Noninvasive assessment of wound-healing angiogenesis with multimeric peptide conjugate (^99mTc-laminin-cRGD)

The ability to depict wounds for the purposes of assessing severity, healing potential and to define the correct treatment for all wound types has largely depends on visual inspection by the experienced clinician only. Advance in emerging techniques include broader evaluation of the wound bed and the quantification of angiogenesis and epithelialization, which is the critical steps in the healing process. Imaging technologies could assess wound severity, healing potential and healing progress in a rapid, objective and noninvasive manner. However only optical technologies are currently in progress for noninvasive wound assessment. Angiognesis plays a crucial role in wound healing by forming new blood vessel from preexisting vessels by invading the wound clot. Growth factor, cytokines and cell adhesive molecule like Laminin and integrin α_vβ₃, plays key roles in regulating the growth of new blood vessels. RGD is the most investigated adhesive peptide known to interact with a number of cell surface receptors belonging to the α_vβ₃ integrin and subtypes. Similarly, Laminin based peptide strongly promote angiogenesis and protease activity via integrin receptor. The rationale of this study was to develop modified ^99mTc-laminin-cRGD as SPECT Imaging probe to access the wound healing progress noninvasively by targeting angiogenesis. The Laminin and cRGD peptide was synthesized by solid phase peptide synthesis through Fmoc chemistry on rink amide resin and chlorotrityl chloride resin respectively. After swelling of resin in dry DMF the F-moc group of resin was deprotected by using 20% (v/v) piperidine solution in rink amide resin and Cl-trityl chloride resin was directly couple with first amino acids. First amino acids was added with HOBT and DIC in dry DMF. All amino acid was coupled in same protocol until the final peptide was achieved. After completion of amino acid coupling the free N-terminal of laminin and c-RGD peptide was coupled through linker. This conjugated peptide cleaved from resin using TFA cocktail solution having scavenger. The conjugate was radiolabeled with ^99mTc in aqueous solution at pH 6.5-7.5. The synthetic yield of multimeric peptide was ~62% yield. Peptide was purified by HPLC and characterized by molecular ion peak at 779.51 (M+ H)+ and 821.58 (M+ H)⁺ in Mass Spectroscopy. In hemolytic studies of multimeric peptide, maximum percentage of erythrocytes lysis was observed to be 4.03 which indicate peptide was stable in blood and nontoxic.The conjugate binds with ^99mTc with high specific activity and efficiency (97.4%). The stability in serum indicated that ^99mTc remained bound to the drug upto 24 h. Further to evaluate the angiogenic effect to monitor the wound progress is under progress. Non-invasive imaging technologies could prove objective assessment modalities and promising advancement in wound assessment, however require further validation. (author)