Application of biotinylated reagents NHS-LC biotin and sulfo-NHS-LC biotin in a new intravascular drug delivery method

NHS-LC Biotin is a long-chain, NHS ester-activated biotinylation reagent for labeling primary amines (such as protein lysine) whose membrane permeability makes it useful for general intracellular labeling. Sulfo NHS-LC Biotin is water-soluble and non-cleavable and can be used as a simple and effective biotinylation reagent for antibodies, proteins, and any other primary amine-containing macromolecules in solution. The specific labeling of cell surface proteins is Another common application for these unique water-soluble and membrane-impermeable reagents.

Katsumi's team published "A Novel Intravascular Drug Delivery Method Utilizing Endothelial Biotinylation and the Avidin-Biotin System" in 2001. In this study, a new intravascular drug delivery system was developed, which will be injected from the catheter To immobilize the drug into the vasculature of the target tissue, the cellular protein of living endothelial cells is first biotinylated directly by a biotinylation reagent, and then bound by an affinity drug or a biotinylated drug using avidin as a linker.

In initial experiments, biotinylation of cultured bovine aortic endothelial cells (BAECs) was studied in vitro by applying a biotinylation reagent (NHS-LC biotin or sulfo-NHS-LC biotin) to washed intact BAEC monolayers and showed that the amount of biotin bound to cells depends on the concentration of biotin reagent applied. Divide the concentration of biotin in the cell lysate by the protein concentration in the lysate (ng biotin/μg protein) to normalize the value to account for possible cell loss during the experiment. Figure 1 shows the relationship between the dose of the biotinylation reagent and the corrected biotin concentration in cell lysates. The concentration of biotin in cell lysates increased with increasing doses of biotinylation reagents. When comparing NHS-LC-biotin and sulfo-NHS-LC-biotin at the same concentration, slightly more cellular proteins were biotinylated by NHS-LC-biotin. For example, cell lysates prepared from 1.8 mM NHS-LC biotin and sulfur-NHS-LC biotin had biotin concentrations of 0.390 and 0.304 ng per 1 g of protein, respectively.

Cell-bound biotin decreases over time after biotinylation, and FITC-avidin is readily available when applied to biotinylated BAEC monolayers. Combine with cells. The LDH release assay showed that the sulfo-NHS-LC biotin was only slightly cytotoxic to BAEC, and the colony formation assay showed only mild adverse effects of the reagents. In vivo studies were performed on the renal arteries of normal rabbits by injecting NHS-LC biotin solution into one kidney through a catheter to biotinylate its vasculature, and injecting the vehicle into the other kidney as a control, followed by perfusion with saline. Finally, a FITC-avidin solution was injected into both kidneys, which were refused with the blood after the catheter was pulled out. In histological sections, more than 85% of glomeruli in biotinylated kidneys were stained with fluorescein, whereas none of the glomeruli in controls were stained. In kidneys collected 2 days after the same surgery, most glomeruli were still brightly stained. Figure 2 shows that after biotinylation, the biotin concentration in ECs decreases over time. When using sulfur-NHS-LC biotin, the concentration of biotinylated protein decreased rapidly over time, with only a small amount remaining after 24 hours. On the other hand, when NHS-LC-Biotin was used, a large number of biotin molecules (60%) were detected within 24 hours, and about 40% were still detected at 48 hours. The biotin half-lives calculated from the data were 38.0 hours (NHS-LC biotin) and 10.8 hours (sulfo-NHS LC-biotin), respectively.

All BAEC were examined by phase-contrast microscopy after 24 hours of biotinylation as controls with 0.18-18 mM sulfo-NHS-LC biotin and solvent exposure, and cells from all groups were negative and did not show any morphological abnormalities. On the other hand, as shown in Figure 3, the group exposed to 18 mM biotinylation reagent showed a slight increase in LDH levels compared to low doses (0, 0.18, and 1.8 mM) of biotinylation (Kruskal-Wo Liss test, p<0.02).

To investigate whether biotinylated inhibitors alter cell viability, their effect on cell proliferation was examined using a colony formation assay. As shown in Figure 4, the number of BAEC colonies decreased with increasing reagent concentration. Inhibition of colony formation was evident at 1.8-5.4 mM (Kruskal-Wallis test, p<0.01).

In this experiment, it was also investigated whether anti-biotinylated drugs could be immobilized on viable biotinylated EC. The results are shown in Figure 5, the concentration of FITC antiviral in cell lysates increased with the increase of the initial dose of FITC antiviral. The correlation coefficient between the initial dose of FITC-avidin and its concentration in cell lysates was 0.987 (p=0.012) when NHS-LC-biotin was used and 0.977 when sulfo-NHS-LC-biotin was used (p=0.026).

In the final experiment, biotinylated kidneys were injected with an anti-biotin solution followed by a fluorescein-biotin solution. Control kidneys were not previously biotinylated but received the same anti-biotin and fluorescein biotin injections as above, and more than 80% of glomeruli were stained in biotinylated kidneys but not in controls. This suggests that biotinylated drugs can be anchored on biotinylated vasculature via anti-biotin without being washed away by blood. No obvious adverse reactions were found in the function of biotinylated kidneys, and this drug delivery system is suitable for the treatment of certain vascular pathological conditions, such as microvascular proliferation in malignant tumors, and continuous drug delivery in certain target organs.

In this study, biotinylation of living endothelial cells was investigated to develop a method to deliver therapeutic drugs to the target area and to anchor the drug to the vascular endothelial cells in the area. Two key elements of this study were the use of catheterization for drug delivery and the use of biotin derivatives of NHS esters for biotinylation. Recent advances in interventional radiology have made it possible to reach many places in the body using vascular catheterization, and using catheterization, we delivered drugs to vascular endothelial cells in the rabbit kidney as a model organ. Several researchers have previously reported the biotinylation of animal tissue cells, such as erythrocytes, lymphocytes, and endothelial cells, using biotin NHS ester derivatives for various purposes. NHS esters are highly reactive with proteins, and compounds with NHS esters can be immobilized on living cells by cellular proteins. Fuente et al. reported the use of isolated lungs to biotinylate the lung endothelium, followed by administration of the biotinylated reagent by intravascular injection. The study by Katsumi's group is the first to use biotinylation of vascular endothelial cells for therapeutic purposes.