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Cell-based therapeutics are one of the most promising and exciting breakthroughs in modern medicine. Modification of the cell surface with ligands, biologics, drugs, and nanoparticles can further enhance the functionality. Previously, we described the synthesis of a dioctadecyl indocarbocyanine Cy3 analog (aminomethyl-DiI) for efficient and stable modification (painting) of mouse erythrocytes with small molecules, enzymes, and biologics. Here, we synthesized a near-infrared aminomethyl dioctadecyl derivative of Cy7 (aminomethyl-DOCy7) and systematically compared it to aminomethyl-DiI as an anchor for the modification of human erythrocytes, Jurkat cells, and primary T cells with immunoglobulin G. To enable copper-free click chemistry modification of cell membranes, we conjugated a methyltetrazine (MTz) group to the amino-indocyanine lipids via a polyethylene glycol (PEG) linker. DOCy7–PEG3400–MTz showed over 99% modification efficiency of human red blood cells (RBCs) at 25 μM. Reaction of trans-cyclooctene (TCO) modified immunoglobulin G (IgG) with DOCy7–PEG4–MTz-modified RBCs (2-step method) resulted in ∼80,000 IgG molecules per erythrocyte, whereas modification with a preconjugated DOCy7–PEG3400–IgG construct (1-step method) resulted in ∼20,000 IgG molecules per erythrocyte as detected by immuno dot-blot. The number of IgG/RBC was controlled by the concentration of IgG. The incubation of RBCs with DiI–PEG3400–MTz resulted in a similar number of IgG/RBC. Modification of the T-lymphocyte cell line Jurkat with IgG resulted in ∼1 × 106IgG/cell with the 1-step and 2-step methods, and the efficiency was similar for DOCy7 and DiI constructs. Finally, we used DOCy7 and DiI constructs to demonstrate efficient modification of primary CD3+T cells from healthy donors. In conclusion, click indocarbocyanine conjugates represent a novel multicolor chemical biology tool kit for efficient surface modification of different cells types and can be used for potential imaging and drug delivery applications involving engineered cells.

Diseases of the joints affect over 10% of the world’s population, resulting in significant morbidity. There is an unmet need in strategies for specific delivery of therapeutics to the joints. Collagen type II is synthesized by chondrocytes and is mainly restricted to the cartilage and tendons. Arthrogen-CIA is a commercially available anticollagen II antibody cocktail that reacts with 5 different epitopes on human, bovine, and mouse collagen II. Arthrogen has been used for induction of experimental rheumatoid arthritis (RA) in mice because of high complement activation on the cartilage surface. Native collagen II might serve as a useful target for potential delivery of therapeutics to the joint. To evaluate the efficiency and specificity of targeting collagen II, Arthrogen was labeled with near-infrared (NIR) dye IRDye 800 or IRDye 680. Using ex vivo NIR imaging, we demonstrate that Arthrogen efficiently and specifically accumulated in the limb joints regardless of the label dye or injection route (intravenous and subcutaneous). After subcutaneous injection, the mean fluorescence of the hind limb joints was 19 times higher than that of the heart, 8.7 times higher than that of the liver, and 3.7 times higher than that of the kidney. Control mouse IgG did not show appreciable accumulation. Microscopically, the antibody accumulated on the cartilage surface of joints and on endosteal surfaces. A monoclonal antibody against a single epitope of collagen II showed similar binding affinity and elimination half-life, but about three times lower targeting efficiency than Arthrogen in vitro and ex vivo, and about two times lower targeting efficiency in vivo. We suggest that an antibody against multiple epitopes of collagen II could be developed into a highly effective and specific targeting strategy for diseases of the joints or spine.

Long blood half-life is one of the advantages of antibodies over small molecule drugs. At the same time, prolonged half-life is a problem for imaging applications or in the case of antibody-induced toxicities. There is a substantial need for antidotes that can quickly clear antibodies from systemic circulation and peripheral tissues. Engineered nanoparticles exhibit intrinsic affinity for clearance organs (mainly liver and spleen). trans-Cyclooctene (TCO) and methyltetrazine (MTZ) are versatile copper-free click chemistry components that are extensively being used for in vivobioorthogonal couplings. To test the ability of nanoparticles to eliminate antibodies, we prepared a set of click-modified, clinically relevant antidotes based on several classes of drug carriers: phospholipid-PEG micelles, bovine serum albumin (BSA), and cross-linked dextran iron oxide (CLIO) nanoparticles. Mice were injected with IRDye 800CW-labeled, click-modified IgG followed by a click-modified antidote or PBS (control), and the levels of the IgG were monitored up to 72 h postinjection. Long-circulating lipid micelles produced a spike in IgG levels at 1 h, decreased IgG levels at 24 h, and did not decrease the area under the curve (AUC) and IgG accumulation in main organs. Long-circulating BSA decreased IgG levels at 1 and 24 h, decreased the AUC, but did not significantly decrease organ accumulation. Long-circulating CLIO nanoworms increased IgG levels at 1 h, decreased IgG levels at 24 h, did not decrease the AUC, and did not decrease the organ accumulation. On the other hand, short-circulating CLIO nanoparticles decreased IgG levels at 1 and 24 h, significantly decreasing the AUC and accumulation in the main organs. Multiple doses of CLIO and BSA were not able to completely eliminate the antibody from blood, despite the click reactivity of the residual IgG, likely due to exchange of IgG between blood and tissue compartments. Pharmacokinetic modeling suggests that short antidote half-life and fast click reaction rate should result in higher IgG depletion efficiency. Short-circulating click-modified nanocarriers are the most effective antidotes for elimination of antibodies from blood. This study sets a stage for future development of antidotes based on nanomedicine.

Accumulation of intravenously injected cytotoxic liposomes in the skin induces serious toxicity. We used single time point and longitudinal intravital microscopy to understand skin accumulation dynamics of non-PEGylated and PEGylated liposomes after systemic injection into mice. Non-PEGylated egg phosphatidylcholine (PC) liposomes showed short circulation half-life (1.3 h) and immediate aggregation in the blood, with some aggregates lodging in skin microvasculature soon after the injection. At 24 h, and more prominently at 48 h postinjection, liposomes appeared in dermal and subdermal cells. PEGylated egg PC liposomes showed long circulation half-life (22 h) and no aggregation in the blood. PEGylated liposomes started to accumulate in the skin microvasculature as soon as 5 min after the injection. Within 3 h postinjection, PEGylated liposomes accumulated in extravascular cells in the dermis and subdermis. Liposomes were present in the skin for at least 7 days postinjection. A regulatory approved PEGylated liposomal doxorubicin (LipoDox) and empty liposomes of the same composition as LipoDox showed similar skin distribution as PEGylated egg PC liposomes, suggesting that this phenomenon is relevant to liposomes of different lipid composition. Decorating liposomes with shorter PEGs (350 or 700) in addition to PEG 2000 did not decrease the deposition. Outside the capillaries, liposomes partially colocalized with CD45-, F4/80+ cells. The accumulation of liposomes was not due to prior neutrophil/platelet binding and transport across endothelium. Moreover, our studies have excluded a role of complement in the skin accumulation of liposomes. Further understanding of mechanisms of this important phenomenon can improve the safety of liposomal nanocarriers.