Cargo-Loaded Red Cell Extracellular Vesicles Production
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Solution Overview
Problem
Current methods for producing red cell extracellular vesicles (RCEVs) loaded with cargo do not effectively ensure that red cells subjected to hypotonic encapsulation can subsequently produce therapeutically effective cargo-loaded RCEVs with a clinically effective amount of the originally encapsulated cargo, as the restorative hypertonic resealing step is often required, limiting the yield and efficiency of vesiculation.
Innovation Solution
Omitting the restorative hypertonic resealing step after hypotonic encapsulation allows red cells to produce a higher quantity of cargo-loaded red cell extracellular vesicles (CLRCEVs) by maintaining them in hypotonic conditions, which increases the number of CLRCEVs produced per cargo-loaded red cell and enhances their uptake by immune cells, such as macrophages, without the need for subsequent resealing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If restorative hypertonic resealing is performed after hypotonic encapsulation, then red cell integrity is maintained, but the yield and efficiency of vesiculation is limited
Solution Approach 1:
The invention extracts the restorative hypertonic resealing step from the standard hypotonic encapsulation protocol. By removing this step, the patent allows red cells to remain in a primed state that dramatically enhances vesiculation efficiency, accepting some loss in individual cell integrity in exchange for massive gains in vesicle production yield
Solution Approach 2:
The invention applies preliminary hypotonic treatment to red cells to prime them for enhanced vesiculation before the actual vesicle formation process. This preliminary action creates a physiological state in the red cells that predisposes them to produce significantly more vesicles, effectively preparing the cells in advance for maximum productivity
2Quantity of substance
If hypotonic encapsulation is followed by hypertonic resealing, then cargo retention in red cells is maintained, but the number of CLRCEVs produced per red cell is reduced
Solution Approach 1:
The invention removes the hypertonic resealing step that would otherwise restore red cell volume and membrane integrity. This extraction allows the cells to maintain a primed state that maximizes vesicle production, accepting some cargo loss in exchange for dramatically increased CLRCEV yield per starting red cell
Solution Approach 2:
The invention changes the osmotic parameter conditions by maintaining red cells in or returning to hypotonic conditions rather than restoring to isotonic conditions. This parameter change keeps the cells in a physiological state that favors vesiculation and maximizes CLRCEV production efficiency
3Stability of the object's composition
If standard hypotonic encapsulation with hypertonic resealing is used, then red cells are restored to normal state, but immune cell uptake of resulting RCEVs is reduced
Solution Approach 1:
The invention applies preliminary hypotonic treatment that primes red cells not only for vesiculation but also for enhanced immune recognition. This preliminary action modifies the red cell state in a way that translates to improved uptake of the resulting CLRCEVs by immune cells like macrophages
Solution Approach 2:
The invention converts what would normally be considered a harmful or destabilizing hypotonic condition into a beneficial state. Rather than restoring red cells to their normal stable state, the patent exploits the hypotonic-primed state to enhance both vesicle production and subsequent immune cell uptake, turning a potential weakness into an advantage
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
This approach significantly increases the production of high-quality CLRCEVs containing the cargo, which are readily taken up by immune cells, particularly in the liver and spleen, making them an effective drug delivery system for penetrating biological barriers and targeting tumors, with enhanced immune modulation capabilities.
Implementation Method 1
red cells subjected to hypotonic encapsulation
Implementation Method 2
restorative hypertonic resealing
Data Source
AI summary
The application relates to the use of loaded red blood cells (e.g. “RBCs”, “red cells” or “erythrocytes”) or red blood cell precursors to produce red cell extracellular vesicles (RCEVs) containing cargos, including cargos comprising biologically active ingredients. Notable red cell precursors include hematopoietic stem cells (HSCs), induced pluripotent stem cells (iPSCs), and reticulocytes. The cargo may comprise nucleic acids, proteins, small molecules, or components of a gene editing system, including CRISPR/Cas9. The RCEVs may be used to treat of diseases and disorders including autoimmune disorders, cancers, cardiovascular diseases, gastrointestinal diseases, genetic disorders, or inflammatory diseases. The RCEVs may also be used to carry antigens and or immune modulator, for use in eliciting immune or immune tolerance responses. Also provided are methods for producing cargo loaded RCEVs (CLRCEVs) by first loading cargo into red cells and then by vesiculating the cargo loaded red cells to yield the CLRCEVs.