Drug delivery using natural biological carriers, especially erythrocytes, is a rapidly developing field. Such erythrocytes can act as carriers that prolong the drug's action due to its gradual release from the carrier; as bioreactors with encapsulated enzymes performing the necessary reactions, while remaining inaccessible to the immune system and plasma proteases; or as a tool for targeted drug delivery to target organs, primarily to cells of the reticuloendothelial system, liver and spleen. To date, erythrocytes have been studied as carriers for a wide range of drugs, such as enzymes, antibiotics, anti-inflammatory, antiviral drugs, etc., and for diagnostic purposes (e.g. magnetic resonance imaging). The review focuses only on drugs loaded inside erythrocytes, defines the main lines of research for erythrocytes with bioactive substances, as well as the advantages and limitations of their application. Particular attention is paid to in vivo studies, opening-up the potential for the clinical use of drugs encapsulated into erythrocytes.

https://www.mdpi.com/1999-4923/12/3/276/pdf

Erythrocytes as Carriers: From Drug Delivery to Biosensors.

Cellular “backpacks” are a new type of anisotropic, nanoscale thickness microparticle that may be attached to the surface of living cells creating a “bio-hybrid” material. Previous work has shown t...

Freely Suspended Cellular “Backpacks” Lead to Cell Aggregate Self-Assembly

Advances on erythrocyte-mimicking nanovehicles to overcome barriers in biological microenvironments

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 Nanoparticles as a Theranostic Agent for Near‐Infrared Fluorescence Imaging and Thrombolysis of Blood Clots

Cell membrane cloaking technique is bioinspired nanotechnology that takes advantage of naturally derived design cues for surface modification of nanoparticles. Unlike modification with synthetic materials, cell membranes can replicate complex physicochemical properties and biomimetic functions of the parent cell source. This technique indeed has the potential to greatly augment existing nanotherapeutic platforms. Here, we provide a comprehensive overview of engineered cell membrane-based nanotherapeutics for targeted drug delivery and biomedical applications and discuss the challenges and opportunities of cell membrane cloaking techniques for clinical translation.

/pdf/cell-membrane-cloaked-nanotherapeutics-for-targeted-drug-fi1fisy9.pdf

Cell Membrane-Cloaked Nanotherapeutics for Targeted Drug Delivery

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.

/pdf/intravital-vascular-phototheranostics-and-real-time-1ts5nkznx1.pdf

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

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 μ M 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.

/pdf/near-infrared-fluorescent-erythrocyte-mimicking-particles-52s7ggxpl7.pdf

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

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.

/pdf/phototheranostics-using-erythrocyte-based-particles-3gumpq5690.pdf

Phototheranostics Using Erythrocyte-Based Particles.

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.

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

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.

Jack C. Tang

Papers

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

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

Phototheranostics Using Erythrocyte-Based Particles.

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

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