Red blood cells (RBCs) attract significant interest as carriers of biomolecules, drugs and nanoparticles. In this regard, versatile technologies to attach molecules and ligands to the RBC surface are of great importance. Reported here is a fast and efficient surface painting strategy to attach ligands to the surface of RBCs, and the factors that control the stability and circulation properties of the modified RBCs in vivo. Distearoyl phosphatidylethanolamine anchor-conjugated immunoglobulin (IgG) efficiently incorporates in the RBC membrane following 15–30 min incubation. The optimized RBCs show prolonged circulation in vivo (70% of the injected dose after 48h) and efficient retention of IgG in the membrane with terminal half-life of 73 hours. The IgG construct is gradually lost from the RBCs mainly due to the transfer to plasma components, liver endothelial cells and Kupffer cells. The ligand retention efficiency is partially dictated by ligand type, anchor type, and ligand concentration in the membrane, while RBC half-life is determined by initial concentration of the ligand in the membrane and presence of PEG linker between the ligand and the anchor. This work provides important guidance for non-covalent surface painting of RBCs as well as other types of blood borne cells for in vivo therapeutic and targeting applications.

Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells.

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There is a great interest in targeting and selective ablation of populations of circulating cells for research or therapeutic purposes. Red blood cells (RBCs) are readily available and fully biocompatible long-circulating intravascular carriers (natural life is 120 days) that are amenable to chemical modifications, drug loading and reinjection. Here we demonstrate that using our previously described lipophilic ligand painting strategy, red blood cells (RBCs) could be in one step converted into targeted entities that selectively seek and bind various cells in vitro and in vivo. In vitro, RBCs modified with lipophilic anti-EpCAM or anti-CD45 antibodies efficiently bound to cancer cells and leukocytes, forming characteristic rosettes. In vivo, intravenously injected RBCs painted with anti-CD45 antibody immediately associated with CD45 positive cells in blood, forming RBC-leukocyte rosettes. Moreover, anti-CD45-modified RBCs, but not the same amount of anti-CD45 antibody or anti-CD45-lipid conjugate (1–2 μg/mouse), depleted over 50% of CD45+ leukocytes from circulation, with main clearance organs of leukocytes being liver and spleen with no visible deposition in kidneys and lungs. Anti-CD20 (Rituximab)-painted RBCs efficiently (over 90%) depleted CD19+/CD20+/CD45+ human lymphoma cells in mantle cell lymphoma (MCL) JeKo-1 model, while the same amount of rituximab-lipid (2 μg/mouse) was much less efficient in lymphoma cell depletion. Treatment of MCL mice with rituximab-modified RBCs carrying only 2 μg of the antibody resulted in a significant prolongation of survival as compared to the same amount of antibody-lipid control. Lipophilic ligand-painted RBCs is a novel tool that can be utilized for targeting blood borne cells for experimental immunology and drug delivery applications.

Lipid monolayer coated microbubbles are currently being developed to identify vascular regions that express certain surface proteins as part of the new technique of ultrasound molecular imaging. The microbubbles are functionalized with targeting ligands which bind to the desired cells holding the microbubbles in place as the remaining unbound microbubbles are eliminated from circulation. Subsequent scanning with ultrasound can detect the highly reflectant microbubbles that are left behind. The ultrasound scanning and detection process results in the destruction of the microbubble, creating lipid fragments from the monolayer. Here we demonstrate that microbubbles targeted to 4T1 murine breast cancer cells and human umbilical cord endothelial cells leave behind adhered fragments of the lipid monolayer after exposure to ultrasound with peak negative pressures of 0.18 and 0.8 MPa. Most of the observed fragments were large enough to be resistant to receptor mediated endocytosis. The fragments were not observed to incorporate into the lipid membrane of the cell over a period of 96 min. They were not observed to break into smaller pieces or significantly change shape but they were observed to undergo translation and rotation across the cell surface as the cells migrated over the substrate. These large fragments will apparently remain on the surface of the targeted cells for significant periods of time and need to be considered for their potential effects on blood flow through the microcapillaries and potential for immune system recognition.