Synaptotagmin C2A Domain Imaging Agent for Cell Death Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current imaging agents for detecting cell death, such as annexin V and C2A domain-based constructs, face limitations including large size, complex production processes, slow renal clearance, and generation of multiple species with varying binding affinities, which restrict their clinical use for molecular imaging of cell death in vivo.
Innovation Solution
A modified synaptotagmin I C2A domain polypeptide with a cysteine residue at position 78, allowing for the attachment of a detectable label, is used to create a molecular imaging agent that binds to phosphatidylserine, enabling homogeneous imaging of cell death with improved clearance and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If annexin V is used as an imaging agent, then cell death detection capability is achieved, but the large size (36 kDa) and slow renal clearance limit clinical use
Solution Approach 1:
The invention extracts and utilizes only the C2A domain of synaptotagmin I, which is the specific phosphatidylserine-binding module, rather than using the entire annexin V protein. This extracted domain is then engineered with a single cysteine residue for label attachment, creating a smaller, faster-clearing imaging agent that retains the essential cell death detection function.
2Measurement precision
If wild type C2A domain is modified on lysine residues, then detectable labeling is achieved, but multiple species with varying binding affinities are generated
Solution Approach 1:
Instead of modifying multiple lysine residues throughout the C2A domain (which creates heterogeneous species), the invention introduces a single cysteine residue at a specific local position (position 78) within the domain. This localized modification approach ensures that all labeled C2A molecules are identical, providing uniform binding affinity and eliminating the complexity of multiple species.
3Measurement precision
If biotinylation is used to label C2A domain, then detectable labeling is achieved, but intermolecular reaction of multiple avidin molecules generates large molecular weight conjugates
Solution Approach 1:
The invention replaces the biotin-avidin labeling system with a more straightforward cysteine-reactive label conjugation approach. This eliminates the need for multiple avidin molecules and their associated intermolecular reactions, resulting in smaller, simpler conjugates that clear faster and are easier to produce without requiring complex purification steps.
4Reliability
If iron oxide nanoparticles are used to label C2A domain, then cell death detection is achieved, but the relatively large size (∼25 nm) restricts extravasation and clearance
Solution Approach 1:
The invention changes the size parameter of the imaging construct by replacing large iron oxide nanoparticle labels with smaller molecular-weight detectable labels attached to the engineered cysteine residue. This parameter change enables better extravasation from vasculature and improved renal clearance while maintaining the phosphatidylserine binding capability for cell death detection.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The modified imaging agent provides enhanced specificity and efficiency in detecting cell death by binding uniformly to phosphatidylserine, allowing for accurate visualization of apoptotic or necrotic cells, thus overcoming the limitations of existing agents.
Implementation Method 1
The C2A domain of synaptotagmin I, which also binds to phosphatidylserine (PS), has been labelled with iron oxide nanoparticles and used to detect cell death in vivo
Data Source
Figure 1
Figure 2
Figure 3~4
AI summary
This invention relates to molecular imaging agents comprising an S78C mutant synaptotagmin I C2A domain. These agents may be useful in detecting or assessing cell death in vitro and in vivo, for example in tumours following cancer treatment.