Transdermal delivery of peptides and other biological macromolecules is limited due to skin's inherent low permeability. Here, the authors report the use of a deep eutectic solvent, choline and geranate (CAGE), to enhance topical delivery of proteins such as bovine serum albumin (BSA, molecular weight: ≈66 kDa), ovalbumin (OVA, molecular weight: ≈45 kDa) and insulin (INS, molecular weight: 5.8 kDa). CAGE enhances permeation of BSA, OVA, and insulin into porcine skin ex vivo, penetrating deep into the epidermis and dermis. Studies using tritium-labeled BSA and fluorescein isothiocyanate labeled insulin show significantly enhanced delivery of proteins into and across porcine skin, penetrating the skin in a time-dependent manner. Fourier transform IR spectra of porcine stratum corneum (SC) samples before and after incubation in CAGE show a reduction in peak area attributed to SC lipid content, suggesting lipid extraction from the SC. Circular dichroism confirms that CAGE does not affect insulin's secondary conformation. In vivo studies in rats show that topical application of 10 U insulin dispersed in CAGE (25 U kg −1 insulin dose) leads to a highly significant 40% drop in blood glucose levels in 4 h that is relatively sustained for 12 h. Taken together, these studies demonstrate that CAGE is a promising vehicle for transdermal delivery of therapeutic proteins; specifically, as a noninvasive delivery alternative to injectable insulin for the treatment of diabetes.
Chronic accumulation of plasma advanced oxidation protein products (AOPPs) promotes renal fibrosis. However, the mechanism at the cellular level has not been clarified. In the present study, endocytic assay of human proximal tubular cells (HK-2 cells) demonstrated that AOPPs-human serum albumin (HSA) (in vitro preparations of chloramine- modified HSA) were significantly endocytosed in a dose-dependent manner at a higher level than HSA. The expression of CD36, a transmembrane protein of the class B scavenger receptor, in HK-2 cells was confirmed in the immunoblot analysis. In a cellular assay using overexpressing human CD36 in Chinese hamster ovary (CHO) cells, AOPPs-HSA were significantly endocytosed by CD36-CHO cells but not by mock-CHO cells. Furthermore, the endocytic association and degradation of AOPPs-HSA by HK-2 cells was significantly inhibited by anti-CD36 antibody treatment, suggesting that CD36 is partly involved in the uptake of AOPPs-HSA by HK-2 cells. AOPPs-HSA upregulated the expression of CD36 in a dose-dependent manner. In addition, AOPPs-HSA upregulated the generation of intracellular reactive oxygen species and the secretion of transforming growth factor (TGF)-beta1 in HK-2 cells, whereas anti-CD36 antibody neutralizes the upregulation of TGF-beta1. These results suggest that AOPPs-HSA may cause renal tubular injury via the CD36 pathway.
To determine the pharmacokinetic properties of advanced oxidation protein products (AOPP), we prepared oxidized human serum albumin (oxi-HSA) using chloramine-T (a hypochlorite analogue) in vitro. The AOPP and dityrosine content of oxi-HSA (AOPP content, 244.3+/-12.3 microM; dityrosine content, 0.7+/-0.11 nmol of dityrosine/mg protein) were similar to those of uremic patients. In structural analysis, the increases in AOPP and dityrosine content of HSA induced slight decreases in its alpha-helical content. In pharmacokinetic analysis, oxi-HSA left the circulation rapidly, and organ distribution of oxi-HSA 30 min after intravenous injection was 51% for the liver, 23% for the spleen, and 9% for the kidney, suggesting that the liver and spleen were the main routes of plasma clearance of oxi-HSA. The liver and spleen uptake clearance of oxi-HSA were significantly greater than those of normal HSA (CLliver, 5058+/-341.6 vs 24+/-4.2 microL/hr [p<0.01]; CLspleen, 2118+/-322.1 vs 32+/-2.7 microL/hr [p<0.01]). However, uptake by other organs was not significantly affected by oxidation. These results suggest that the liver and spleen play important roles in elimination of AOPP.
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