Non-invasive Evaluation of Laminin-peptide (PGPIKVAV) Hydrogel for wound Imaging

Introduction: Wound healing is a complex yet dynamic and well-organized process consisting of four continuous, overlapped and precisely programmed phases. These phase required several cells types, soluble mediators, various growth/chemokine factors and their signaling cascades. Healing is dependent on effective synchronization of these phases in stipulated time frame. Topical administration of hydrogel offer many advantages for wound healing due to their capability to incorporate bioactive agents which can be delivered precisely on the wound site. Laminin, a structural component of the basement membrane can interact directly with integrin receptors on epithelial and endothelial cells and helps in cell migration, proliferation, differentiation and angiogenesis. Imaging could examine the wound severity and healing potential and progress non-invasively which mostly depend on visual inspection only.

The rational of this study was to synthesized ^99mTc-PGPIKVAV- hydrogel as a SPECT probe for non-invasive assessment of healing.

Methods: The peptide was synthesized on rink amide resin using Fmoc chemistry by employing SPPS by coupling (HOBT/DIC) and deprotection (20% (v/v) piperidine). The peptide conjugate was cleaved from resin by TFA cocktail. The peptide-hydrogel formulation was developed using a polymer sodium carboxymethylcellulose (CMC). Various analytical techniques were used to evaluate the formation & efficacy of peptide hydrogel formulation. The biocompatibility of synthezied peptide was evaluated by hemolytic, cell integrity and MTT studies. The in vitro cell proliferation study was performed in HUVEC cell to evaluate the proliferation ability. The peptide hydrogel radiolabelling was optimized with ^99mTc in aqueous solution at pH 4-7.

Results: The synthesized peptide conjugate was purified by HPLC and characterized by molecular ion peak at 779.3451 [M+H]⁺ in mass spectrometry. The physiochemical characterization of peptide hydrogel formulation i.e. swelling ratio (96%), viscosity (10000 cps), spreadibility (10 mm), etc had shown the potential for topical application. The biocompatibility (erythrocytes lysis was observed to be 4.71%, No cellular cytotoxicity upto 100nm concentration on HUVEC cell) and cell proliferation studies reveal the safety and stability of peptide with significant rate of proliferation upto 100nm concentration. The peptide hydrogel formulation binds with ^99mTc with high specific activity & efficiency (~80%). The preliminary SPECT images showed peptide hydrogel remains at the wound site for specific time frame.

Conclusions: The in-vitro results indicate that peptide hydrogel (PGPIKVAV) has good wound healing potential. The imaging techniques showed that topically applied peptide hydrogel remains at the administered area for 24h. This preliminary study could add the added advantage to examine the wound severity and healing potential non-invasively.