Extracellular Vesicle Protein Loading for Reliable Intracellular Delivery
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Solution Overview
Problem
Current intracellular protein delivery systems face challenges in achieving high efficiency and stability, particularly due to the large size, hydrophilicity, and membrane impermeability of proteins, leading to low loading and lysosome escape rates, and engineered extracellular vesicles (EVs) require improvements for therapeutic efficacy and safety.
Innovation Solution
A dual-plasmid co-transfection method using a CAG promoter-gene of interest-containing plasmid and a pMD2.G plasmid to load target proteins or polypeptides into EVs, specifically utilizing HEK293F cells to produce EVs that can efficiently deliver biologically-active proteins, such as cGAS and Cre, with targeted modification strategies for improved cell and tissue specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional nanocarriers (liposomes, polymers, inorganic nanomaterials) are used for intracellular protein delivery, then protein loading is achieved, but the loading efficiency and lysosome escape efficiency are low
Solution Approach 1:
The patent uses extracellular vesicles (EVs) as an intermediary carrier to deliver proteins into cells. EVs naturally interact with cell membranes through endocytosis or membrane fusion, serving as a bridge between the protein cargo and the target cell, thereby improving both loading efficiency and delivery reliability compared to traditional nanocarriers
Solution Approach 2:
The patent modifies the surface properties of EVs by conjugating targeting ligands (such as antibodies, peptides, or small molecules) to the EV surface. This changes the physical-chemical parameters of the carrier, enabling active targeting to specific cell types and improving delivery efficiency and reliability
2Reliability
If proteins are delivered intracellularly, then therapeutic effect is achieved, but proteins face challenges due to large size, hydrophilicity, and membrane impermeability
Solution Approach 1:
The patent encapsulates proteins within extracellular vesicles, nesting the protein cargo inside the EV structure. This protects the protein from degradation and facilitates its transport across cellular barriers, making intracellular delivery feasible while maintaining therapeutic efficacy
Solution Approach 2:
EVs serve as a mediator that overcomes the membrane impermeability issue of proteins. The natural ability of EVs to fuse with or be endocytosed by cell membranes allows proteins to be delivered intracellularly without requiring the proteins themselves to cross membranes directly
3Object-affected harmful factors
If engineered EVs are used for delivery, then biocompatibility is improved, but therapeutic concentration and stability require improvement
Solution Approach 1:
The patent applies local quality modification by conjugating targeting ligands to the EV surface, which directs the carrier to specific target cells or tissues. This localized targeting increases the concentration of the therapeutic agent at the disease site while reducing systemic distribution and toxicity
Solution Approach 2:
The patent engineering EVs with multiple functions: they provide passive targeting through the enhanced permeability and retention (EPR) effect for tumor accumulation, active targeting through surface ligands for specific cell recognition, and protection of cargo stability. This multi-functionality achieves both high therapeutic concentration at target sites and reduced systemic toxicity
Data Source
AI summary
A plasmid combination for preparing an extracellular vesicle loaded with a target protein or polypeptide, including a pCAG-GOI plasmid and a pMD2.G plasmid, where GOI refers to a gene sequence corresponding to the target protein or the polypeptide. The pCAG-GOI plasmid is a pCAG-cyclic GMP-AMP synthase (cGAS) gene plasmid, a pCAG-cyclization recombinase (Cre) gene plasmid and a combination thereof. An extracellular vesicle loaded with cyclic GMP-AMP synthase, cyclization recombinase or a combination thereof is provided, which is prepared by transfecting a cell with the plasmid combination. An application of the extracellular vesicle in the prevention or treatment of a cancer is further provided.