Synthesis, characterization andin vitro evaluation of triptolide-lysozyme conjugate for renal targeting delivery of triptolide

Abstract

A triptolide-lysozyme (TP-LZM) conjugate was synthesized to achieve renal specific delivery and to reduce the side effects of triptolide. Triptolide was coupled to lysozyme through succinic via an ester bond with an average coupling degree of 1 mol triptolide per 1 mol lysozyme. The lysozyme can specifically accumulate in the proximal tubular cells of the kidney, making it a potential carrier for targeting drugs to the kidney. The structure of triptolide succinate (TPS) was confirmed by IR, 1H-NMR, MS and UV. The concentrations of triptolide in various samples were determined by reversed-phase high-performance liquid chromatography (HPLC). In this study, the physicochemical and stability profiles of TP-LZM under various conditions were investgated the stability and releasing profiles of triptolide-lysozyme (TP-LZM) under various conditions. In vitro release trails showed triptolide-lysozyme was relatively stable in plasma (less than 30% of free triptolide released) and could release triptolide quickly in lysosome (more than 80% of free triptolide released) at 37 degrees C for 24 h. In addition, the biological activities of the conjugate on normal rat kidney proximal tubular cells (NRK52E) were also tested. The conjugate can effectively reduce NO production in the medium of NRK52E induced by lipopolysaccharide (LPS) but with much lower toxicity. These studies suggest the possibility to promote curative effect and reduce its extra-renal toxicity of triptolide by TP-LZM conjugate.

The targeting of 14-succinate triptolide-lysozyme conjugate to proximal renal tubular epithelial cells

“…Endogenous low molecular weight proteins (LMWP), lysozyme, was utilized as renal-specific drug carriers to form 14-succinate triptolide-lysozyme (TPS-LZM) conjugate [14][15][16]. Our data demonstrated the efficacy and safety of this PTEC specific TP therapeutics as well as its potential clinical applications as a local immunosuppressant.…”

“…14-Succinate TP was synthesized and coupled to LZM and labeling of TPS-LZM with FITC was carried out as described previously, FITC was conjugated to LZM [14,17]. Briefly, 1 mg of TPS-LZM was dissolved in carbonate buffer solution (PBS, pH 7.2).…”

“…TPS-LZM conjugate was prepared as previously described [14]. In attempt to ease the track of cell binding, uptake, processing of TPS-LZM in vitro and its PTEC specific targeting and biodistribution in vivo, this conjugate was labeled with fluorescein isothiocyanate (FITC) (Fig.…”

Kinetic release of triptolide after injection of renal‐targeting 14‐succinyl triptolide‐lysozyme in a rat kidney study by liquid chromatography/mass spectrometry

“…The stability of TP under the following conditions has been studied: (1) the stability of TPS-LZM in the sample preparation process; and (2) the stability of TP after extraction from the kidney homogenate samples. Our preliminary study showed that it is the lysosomal enzymes that are responsible for the hydrolysis of TPS-LZM in the kidney (Zheng et al, 2007). To avoid enzymatic hydrolysis and subsequent release of free TP during the sample storage and preparation, we adopted rapid liquid-nitrogen freezing and organic solvent extraction.…”

“…To address these issues with the potential eventual clinical applications, we designed and prepared the renaltargeting 14-succinyl triptolide-lysozyme (TPS-LZM, Fig. 1, III) (Fransen et al, 1991;Haas et al, 1993Haas et al, , 1997Zheng et al, 2007). Although many studies have been carried out on the immunosuppressant effects of TP and its derivatives in renal diseases, most of them have focused on pharmacological features.…”