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. 2025 Dec;63(1):411-427.
doi: 10.1080/13880209.2025.2511807. Epub 2025 Jun 5.

Hyaluronic acid-engineered milk extracellular vesicles to target triple negative breast cancer through CD44

Affiliations

Hyaluronic acid-engineered milk extracellular vesicles to target triple negative breast cancer through CD44

Filipa A Soares et al. Pharm Biol. 2025 Dec.

Abstract

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.

Keywords: Bovine milk; CD44; breast cancer; hyaluronic acid; selective targeting; small extracellular vesicles.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Physicochemical characterization of the isolated sEVs: (a) TEM image for morphological evaluation (100 000x); (b) Nanoparticle tracking analysis size distribution of a representative sample; detection of (C1) TSG101 and (C2) CD81 in the isolates (F2 and F3, in duplicate), by Western blot (cropped gels), compared to a positive control of sEVs obtained from goat milk.
Figure 2.
Figure 2.
(a) Schematic representation of the reaction of activation of HA carboxylic acid groups using carbodiimide chemistry. (b) FTIR spectra of activated HA (HA-NHS-ester), HA and NHS. The characteristic absorption peaks are indicated with arrows; the red arrow highlights the new peak corresponding to the formation of an ester bond.
Figure 3.
Figure 3.
In Vitro metabolic activity evaluation with (a) MCF-7 and (b) MDA-MB-231, determined by the resazurin assay. Both cell lines were treated with different concentrations of sEVs, sEVs-SCy5, sEVs-SCy5@20-60kDa, sEVs-SCy5@250kDa and sEVs-SCy5@1000–1600kDa, for 24h. Reference data is represented by the results with untreated cells (0 µg/ml of protein). Statistical analysis was performed using the two-way ANOVA and Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).
Figure 4.
Figure 4.
Immunofluorescence of CD44 protein (FITC - green) on (a2) MCF-7 and (b2) MDA-MB-231 BC cell lines. CLSM control images of (a1) MCF-7, (b1) MDA-MB-231 cells. Cell nuclei were stained with Hoechst 33,342 dye (blue). (c) Quantification of the green channel intensity per number of cells. The analysis was carried out in 8 images of each cell line, with a minimum of 5 cells per image. Statistical analysis was performed using unpaired t-test (****p ≤ 0.0001).
Figure 5.
Figure 5.
(A) Evaluation of sEVs (functionalized and non-functionalized) uptake by MDA-MB-231 cell line after (a1) 30 min and (a2) 2h; and MCF-7 cell line after (a3) 30 min and (a4) 2h. Quantification of fluorescent sEVs on CLSM images (red channel intensity per number of cells). The analysis was carried out in 8 images of each cell line, with a minimum of 5 cells per image. Statistical analysis was performed using the two-way ANOVA and Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). (B) CLSM images of MDA-MB-231 (b1) and MCF-7 (b2) cell lines after 2h of incubation with sEVs-SCy5@1000–1600kDa. Cell nuclei were stained with Hoechst 33342 dye (blue) and functionalized and non-functionalized sEVs were labeled with SCy5 (red). Zoomed-in cells are presented on the right side of each image to facilitate visualization of the sEVs distribution on cells.
Figure 6.
Figure 6.
Evaluation of sEVs (functionalized and non-functionalized) uptake by MCF-7 and MDA-MB-231 BC cell lines, after 24h of incubation. (a) CLSM images of BC cell lines after 24h of incubation with 10 μg/ml of sEV-SCy5, sEVs-SCy5@20-60kDa, sEVs-SCy5@250kDa and sEVs-SCy5@1000–1600kDa. Cell nuclei were stained with Hoechst 33,342 dye (blue) and functionalized and non-functionalized sEVs were labeled with SCy5 (red). (b) Quantification of fluorescent sEVs on CLSM images (red channel intensity per number of cells). The analysis was carried out in 8 images of each cell line, with a minimum of 5 cells per image. Statistical analysis was performed using the two-way ANOVA and Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).
Figure 7.
Figure 7.
Evaluation of sEVs-SCy5, sEVs-SCy5@20-60kDa, sEVs-SCy5@250kDa, sEVs-SCy5@1000–1600kDa internalization by MDA-MB-231 and MCF-7 cell lines, after trypsinization. Flow cytometry results corresponding to incubation times of (a) 30 min, (b) 2h and (c) 24h, of each type of sEVs (10 µg/ml). Statistical analysis was performed using the two-way ANOVA and tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).
Figure 8.
Figure 8.
Assessment of cellular internalization pathways of sEVs-SCy5 and sEVs-SCy5@1000–1600kDa on MDA-MB-231 and MCF-7 cell lines, by flow cytometry. Cells were pre-incubated with the respective pathway inhibitor (chlorpromazine and HA 1000–1600kDa) or incubated at 4 °C for 30 min before incubation with sEVs-SCy5/sEVs-SCy5@1000–1600kDa for 1 h. Fluorescence values were normalized to those of cells (of the same cell line) without any inhibition. Statistical analysis was performed using the two-way ANOVA and Tukey’s multiple comparisons test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

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