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.

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.

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.

The human secretome atlas initiative: implications in health and disease conditions.

Proteomic analysis of human body fluids is highly challenging, therefore many researchers are redirecting efforts toward secretome profiling. The goal is to define potential biomarkers and therapeutic targets in the secretome that can be traced back in accessible human body fluids. However, currently there is a lack of secretome profiles of normal human primary cells making it difficult to assess the biological meaning of current findings. In this study we sought to establish secretome profiles of human primary cells obtained from healthy donors with the goal of building a human secretome atlas. Such an atlas can be used as a reference for discovery of potential disease associated biomarkers and eventually novel therapeutic targets. As a preliminary study, secretome profiles were established for six different types of human primary cell cultures and checked for overlaps with the three major human body fluids including plasma, cerebrospinal fluid and urine. About 67% of the 1054 identified proteins in the secretome of these primary cells occurred in at least one body fluid. Furthermore, comparison of the secretome profiles of two human glioblastoma cell lines to this new human secretome atlas enabled unambiguous identification of potential brain tumor biomarkers. These biomarkers can be easily monitored in different body fluids using stable isotope labeled standard proteins. The long term goal of this study is to establish a comprehensive online human secretome atlas for future use as a reference for any disease related secretome study. This article is part of a Special Issue entitled: An Updated Secretome.

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.

Erythrocyte-Derived Optical Nanoprobes Doped with Indocyanine Green-Bound Albumin: Material Characteristics and Evaluation for Cancer Cell Imaging.

Nanosize structures activated by near-infrared (NIR) photoexcitation can provide an optical platform for the image-guided removal of small tumor nodules. We have engineered nanoparticles derived from erythrocytes that can be doped with NIR fluorophore indocyanine green (ICG). We refer to these constructs as NIR erythrocyte-derived transducers (NETs). The objective of this study was to determine if ICG-bound albumin (IbA), as the doping material, could enhance the fluorescence emission of NETs, and evaluate the capability of these nanoprobes in imaging cancer cells. Erythrocytes were isolated from bovine whole blood and depleted of hemoglobin to form erythrocyte ghosts (EGs). EGs were then extruded through nanosize porous membranes in the presence of 10-100 µm ICG or Iba (1:1 molar ratio) to form ICG- or IbA-doped NETs. The resulting nanosize constructs were characterized for their diameters, zeta-potentials, absorption, and fluorescence emission spectra. We used fluorescence microscopic imaging to evaluate the capability of the constructs in imaging SKOV3 ovarian cancer cells. Based on dynamic light-scattering measurements, ICG- and IbA-doped NETs had similar diameter distributions (Z-average diameter of 236 and 238 nm, respectively) in phosphate-buffered saline supplemented with 10% fetal bovine serum, which remained nearly constant over the course of 2 h at 37 °C. Despite a much-lower loading efficiency of IbA (∼0.7-8%) as compared to ICG (10-45%), the integrated normalized fluorescence emission of IbA-NETs was 2- to 6-fold higher than ICG-doped NETs. IbA-NETs also demonstrated an enhanced capability in fluorescence imaging of SKOV3 ovarian cancer cells, and can serve as potentially effective nanoprobes for the fluorescence imaging of cancerous cells.

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.

Erythrocyte-derived nano-probes functionalized with antibodies for targeted near infrared fluorescence imaging of cancer cells.

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.

Photoinduced dynamics of a cyanine dye: parallel pathways of non-radiative deactivation involving multiple excited-state twisted transients.

Cyanine dyes are broadly used for fluorescence imaging and other photonic applications. 3,3'-Diethylthiacyanine (THIA) is a cyanine dye composed of two identical aromatic heterocyclic moieties linked with a single methine, -CH[double bond, length as m-dash]. The torsional degrees of freedom around the methine bonds provide routes for non-radiative decay, responsible for the inherently low fluorescence quantum yields. Using transient absorption spectroscopy, we determined that upon photoexcitation, the excited state relaxes along two parallel pathways producing three excited-state transients that undergo internal conversion to the ground state. The media viscosity impedes the molecular modes of ring rotation and preferentially affects one of the pathways of non-radiative decay, exerting a dominant effect on the emission properties of THIA. Concurrently, the polarity affects the energy of the transients involved in the decay pathways and further modulates the kinetics of non-radiative deactivation.

Correction: Photoinduced dynamics of a cyanine dye: parallel pathways of non-radiative deactivation involving multiple excited-state twisted transients.

[This corrects the article DOI: 10.1039/C4SC02881C.].