A kidney-selective biopolymer for targeted drug delivery

Abstract

Improving drug delivery to the kidney using renal-targeted therapeutics is a promising but underdeveloped area. We aimed to develop a kidney-targeting construct for renal-specific drug delivery. Elastin-like polypeptides (ELPs) are nonimmunogenic protein-based carriers that can stabilize attached small-molecule and peptide therapeutics. We modified ELP at its NH₂-terminus with a cyclic, seven-amino acid kidney-targeting peptide (KTP) and at its COOH-terminus with a cysteine residue for tracer conjugation. Comparative in vivo pharmacokinetics and biodistribution in rat and swine models and in vitro cell binding studies using human renal cells were performed. KTP-ELP had a longer plasma half-life than ELP in both animal models and was similarly accumulated in kidneys at levels fivefold higher than untargeted ELP, showing renal levels 15- to over 150-fold higher than in other major organs. Renal fluorescence histology demonstrated high accumulation of KTP-ELP in proximal tubules and vascular endothelium. Furthermore, a 14-day infusion of a high dose of ELP or KTP-ELP did not affect body weight, glomerular filtration rate, or albuminuria, or induce renal tissue damage compared with saline-treated controls. In vitro experiments showed higher binding of KTP-ELP to human podocytes, proximal tubule epithelial, and glomerular microvascular endothelial cells than untargeted ELP. These results show the high renal selectivity of KTP-ELP, support the notion that the construct is not species specific, and demonstrate that it does not induce acute renal toxicity. The plasticity of ELP for attachment of any class of therapeutics unlocks the possibility of applying ELP technology for targeted treatment of renal disease in future studies.

Keywords: biopolymer; drug delivery; elastin-like polypeptide; kidney targeting peptide; renal disease.

Fig. 1.

Overview of polypeptides used in this study. The sequence of the targeting agents and overview of the ELP structure for all polypeptides used in this study are shown.

Fig. 2.

Enhancement of kidney specificity using kidney targeting peptides. A: rats were administered fluorescently labeled ELP, SynB1-ELP, Tat-ELP, or KTP-ELP, and organ biodistribution was determined by ex vivo fluorescence imaging. Quantitative analysis showed that the highest accumulation of all peptides was in the kidney (B), and the targeting agents significantly increased kidney specificity as assessed by kidney-to-liver (C) and kidney-to-heart ratios (D). Values are means ± SE. *^,#Statistically significant difference between indicated groups as assessed by a one-way ANOVA with post hoc Tukey’s multiple comparison, P < 0.05. **Polypeptide levels undetectable over autofluorescence.

Fig. 3.

Plasma and tissue pharmacokinetics of KTP-ELP and ELP. Blood was sampled, and whole body in vivo images were collected at various time points up to 24 h after bolus intravenous injection. A: plasma data were fit to a two-compartment pharmacokinetic model. B: in vivo images were quantified to determine the mean fluorescence radiant efficiency at each time point to elucidate peak tissue levels and whole body clearance kinetics. Effects of fluorophor loss from polypeptides were assessed by measuring the plasma fluorescence before and after precipitation of the proteins with trichloroacetic acid (TCA). C: fluorescence levels after TCA were corrected for dilution and compared with the pre-precipitation fluorescence to calculate the percentage of free dye at each time point. Values are means ± SD in A and C. Values are means ± SE in B.

Fig. 4.

Intrarenal distribution of renally targeted ELPs. Four hours after intravenous infusion of fluorescently labeled ELP, SynB1-ELP, Tat-ELP, or KTP-ELP, rat kidneys were rapidly frozen and cut into 20-μm sections. A: slides were scanned using a fluorescence slide scanner. Identical scan settings were used to directly compare the total kidney levels. B and C: slides were stained with CD31 to mark blood vessels (green; B) or synaptopodin to mark podocytes (green; C), and KTP-ELP-rhodamine (red) was imaged using confocal microscopy (scale bar = 50 μm). D: specificity of antibody cell type markers was confirmed by lack of signal when the primary antibody was omitted from the staining protocol.

Fig. 5.

Chronic infusion of ELP and KTP-ELP do not have adverse effects on kidney function or development of renal injury. A: during a 14-day infusion of saline, ELP, or KTP-ELP (10 mg·kg⁻¹·day⁻¹), daily body weights were recorded. B: on the final day of infusion, renal function was measured by the FITC-sinistrin clearance method. Values are means ± SD of 8 rats per group, with individual animals’ values shown by the markers. C: representative renal cross section (trichrome, ×40) showing the absence of renal fibrosis and preserved renal parenchyma in ELP and KTP-ELP-treated kidneys compared with saline-treated controls.

Fig. 6.

KTP enhances ELP deposition in the swine kidney after intravenous administration. Pigs were given fluorescently labeled ELP or KTP-ELP by intravenous injection. A: plasma was sampled intermittently for determination of polypeptide clearance, and plasma clearance data were fit using a two-compartment pharmacokinetic model. B: the presence of unbound dye in the plasma at each time point was assessed by TCA precipitation and fluorescence measurement. C: kidney distribution was determined 4 h after injection by ex vivo whole organ fluorescence imaging in both intact and vertically sliced kidneys (arrows indicated major vascular areas). D: whole organ ex vivo fluorescence of kidneys and other major organs were quantified relative to standard curves of each agent. Values are means ± SD in A and B and mean ± SE in D. *P < 0.05. E: intrarenal distribution was determined by confocal microscopy (scale bar = 100 μm).

Fig. 7.

KTP-ELP enhances ELP binding to human renal cells. Cell binding (A–C) and cell survival (D–F) of SynB1-ELP, Tat-ELP, and KTP-ELP relative to ELP control were determined by flow cytometry and a cell viability assay, respectively, in primary human glomerular microvascular endothelial cells (A and D), primary human podocytes (B and E), and primary human proximal tubule epithelial cells (C and F). Values are means ± SE. *P < 0.05.