS) from the inner to the outer membrane leaflet normally occurs during the fabrication of such particles. PS externalization is a signal for phagocytic removal of the particles from circulation. Herein, we demonstrate that membrane cholesterol enrichment can mitigate the outward display of PS on microparticles engineered from RBCs. Our in-vitro results show that the phagocytic uptake of cholesterol-enriched particles by murine macrophages takes place at a lowered rate, resulting in reduced uptake as compared to RBC-derived particles without cholesterol enrichment. When administered via tail-vein injection into healthy mice, the percent of injected dose (ID) per gram of extracted blood for cholesterol-enriched particles was ∼1.5 and 1.8 times higher than the particles without cholesterol enrichment at 4 and 24 h, respectively. At 24 h, ∼43% ID/g of the particles without cholesterol enrichment was eliminated or metabolized while ∼94% ID/g of the cholesterol-enriched particles were still retained in the body. These results indicate that membrane cholesterol enrichment is an effective method to reduce PS externalization on the surface of RBC-derived particles and increase their longevity in circulation.

Near Infrared Fluorescence Imaging of Intraperitoneal Ovarian Tumors in Mice Using Erythrocyte-Derived Optical Nanoparticles and Spatially-Modulated Illumination.

Ovarian cancer is the deadliest gynecological cancer. Cytoreductive surgery to remove primary and intraperitoneal tumor deposits remains as the standard therapeutic approach. However, lack of an intraoperative image-guided approach to enable the visualization of all tumors can result in incomplete cytoreduction and recurrence. We engineered nano-sized particles derived from erythrocytes that encapsulate the near infrared (NIR) fluorochrome, indocyanine green, as potential imaging probes for tumor visualization during cytoreductive surgery. Herein, we present the first demonstration of the use of these nanoparticles in conjunction with spatially-modulated illumination (SMI), at spatial frequencies in the range of 0-0.5 mm-1, to fluorescently image intraperitoneal ovarian tumors in mice. Results of our animal studies suggest that the nanoparticles accumulated at higher levels within tumors 24 h post-intraperitoneal injection as compared to various other organs. We demonstrate that, under the imaging specifications reported here, use of these nanoparticles in conjunction with SMI enhances the fluorescence image contrast between intraperitoneal tumors and liver, and between intraperitoneal tumors and spleen by nearly 2.1, and 3.0 times, respectively, at the spatial frequency of 0.2 mm-1 as compared to the contrast values at spatially-uniform (non-modulated) illumination. These results suggest that the combination of erythrocyte-derived NIR nanoparticles and structured illumination provides a promising approach for intraoperative fluorescence imaging of ovarian tumor nodules at enhanced contrast.

Phototheranostics Using Erythrocyte-Based Particles.

There has been a recent increase in the development of delivery systems based on red blood cells (RBCs) for light-mediated imaging and therapeutic applications. These constructs are able to take advantage of the immune evasion properties of the RBC, while the addition of an optical cargo allows the particles to be activated by light for a number of promising applications. Here, we review some of the common fabrication methods to engineer these constructs. We also present some of the current light-based applications with potential for clinical translation, and offer some insight into future directions in this exciting field.

Virus-Mimicking Nanoparticles for Targeted Near Infrared Fluorescence Imaging of Intraperitoneal Ovarian Tumors in Mice.

Ovarian cancer is the most lethal malignancy affecting the female reproductive system. Identification and removal of all ovarian intraperitoneal tumor deposits during the intraoperative surgery is important towards preventing cancer recurrence and ultimately improving patient survival. Herein, we investigate the effectiveness of virus mimicking nanoparticles, derived from genome-depleted plant-infecting brome mosaic virus, and doped with near infrared (NIR) brominated cyanine dye BrCy106-NHS, for targeted NIR fluorescence imaging of intraperitoneal ovarian tumors. We refer to these nanoparticles as optical viral ghosts (OVGs). We functionalized the OVGs with antibodies against HER2 receptor, a biomarker over-expressed in ovarian cancers. We injected functionalized OVGs, non-functionalized OVGs, and non-encapsulated BrCy106-NHS intravenously in mice implanted with ovarian intraperitoneal tumors. Tumors were extracted at 2, 6, and 24 h post-injection, and quantitatively analyzed using NIR fluorescence imaging. Fluorescence emission from tumors associated with the injection of the functionalized OVGs continued to increase between 2 and 24 h post-injection. At 24 h timepoint, the average spectrally-integrated fluorescence emission from homogenized tumors containing functionalized-OVGs was about 3.5 and 19.5 times higher than those containing non-functionalized OVGs or non-encapsulated BrCy106-NHS, respectively. Similarly, by using the functionalized-OVGs, the imaging signal-to-noise ratio at 24 h timepoint was enhanced by approximately threefold and sevenfold as compared to non-functionalized OVGs and the non-encapsulated dye, respectively. These functionalized virus-mimicking NIR nano-constructs could potentially be used for intraoperative visualization of ovarian tumors implants.

Author Correction: Non-Invasive Photoacoustic Imaging of In Vivo Mice with Erythrocyte Derived Optical Nanoparticles to Detect CAD/MI.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Acute Immune Response of Micro- and Nanosized Erythrocyte-Derived Optical Particles in Healthy Mice.

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.

RBC-Derived Optical Nanoparticles Remain Stable After a Freeze-Thaw Cycle.

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.

Non-Invasive Photoacoustic Imaging of In Vivo Mice with Erythrocyte Derived Optical Nanoparticles to Detect CAD/MI.

Coronary artery disease (CAD) causes mortality and morbidity worldwide. We used near-infrared erythrocyte-derived transducers (NETs), a contrast agent, in combination with a photoacoustic imaging system to identify the locations of atherosclerotic lesions and occlusion due to myocardial-infarction (MI). NETs (≈90 nm diameter) were fabricated from hemoglobin-depleted mice erythrocyte-ghosts and doped with Indocyanine Green (ICG). Ten weeks old male C57BL/6 mice (n = 9) underwent left anterior descending (LAD) coronary artery ligation to mimic vulnerable atherosclerotic plaques and their rupture leading to MI. 150 µL of NETs (20 µM ICG,) was IV injected via tail vein 1-hour prior to photoacoustic (PA) and fluorescence in vivo imaging by exciting NETs at 800 nm and 650 nm, respectively. These results were verified with histochemical analysis. We observed ≈256-fold higher PA signal from the accumulated NETs in the coronary artery above the ligation. Fluorescence signals were detected in LAD coronary, thymus, and liver. Similar signals were observed when the chest was cut open. Atherosclerotic lesions exhibited inflammatory cells. Liver demonstrated normal portal tract, with no parenchymal necrosis, inflammation, fibrosis, or other pathologic changes, suggesting biocompatibility of NETs. Non-invasively detecting atherosclerotic plaques and stenosis using NETs may lay a groundwork for future clinical detection and improving CAD risk assessment.

Intravital Vascular Phototheranostics and Real-Time Circulation Dynamics of Micro- and Nanosized Erythrocyte-Derived Carriers.

Erythrocyte-based carriers can serve as theranostic platforms for delivery of imaging and therapeutic payloads. Engineering these carriers at micro- or nanoscales makes them potentially useful for broad clinical applications ranging from vascular diseases to tumor theranostics. Longevity of these carriers in circulation is important in delivering a sufficient amount of their payloads to the target. We have investigated the circulation dynamics of micro (∼4.95 µm diameter) and nano (∼91 nm diameter) erythrocyte-derived carriers in real time using near-infrared fluorescence imaging, and evaluated the effectiveness of such carrier systems in mediating photothermolysis of cutaneous vasculature in mice. Fluorescence emission half-lives of micro- and nanosized carriers in response to a single intravenous injection were ∼49 and ∼15 min, respectively. A single injection of microsized carriers resulted in a 3-fold increase in signal-to-noise ratio that remained nearly persistent over 1 h of imaging time. Our results also suggest that a second injection of the carriers 7 days later can induce a transient inflammatory response, as manifested by the apparent leakage of the carriers into the perivascular tissue. The administration of the carriers into the mice vasculature reduced the threshold laser fluence to induce photothermolysis of blood vessels from >65 to 20 J/cm2. We discuss the importance of membrane physicochemical and mechanical characteristics in engineering erythrocyte-derived carriers and considerations for their clinical translation.

Functionalized erythrocyte-derived optical nanoparticles to target ephrin-B2 ligands.

Over- or under-expression of erythropoietin-production human hepatocellular receptors (Eph) and their ligands are associated with various diseases. Therefore, these molecular biomarkers can potentially be used as binding targets for the delivery of therapeutic and/or imaging agents to cells characterized by such irregular expressions. We have engineered nanoparticles derived from erythrocytes and doped with the near-infrared (NIR) FDA-approved dye, indocyanine green. We refer to these nanoparticles as NIR erythrocyte-derived transducers (NETs). We functionalized the NETs with the ligand-binding domain of a particular Eph receptor, EphB1, to target the genetically modified human dermal microvascular endothelial cells (hDMVECs) with coexpression of EphB1 receptor and its ligand ephrin-B2. This cell model mimics the pathological phenotypes of lesional endothelial cells (ECs) in port wine stains (PWSs). Our quantitative fluorescence imaging results demonstrate that such functionalized NETs bind to the ephrin-B2 ligands on these hDMVECs in a dose-dependent manner that varies sigmoidally with the number density of the particles. These nanoparticles may potentially serve as agents to target PWS lesional ECs and other diseases characterized with over-expression of Eph receptors or their associated ligands to mediate phototherapy.

Biodistribution and toxicological evaluation of micron- and nano-sized erythrocyte-derived optical particles in healthy Swiss Webster mice.

Particle-based systems provide a capability for the delivery of imaging and/or therapeutic payloads. We have engineered constructs derived from erythrocytes, and doped with the FDA-approved near infrared dye, indocyanine green (ICG). We refer to these optical particles as NIR erythrocyte-mimicking transducers (NETs). A particular feature of NETs is that their diameters can be tuned from micron- to nano-scale. Herein, we investigated the effects of micron- (≈2.6 µm diameter), and nano- (≈145 nm diameter) sized NETs on their biodistribution, and evaluated their acute toxicity in healthy Swiss Webster mice. Following tail vein injection of free ICG and NETs, animals were euthanized at various time points up to 48 hours. Fluorescence analysis of blood showed that nearly 11% of the injected amount of nano-sized NETs (nNETs) remained in blood at 48 hours post-injection as compared to ≈5% for micron-sized NETs (µNETs). Similarly, at this time point, higher levels of nNETs were present in various organs including the lungs, liver, and spleen. Histological analyses of various organs, extracted at 24 hours post-injection of NETs, did not show pathological alterations. Serum biochemistry profiles, in general, did not show elevated levels of the various analyzed biomarkers associated with liver and kidney functions. Values of various hematological profiles remained within the normal ranges following the administration of µNETs and nNETs. Results of this study suggest that erythrocyte-derived particles can potentially provide a non-toxic platform for delivery of ICG.

Near-Infrared-Fluorescent Erythrocyte-Mimicking Particles: Physical and Optical Characteristics.

Exogenous fluorescent materials activated by near-infrared (NIR) light can offer deep optical imaging with subcellular resolution, and enhanced image contrast. We have engineered NIR particles by doping hemoglobin-depleted erythrocyte ghosts (EGs) with indocyanine green (ICG). We refer to these optical particles as NIR erythrocyte-mimicking transducers (NETs). A particular feature of NETs is that their diameters can be tuned from micrometer to nanometer scale, thereby, providing a capability for broad NIR biomedical imaging applications. Herein, we investigate the effects of ICG concentration on key material properties of micrometer-sized NETs, and nanometer-sized NETs fabricated by either sonication or mechanical extrusion of EGs. The zeta potentials of NETs do not vary significantly with ICG concentration, suggesting that ICG is encapsulated within NETs regardless of particle size or ICG concentration. Loading efficiency of ICG into the NETs monotonically decreases with increasing values of ICG concentration. Based on quantitative analyses of the fluorescence emission spectra of the NETs, we determine that 20 µM ICG utilized during fabrication of NETs presents an optimal concentration that maximizes the integrated fluorescence emission for micrometer- and nanometer-sized NETs. Encapsulation of the ICG in these constructs also enhances the fluorescence stability and quantum yield of ICG. These results guide the engineering of NETs with maximal NIR emission for imaging applications such as fluorescence-guided tumor resection and real-time angiography.

Photothermal treatment of port-wine stains using erythrocyte-derived particles doped with indocyanine green: a theoretical study.

Pulsed dye laser irradiation in the wavelength range of 585 to 600 nm is currently the gold standard for treatment of port-wine stains (PWSs). However, this treatment method is often ineffective for deeply seated blood vessels and in individuals with moderate to heavy pigmentation. Use of optical particles doped with the FDA-approved near-infrared (NIR) absorber, indocyanine green (ICG), can potentially provide an effective method to overcome these limitations. Herein, we theoretically investigate the effectiveness of particles derived from erythrocytes, which contain ICG, in mediating photothermal destruction of PWS blood vessels. We refer to these particles as NIR erythrocyte-derived transducers (NETs). Our theoretical model consists of a Monte Carlo algorithm to estimate the volumetric energy deposition, a finite elements approach to solve the heat diffusion equation, and a damage integral based on an Arrhenius relationship to quantify tissue damage. The model geometries include simulated PWS blood vessels as well as actual human PWS blood vessels plexus obtained by the optical coherence tomography. Our simulation results indicate that blood vessels containing micron- or nano-sized NETs and irradiated at 755 nm have higher levels of photothermal damage as compared to blood vessels without NETs irradiated at 585 nm. Blood vessels containing micron-sized NETs also showed higher photothermal damage than blood vessels containing nano-sized NETs. The theoretical model presented can be used in guiding the fabrication of NETs with patient-specific optical properties to allow for personalized treatment based on the depth and size of blood vessels as well as the pigmentation of the individual's skin.

Erythrocyte-Derived Theranostic Nanoplatforms for Near Infrared Fluorescence Imaging and Photodestruction of Tumors.

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/cm2 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.

Erythrocyte-Derived Nanoparticles as a Theranostic Agent for Near-Infrared Fluorescence Imaging and Thrombolysis of Blood Clots.

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.