Peptide interfacial biomaterials improve endothelial cell adhesion and spreading on synthetic polyglycolic acid materials.

Resorbable scaffolds such as polyglycolic acid (PGA) are employed in a number of clinical and tissue engineering applications owing to their desirable property of allowing remodeling to form native tissue over time. However, native PGA does not promote endothelial cell adhesion. Here we describe a novel treatment with hetero-bifunctional peptide linkers, termed "interfacial biomaterials" (IFBMs), which are used to alter the surface of PGA to provide appropriate biological cues. IFBMs couple an affinity peptide for the material with a biologically active peptide that promotes desired cellular responses. One such PGA affinity peptide was coupled to the integrin binding domain, Arg-Gly-Asp (RGD), to build a chemically synthesized bimodular 27 amino acid peptide that mediated interactions between PGA and integrin receptors on endothelial cells. Quartz crystal microbalance with dissipation monitoring (QCMD) was used to determine the association constant (K (A) 1 x 10(7) M(-1)) and surface thickness (~3.5 nm). Cell binding studies indicated that IFBM efficiently mediated adhesion, spreading, and cytoskeletal organization of endothelial cells on PGA in an integrin-dependent manner. We show that the IFBM peptide promotes a 200% increase in endothelial cell binding to PGA as well as 70-120% increase in cell spreading from 30 to 60 minutes after plating.

D-Amino acid peptide residualizing agents bearing N-hydroxysuccinimido- and maleimido-functional groups and their application for trastuzumab radioiodination.

INTRODUCTION: Proteins that undergo receptor-mediated endocytosis are subject to lysosomal degradation, requiring radioiodination methods that minimize loss of radioactivity from tumor cells after this process occurs. To accomplish this, we developed the residualizing radioiodination agent N(ϵ)-(3-[(*)I]iodobenzoyl)-Lys(5)-N(α)-maleimido-Gly(1)-D-GEEEK (Mal-D-GEEEK-[(*)I]IB), which enhanced tumor uptake but also increased kidney activity and necessitates generation of sulfhydryl moieties on the protein. The purpose of the current study was to synthesize and evaluate a new D-amino acid based agent that might avoid these potential problems. METHODS: N(α)-(3-iodobenzoyl)-(5-succinimidyloxycarbonyl)-D-EEEG (NHS-IB-D-EEEG), which contains 3 D-glutamates to provide negative charge and a N-hydroxysuccinimide function to permit conjugation to unmodified proteins, and the corresponding tin precursor were produced by solid phase peptide synthesis and subsequent conjugation with appropriate reagents. Radioiodination of the anti-HER2 antibody trastuzumab using NHS-IB-D-EEEG and Mal-D-GEEEK-IB was compared. Paired-label internalization assays on BT474 breast carcinoma cells and biodistribution studies in athymic mice bearing BT474M1 xenografts were performed to evaluate the two radioiodinated D-peptide trastuzumab conjugates. RESULTS: NHS-[(131)I]IB-D-EEEG was produced in 53.8%±13.4% and conjugated to trastuzumab in 39.5%±7.6% yield. Paired-label internalization assays with trastuzumab-NHS-[(131)I]IB-D-EEEG and trastuzumab-Mal-D-GEEEK-[(125)I]IB demonstrated similar intracellular trapping for both conjugates at 1h ((131)I, 84.4%±6.1%; (125)I, 88.6%±5.2%) through 24h ((131)I, 60.7%±6.8%; (125)I, 64.9%±6.9%). In the biodistribution experiment, tumor uptake peaked at 48 h (trastuzumab-NHS-[(131)I]IB-D-EEEG, 29.8%±3.6%ID/g; trastuzumab-Mal-D-GEEEK-[(125)I]IB, 45.3%±5.3%ID/g) and was significantly higher for (125)I at all time points. In general, normal tissue levels were lower for trastuzumab-NHS-[(131)I]IB-D-EEEG, with the differences being greatest in kidneys ((131)I, 2.2%±0.4%ID/g; (125)I, 16.9%±2.8%ID/g at 144 h). CONCLUSION: NHS-[(131)I]IB-D-EEEG warrants further evaluation as a residualizing radioiodination agent for labeling internalizing antibodies/fragments, particularly for applications where excessive renal accumulation could be problematic.