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

Abstract

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.

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

“…Our recent in vivo study indicates that ∼35% of the injected amount of RBC ghosts ICG remained in the blood for 24 h following intravenous injection in healthy mice. 11 Spleen, liver, and lungs, organs of the reticuloendothelial system rich in macrophages, contained ∼40% of the injected RBC ghosts ICG at 24 h. 11 Changes in the mechanical characteristic may also affect the interaction of the particles with macrophages. In particular, rigidity and shape of the RBC are thought to override the signaling by CD47, with rigidified RBCs completely engulfed by macrophages in a shorter time than native RBCs.…”

“…Flipping of PS to the outer leaflet, either as a result of RBCs' senescence or induced during the fabrication of RBC-based carrier systems serves as a signal for phagocytosis by splenic macrophages. 10,11 While the importance of membrane biochemical characteristics in relation to the longevity of RBC-based carriers has been recognized, the morphological characteristics and membrane mechanical properties of such carriers have not been extensively studied. The normal biconcave discoidal shape and the extensive membrane deformability allow for the repeated passage of RBCs through narrow capillaries and nanosized (∼200−400 nm) splenic endothelial slits during their lifetime.…”

“…Another important mechanism is related to the role of the membrane phospholipid, phosphatidylserine (PS), which is normally confined to the inner leaflet of the membrane. Flipping of PS to the outer leaflet, either as a result of RBCs’ senescence or induced during the fabrication of RBC-based carrier systems serves as a signal for phagocytosis by splenic macrophages. , …”

Recent Progress in Red Blood Cells-Derived Particles as Novel Bioinspired Drug Delivery Systems: Challenges and Strategies for Clinical Translation

“…It was reported that the sialic acid was persistent post hypotonic treatment and mechanical extrusion imparting negative charge to the RBC membrane. But sonication presumably results in the degradation of the sialic acid content of the RBC membrane as a decrease in zeta potential of the RBCs and the engineered RBCs was noted in a study ( Tang et al, 2022 ). Hence, the surface charges of both the RBC carriers and RBC-camouflaged nanoparticles have been noted to be negative owing to the negative surface charge of the native RBC membrane.…”

“…During the fabrication of RBC-based particles, there is PS flipping from the inner to the outer leaflet, signaling phagocytic clearance of the particles from the circulation due to macrophage recognition. Researchers have reported a cholesterol enrichment method for the RBC membrane that has successfully reduced PS externalization to enhance the longevity of the RBC-based micron-sized particles in circulation ( Tang et al, 2022 ).…”