Milk-Derived Extracellular Vesicle Isolation for High Purity
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Solution Overview
Problem
Current methods for isolating extracellular vesicles (EVs) from natural sources like milk face challenges due to high contamination levels and are difficult to scale up, leading to variable yields and qualities, and existing drug loading methods are inefficient for oral delivery.
Innovation Solution
A process involving enzyme-based casein-coagulation, thermal treatment, and ultrafiltration with specific pore size membranes, followed by ion-strength controlled dialysis, to isolate high-purity EVs suitable for drug loading and systemic delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional isolation methods (ultracentrifugation, precipitation) are used to obtain EVs from milk, then EV isolation can be achieved, but high contamination levels with proteins and other milk components occur
Solution Approach 1:
The isolation process is divided into multiple sequential steps: (1) casein micelle removal by acidification to pH 4.6, (2) lipoprotein removal by organic solvent extraction, and (3) EV concentration by ultrafiltration. This segmentation allows each step to target specific contaminants, achieving high purity while maintaining scalability for industrial production
Solution Approach 2:
The patent extracts and removes specific interfering components (casein micelles, lipoproteins) from the milk matrix before EV isolation. By taking out these contaminants in dedicated extraction steps, the final EV preparation achieves superior purity without relying on contamination-prone ultracentrifugation
2Manufacturing precision
If laboratory-scale isolation methods are used, then EV purity can be achieved, but the process is difficult to scale up for industrial production
Solution Approach 1:
The patent replaces ultracentrifugation (complex mechanical system requiring specialized equipment) with acidification and ultrafiltration (simpler chemical and physical processes). This substitution maintains high purity while enabling scalable industrial production using standard equipment
Solution Approach 2:
The process utilizes parameter changes (pH adjustment to 4.6, temperature control during ultrafiltration) to drive separation and concentration. These parameter-based approaches are easily scalable and do not require the complex mechanical systems needed for ultracentrifugation
3Quantity of substance
If existing drug loading methods are used, then EVs can be loaded with drugs, but the efficiency is low and oral delivery is not achieved
Solution Approach 1:
The patent performs preliminary action by isolating highly pure EVs free from contaminating proteins before drug loading. This preliminary purification ensures that subsequent drug loading occurs on intact, functional EVs, dramatically improving loading efficiency and enabling effective oral delivery
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 method achieves scalable, high-purity EV isolation maintaining biological function, enabling efficient systemic delivery and drug loading, particularly with Amphotericin B, suitable for treating fungal infections.
Implementation Method 1
enzyme-based casein-coagulation
Implementation Method 2
ultrafiltration with specific pore size membranes
Implementation Method 3
ion-strength controlled dialysis
Implementation Method 4
thermal treatment
Data Source
AI summary
The present invention relates to the field of biotechnology, and particularly to milk derived extracellular vesicles, and provides a process for isolating such extracellular vesicles from milk and milk related fluids. The present invention is also related to compositions containing said extracellular vesicles derived from milk, particularly suitable for use in pharmaceutical, veterinary, cosmetic and/or nutraceutical applications.