Peptide-Based Fusion Proteins for Monitoring, Modulating, and Imaging Fibronectin Matrix Assembly in Fibrotic Diseases

Background

Fibrotic diseases, such as diabetic retinopathy (DR), are characterized by the excessive accumulation of extracellular matrix (ECM) proteins, which leads to tissue dysfunction and significant clinical complications. In the context of DR, high glucose levels trigger abnormal fibronectin (FN) and collagen deposition in the retinal basement membrane, resulting in thickening and loss of vascular integrity. This pathological ECM expansion disrupts the inner blood-retinal barrier (BRB), causing leakage, loss of pericytes and endothelial cells, and ultimately vision impairment. Despite the prevalence and severity of these fibrotic complications, there are currently no therapies that directly target or reverse the underlying fibrotic processes, highlighting a critical unmet need in the management of DR and related disorders.

Current approaches to studying and intervening in ECM assembly and fibrosis are limited by several factors. Traditional methods rely on endpoint assays, such as immunoblotting or immunofluorescence, which only provide static snapshots of matrix composition and do not allow for real-time monitoring of dynamic assembly processes. Additionally, existing pharmacological inhibitors, such as the FUD peptide or PKC inhibitors, lack specificity and can have off-target effects, making it difficult to dissect the precise roles of individual ECM components in disease progression. Genetic approaches, like siRNA knockdown, are labor-intensive and not easily adaptable for high-throughput screening or live-cell imaging. These limitations hinder the development of targeted anti-fibrotic therapies and impede a detailed understanding of how specific ECM proteins, such as FN, contribute to barrier dysfunction and disease pathology.

Technology

This technology centers on the development of specialized fusion proteins that enable real-time monitoring and modulation of fibronectin (FN) matrix assembly, a key process in fibrotic diseases such as diabetic retinopathy. Each fusion protein is engineered with a central peptide—such as S2, S3, or S20—identified via phage display for their specific interactions with FN, flanked by a purification tag (like GST) and a fluorescent tag (such as GFP), resulting in a molecule with a molecular weight between 30-100 kDa. The S2-based fusion proteins bind specifically to FN fibrils during their assembly, allowing live visualization of matrix dynamics in complex cell culture models. Meanwhile, S3 and S20 peptides are designed to inhibit and enhance FN matrix formation, respectively. These fusion proteins can be used for live imaging, high-throughput screening of matrix assembly inhibitors, and as targeted diagnostic or therapeutic agents, with applications ranging from basic research to the development of anti-fibrotic therapies. What differentiates this technology is its comprehensive and versatile approach to both probing and intervening in pathological matrix assembly. Unlike existing tools, these fusion proteins not only enable high-resolution, real-time imaging of FN matrix dynamics in physiologically relevant co-culture systems but also provide direct means to modulate matrix assembly—either inhibiting or enhancing it as needed. The use of a 3-cell co-culture model (endothelial cells, pericytes, and astrocytes) closely mimics the retinal environment, allowing for precise dissection of cell-specific contributions and matrix interactions under disease-relevant conditions. The modular design of the fusion proteins, with customizable tags and the ability to incorporate therapeutic or imaging moieties, further expands their utility for targeted drug delivery and diagnostic imaging. This integrated platform thus addresses critical gaps in both the mechanistic understanding and therapeutic targeting of fibrotic diseases, offering a powerful toolkit for research and clinical translation.

Benefits

• Enables real-time visualization and monitoring of fibronectin (FN) matrix assembly and remodeling in live cells
• Provides tools for high-throughput screening of inhibitors targeting FN matrix assembly, addressing unmet needs in anti-fibrotic drug development
• Allows modulation of FN matrix formation through peptides that inhibit or enhance assembly, offering therapeutic potential
• Facilitates targeted delivery of diagnostics or therapeutics to FN-rich pathological sites such as fibrotic tissues or tumors
• Supports detailed study of extracellular matrix dynamics in disease models, including diabetic retinopathy
• Offers stable and specific binding to FN fibrils, enabling long-term imaging and analysis even after sample processing
• Utilizes a physiologically relevant 3-cell co-culture model to mimic complex tissue environments for accurate assessment

Applications

• Live imaging of fibronectin assembly
• High-throughput screening for anti-fibrotics
• Targeted delivery to fibrotic tissues
• Diagnostic imaging of fibrotic sites
• Therapeutic modulation of ECM formation

Additional info

These fusion proteins comprise a central peptide (S2, S3, or S20) flanked by a GST purification tag and a fluorescent marker like GFP. Weighing 30–100 kDa, they specifically interact with fibronectin. S2-based constructs enable real-time visualization of matrix assembly, while S3 and S20 respectively inhibit or enhance fibril formation. These molecular tools support high-throughput screening for anti-fibrotic therapeutics and targeted diagnostic imaging for pathologies like diabetic retinopathy.

Publication: Sci Rep 16, 3581 (2026).

Intellectual Property & Development Status:

Patent protection is pending, 18/804,806

Princeton is currently seeking commercial partners for the further development and commercialization of this opportunity.