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Figure 5 In vivo recovery of integrin-binding cyclic Arg-Gly-Asp peptide (cRGD) from vascular tissues surrounding cRGD-loaded stents and quantification of cRGD vascular tissue levels. (A) The high-performance liquid chromatography (HPLC) chromatogram obtained from a vascular tissue lysate surrounding an unloaded polymer stent implanted for 4 weeks and compared with a tissue lysate that was spiked with cRGD after removal from the stent. The spectrum from the cRGD-containing and cRGD-free tissue lysates are essentially superimposable. In the preparation from the cRGD-spiked tissue lysate (blue), a peak is visible at an elution time around 16.2 min that precisely co-elutes with the standard cRGD peak (black). As an HPLC control, pure cRGD peptide alone underwent chromatography (red). Only the relevant part of the spectrum is shown, but overall, only a limited number of substances with a relative molecular weight (Mr) < 3,000 Da was obtained (latter not shown). (B) Recovery of cRGD from the surrounding vascular tissue 4 weeks after stent implantation. The HPLC chromatogram obtained from a vascular tissue lysate surrounding the cRGD-loaded stent implanted for 4 weeks (green). After separation of the tissue from the stent, the tissue was lysed and spiked with additional cRGD (blue). Pure cRGD underwent chromatography as an HPLC standard (red). The two lysates show a peak that elutes at the same time (at 16 min) as the standard cRGD. Spiking the tissue with cRGD leads to a higher peak and shows the chromatographic identity of the spiked cRGD species with that loaded onto the stent. The cRGD peaks are indicated by arrows. (C) Identification of the recovered implanted cRGD by electrospray ionization mass spectrometry analysis. The cRGD from tissue lysate surrounding the cRGD-loaded 4-week implanted stent was detected by HPLC (see B), the assumed cRGD-containing HPLC peak at 16 min (green spectrum in B) was isolated and lyophilized, and the cRGD identity was verified by MS analysis. The M+2 peak of cRGD was clearly detectable as indicated. (D) Confirmation of stent-derived cRGD identification by ESI MS of the spiked tissue lysate. The tissue lysate was spiked with additional cRGD before work-up (which was identical to that as described in panel C) and HPLC. The M+2 peak appears at a markedly enhanced intensity but at the same Mr as in (C).