Phage Display In Vivo: Protocols for Identifying Brain-Targeting Peptides in Mouse Models of Neurodegeneration

In vivo phage display is a versatile strategy for an unbiased identification of peptide ligands that selectively interact with molecular targets, in vitro, in cell, and in vivo, in living organisms. In a previous study, we validated this technology to identify a brain targeting peptide. Here, we detail the protocol for selection using phage display in vivo of peptide targeting the mouse brain, in health and disease. Given the need of alternative models to study degenerative disease, such as Alzheimer's disease, we elected the murine model of neuroinflammation and neurodegeneration as an example. This model utilizes the stereotaxic intracerebroventricular administration of streptozotocin (STZ) to induce neuroinflammation, an event that recapitulates key pathological hallmarks of Alzheimer's disease. Following the establishment of the disease model, a randomized phage display peptide library-a collection of billions of phages, each displaying a different peptide sequence on their surface-is administered intravenously into healthy and diseased mice. This allows selective interaction of individual phage with vascular, tissue-specific receptors under physiological or in a pathological condition with which a compromised blood-brain barrier may alter brain homeostasis. After systemic circulation, animals undergo perfusion and organs are harvested for phage recovery and amplification, followed by iterative rounds of biopanning to enrich for high-affinity ligands. This approach offers a robust platform for the discovery of novel diagnostic probes, therapeutic ligands, and molecular markers relevant to brain biology, in health, neuroinflammation and neurodegeneration. Most importantly, the adaptability of this technology enables its application across diverse murine models of human disease, thereby establishing an easy and systematic approach to facilitate the translation of laboratory discoveries into a wide spectrum of human diseases.