Multistage Intracellular Delivery via Segmented Permeabilization and Active Insertion
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Solution Overview
Problem
Current methods for intracellular delivery and transfection, such as sonoporation and electroporation, are inefficient as they primarily focus on permeabilizing the cell membrane without ensuring effective transport of DNA into the cell, leading to suboptimal transfection rates and potential DNA degradation in the cytoplasm.
Innovation Solution
A multistage procedure involving sequential permeabilization of the cell membrane using mechanical, electrical, or thermal techniques followed by active insertion of substances through electrophoresis, magnetophoresis, or acoustophoresis to ensure efficient delivery of charged molecules like DNA into the cell interior.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional physical transfection methods (sonoporation, electroporation) are used to permeabilize the cell membrane, then membrane permeability is improved, but transfection efficiency deteriorates due to insufficient DNA transport and increased DNA degradation in cytoplasm
Solution Approach 1:
The transfection process is divided into distinct stages: first permeabilization of the cell membrane using physical methods (sonoporation or electroporation), then active transport of DNA into the cell interior using a separate mechanism (acoustophoresis or electrophoresis). This segmentation allows optimization of each step independently, ensuring both membrane permeability and effective DNA transport are achieved.
Solution Approach 2:
The patent introduces intermediary physical fields (acoustic fields for acoustophoresis or electric fields for electrophoresis) that act as mediators to transport DNA through the permeabilized membrane into the cell interior. These intermediary mechanisms bridge the gap between membrane permeabilization and successful DNA delivery, preventing DNA degradation in the cytoplasm.
2Reliability
If strong physical fields are applied for membrane permeabilization, then membrane permeability is improved, but cell mortality increases
Solution Approach 1:
The patent employs periodic or pulsed application of physical fields (ultrasonic pulses for sonoporation, electric pulses for electroporation) rather than continuous exposure. This periodic action allows the membrane to recover between pulses, reducing cell mortality while maintaining effective permeability for DNA delivery.
Solution Approach 2:
The patent optimizes parameters of the physical fields (intensity, duration, frequency, pulse timing) to achieve the minimum effective dose for membrane permeabilization. By carefully controlling these parameters, the patent reduces harmful effects on cells while maintaining sufficient permeability for DNA transport.
3Ease of manufacture
If passive diffusion is used for DNA entry after permeabilization, then process simplicity is maintained, but transfection efficiency deteriorates due to DNA degradation and slow transport
Solution Approach 1:
The patent replaces passive diffusion (a passive mechanical process) with active transport mechanisms driven by acoustic fields (acoustophoresis) or electric fields (electrophoresis). This substitution actively propels DNA into the cell interior, preventing degradation and ensuring efficient delivery, while the overall process remains relatively simple to implement.
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 enhances transfection efficiency by decoupling permeabilization and insertion steps, reducing cell mortality, and achieving higher delivery rates of large molecules, including DNA, into specific intracellular targets, as demonstrated by improved GFP expression and biomolecule delivery in various cell types.
Implementation Method 1
permeabilization includes permeabilization of the cell membrane using a technique selected from: mechanical poration, electrical poration, thermal poration
Implementation Method 2
permeabilization includes permeabilization of the cell membrane using a technique selected from: mechanical poration, electrical poration, thermal poration
Implementation Method 3
permeabilization includes permeabilization of the cell membrane using a technique selected from: mechanical poration, electrical poration, thermal poration
Implementation Method 4
the insertion is conducted using an energetic technique selected from: an electrophoretic technique, a magnetophoretic technique, an acoustophoretic technique
Implementation Method 5
the insertion is conducted using an energetic technique selected from: an electrophoretic technique, a magnetophoretic technique, an acoustophoretic technique
Implementation Method 6
the insertion is conducted using an energetic technique selected from: an electrophoretic technique, a magnetophoretic technique, an acoustophoretic technique
Data Source
AI summary
Embodiments of the present disclosure provide a multistage procedure for treatment of biological samples (e.g., living cells with membranes, and the like) with a substance (e.g., a drug, DNA, RNA, plasmids, and other biomolecules or materials) to achieve more efficacious intracellular delivery and transfection.