# Labeling Peptides with Radioiodine: An Overview of Traditional and Emerging Techniques
## 6 Modern Examples of Biological Applications of Radioiodinated Peptides  
Building on the radioiodination methodologies discussed above, we next highlight several recent applications starting with a recent one in the field of systemic amyloidosis imaging and therapy. Systemic amyloidosis is a progressive disorder marked by the extra cellular deposition of amyloid fibrils in organs and tissues, leading to organ dysfunction. Current treatments primarily focus on reducing the production of amyloid precursor proteins. However, a curative approach likely necessitates the removal of existing amyloid deposits. A recent study describes the development and characterization of a prototypic pan-amyloid binding peptide-antibody fusion molecule, murine Igp5 (mlgp5), designed to enhance macrophage uptake of amyloid [74]. Radiolabeling was fundamental for achieving the results. The mlgp5 molecule is a murine IgG1-IgG2a hybrid immunoglobulin with a pan amyloid-reactive peptide, p5, genetically fused to the \(N\)-terminal of the immunoglobulin light chain. The p5 peptide is a synthetic polybasic l-amino acid reagent known to bind amyloid fibrils and associated heparan sulfate glycosaminoglycans through electrostatic interactions. The researchers radiolabeled mlgp5, along with m11-1F4 and peptide p5, with iodine-125 (\({}^{125}\)I). This was achieved using chloramine T as an oxidant. The radioiodinated products served several critical purposes in the study. Firstly, they were used in in vitro binding studies, specifically a suspension-phase pulldown assay, to assess differences in solution phase reactivity with amyloid-like fibrils and patient extracts. This also functioned as a quality control step to ensure the radiolabeling process did not compromise the binding capabilities of the molecules. Secondly, and perhaps most significantly for in vivo investigations, \({}^{125}\)I-mlgp5 was used in biodistribution measurements in a murine model of systemic AA amyloidosis and in healthy wild-type control mice. This involved small animal SPECT/CT imaging to visualize the distribution of radioactivity over time. Measurement of tissue radioactivity in harvested organs was also studied, expressed as percent injected dose per gram of tissue (%ID/g), to quantify the uptake and retention of \({}^{125}\)I-mlgp5 in different organs. Finally, microautoradiography (ARG) of tissue sections was used to determine the microdistribution of \({}^{125}\)I-mlgp5 at a cellular level and to assess its co-localization with amyloid deposits identified by Congo red staining. These in vivo studies demonstrated that \({}^{125}\)I-mlgp5 bound rapidly and specifically to amyloid deposits in organs such as the liver and spleen of mice with AA amyloidosis, with sustained retention for up to 72 h. Importantly, there was minimal non-specific uptake in healthy tissues. In contrast, the control molecule \({}^{125}\)I-mlgp5G, with a mutated peptide sequence, showed no significant retention in amyloid-laden organs, highlighting the specificity of mIgp5 for amyloid. The findings from the autoradiography precisely correlated the presence of the radioactive tracer with the amyloid deposits, further confirming the in vivo amyloid-targeting ability of mIgp5. Radioiodination of the stapled peptide VIP116 was successfully achieved using a dehalogenation-resistant radioiodination prosthetic agent SIB [75]. The conjugation of the [**\"**]SIB prosthetic agent was specifically directed to a lysine residue present on the VIP116 peptide. This lysine residue is connected via an aminohexanoic spacer at the \(N\)-terminus of the peptide. The synthesis process involved two main steps, similar to the preparation of the non-radioactive analogue: (1) Synthesis of SIB (the radioiodinated [**\"**]SIB itself was first synthesized). This was accomplished using a tributytlin precursor and \(N\)-chlorosuccinimide as the oxidizing reagent with a yield of \(75.9\pm 7.8\%\).