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Updated: Jan 23, 2026
Rapid Isolation of Human Breast Milk-Derived Extracellular Vesicles
Published on: November 14, 2025
Hyaluronic acid-engineered milk extracellular vesicles to target triple negative breast cancer through CD44
Filipa A Soares1,2, Beatriz Salinas3,4,5,6, Salette Reis1
1LAQV, REQUIMTE, Departamento de Ciências Químicas, Universidade Do Porto, Porto, Portugal.
Context:
Cancer therapy remains a challenge in healthcare, particularly in the context of triple-negative breast cancer (TNBC), where targeted therapies are still scarce.
Objective:
Addressing this issue, our study explores a novel targeting approach using small extracellular vesicles (sEVs) isolated from cow milk, functionalized with hyaluronic acid (HA) to target the overexpressed cluster of differentiation 44 (CD44) cell surface receptor in TNBC cells.
Materials & Methods:
A method for isolating sEVs from cow milk was optimized, and the obtained sEVs were fully characterized in terms of size, morphology, and protein markers. Subsequently, milk-derived sEVs were covalently bound with HA of varying molecular weights (MW, 20-60 kDa, 250 kDa, 1000-1600 kDa) and binding and internalization dynamics were investigated. Breast cancer cell lines, MDA-MB-231 (TNBC and CD44+) and MCF-7 (CD44-), were used as in vitro models to evaluate CD44 selectivity.
Results:
The binding and internalization studies unveiled enhanced selectivity of functionalized sEVs for CD44-overexpressing cells compared to non-functionalized sEVs. Notably, higher MW HA exhibited enhanced binding capacity, with partial internalization occurring through CD44 endocytic mechanisms.
Discussion And Conclusion:
In summary, this work introduces a sEVs isolation method and sheds light on the role of HA MW in enhancing cellular uptake of CD44 overexpressing cancer cells.
Insights
Researchers developed a novel method to isolate and functionalize small extracellular vesicles (sEVs) from cow milk with hyaluronic acid (HA). This approach enhances targeted delivery to triple-negative breast cancer (TNBC) cells by utilizing the CD44 receptor.
Area of Science:
- Biomedical Engineering
- Nanotechnology
- Cancer Research
Background:
- Triple-negative breast cancer (TNBC) lacks effective targeted therapies.
- Targeting strategies are crucial for improving cancer treatment efficacy.
- Cluster of differentiation 44 (CD44) is a cell surface receptor often overexpressed in TNBC.
Purpose of the Study:
- To develop a novel targeting approach for TNBC using milk-derived small extracellular vesicles (sEVs).
- To functionalize sEVs with hyaluronic acid (HA) to target CD44 receptors on TNBC cells.
- To investigate the impact of HA molecular weight on sEVs binding and internalization.
Main Methods:
- Optimized isolation of sEVs from cow milk and characterized their properties.
- Covalently conjugated HA of various molecular weights (MW) to milk-derived sEVs.
- Evaluated CD44 selectivity using TNBC (MDA-MB-231) and non-TNBC (MCF-7) cell lines in vitro.
Main Results:
- Functionalized sEVs demonstrated enhanced binding selectivity for CD44-overexpressing TNBC cells.
- Higher molecular weight HA showed increased binding capacity.
- Partial internalization of functionalized sEVs occurred via CD44-mediated endocytosis.
Conclusions:
- A robust method for isolating and functionalizing milk-derived sEVs was established.
- HA functionalization significantly enhances the targeting of CD44-positive TNBC cells.
- This study provides a promising nanocarrier system for targeted cancer therapy.
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