A Phospholipid−PEG2000 Conjugate of a Vascular Endothelial Growth Factor Receptor 2 (VEGFR2)-Targeting Heterodimer Peptide for Contrast-Enhanced Ultrasound Imaging of Angiogenesis

Abstract

The transition of a targeted ultrasound contrast agent from animal imaging to testing in clinical studies requires considerable chemical development. The nature of the construct changes from an agent that is chemically attached to microbubbles to one where the targeting group is coupled to a phospholipid, for direct incorporation to the bubble surface. We provide an efficient method to attach a heterodimeric peptide to a pegylated phospholipid and show that the resulting construct retains nanomolar affinity for its target, vascular endothelial growth factor receptor 2 (VEGFR2), for both the human (kinase insert domain-containing receptor - KDR) and the mouse (fetal liver kinase 1 - Flk-1) receptors. The purified phospholipid-PEG-peptide isolated from TFA-based eluents is not stable with respect to hydrolysis of the fatty ester moieties. This leads to the time-dependent formation of the lysophospholipid and the phosphoglycerylamide derived from the degradation of the product. Purification of the product using neutral eluent systems provides a stable product. Methods to prepare the lysophospholipid (hydrolysis product) are also included. Biacore binding data demonstrated the retention of binding of the lipopeptide to the KDR receptor. The phospholipid-PEG2000-peptide is smoothly incorporated into gas-filled microbubbles and provides imaging of angiogenesis in a rat tumor model.

Paramagnetic Phospholipid-Based Micelles Targeting VCAM-1 Receptors for MRI Visualization of Inflammation

“…The activated NHS moiety allowed for the conjugation of DSPE-PEG2000-NH 2 to form DSPE-PEG2000-VCAM1-pep and DSPE-PEG2000-scr-pep with a yield of about 50 %. 26 The formation of the products was monitored by analytical HPLC, whereas purification was carried out by preparative HPLC using a buffered solution of ammonium acetate as eluent. The characterization of the final products was performed by 1 H-NMR and Mass spectrometry (see Supplementary material).…”

Advanced Microbubbles as a Multifunctional Platform Combining Imaging and Therapy

“…Pillai purified phospholipid-PEG2000-peptide which has nanomolar affinity for VEGFR2, and incorporated it into gas-filled microbubbles. In vivo experiments showed the VEGFR2-targeted microbubbles (BR55) greatly provided imaging of angiogenesis [36] (Figure 2). In a further study, Sorace established triple-targeted MBs with antibodies against mouse αvβ3 integrin, P-selectin and VEGFR2 for evaluating the early response to antiangiogenic therapy.…”

Synthesis of VIP-Lipopeptide Using a New Linker to Modify Liposomes: Towards the Development of a Drug Delivery System for Active Targeting

“…3,4) Peptides are generally used to modify liposomes in order to recognize the target receptor on the cell membrane. [5][6][7][8][9] It is easiest to modify the liposome surface when the functional groups of a pre-formed liposome are reacted with peptides in an aqueous environment. 10) However, such methods have several limitations 5,6) : 1) steric hindrance of the liposome leads to low yield, 2) purification of the liposomes is very difficult due to by-products and unreacted reagents, and 3) the amino acid sequence of the peptide can hinder reactivity.…”

“…Although a promising approach to solving this problem is to produce a lipid-protein/peptide conjugate molecule by total synthesis and use it to make liposomes, the modification of the phospholipid requires many protection and deprotection steps of the phosphate group, resulting in poor yields due to hydrolysis in aqueous media. 7,8) We therefore designed 11,12) a novel lipid analog mimicking the structural features of phospholipids that is easily synthesized using a peptide synthesizer (Fig. 1).…”