Enhancing Gene Delivery to Breast Cancer with Highly Efficient siRNA Loading and pH-Responsive Small Extracellular Vesicles

Academic Background

In recent years, small extracellular vesicles (sEVs) have become a hot topic in the field of drug delivery due to their natural origin and inherent homing properties. sEVs are lipid nanoparticles secreted by most eukaryotic cells, typically ranging from 50 to 150 nanometers in diameter. They carry various biomolecules and can transmit information through intercellular communication, demonstrating excellent structural and physiological stability in vivo. These characteristics make sEVs highly promising for treating a wide range of diseases, particularly in drug delivery.

However, despite the broad prospects of sEVs in drug delivery, their clinical application still faces numerous challenges. First, the large-scale production and efficient purification techniques for sEVs are not yet mature, limiting their widespread clinical use. Second, the heterogeneity of sEVs makes their in vivo effects difficult to predict. Additionally, traditional drug-loading methods are inefficient and struggle to achieve precise cell-targeted delivery. This is especially true for larger biomolecules such as RNA, where the loading efficiency of sEVs is particularly inadequate. These issues severely hinder the application of sEVs in gene therapy.

To address these challenges, researchers have developed a novel gene delivery system that significantly improves the siRNA loading efficiency and targeted delivery capability of sEVs by integrating chiral graphene quantum dots (GQDs) and pH-responsive peptides. This research not only provides new insights into the application of sEVs in gene therapy but also offers a potential solution to overcome chemotherapy resistance in breast cancer.

Source of the Paper

This paper was co-authored by Gaeun Kim, Runyao Zhu, Sihan Yu, Bowen Fan, Hyunsu Jeon, Jennifer Leon, Matthew J. Webber, and Yichun Wang from the Department of Chemical and Biomolecular Engineering at the University of Notre Dame. The study was published on December 23, 2024, in the journal ACS Biomaterials Science & Engineering, titled Enhancing Gene Delivery to Breast Cancer with Highly Efficient siRNA Loading and pH-Responsive Small Extracellular Vesicles.

Research Process and Results

1. Isolation and Characterization of sEVs

The study began by isolating sEVs from human breast cancer cells (MCF-7) and conducting detailed characterization. Transmission electron microscopy (TEM) confirmed the spherical structure and integrity of the sEVs. Nanoparticle tracking analysis (NTA) revealed a size distribution of 127.9 ± 47.0 nanometers and a surface charge of -12.93 ± 0.85 millivolts, indicating good colloidal stability. Additionally, Western blot analysis confirmed the presence of sEV markers (CD9, CD63, CD81, and HSP70), further validating the purity of the sEVs.

2. Synthesis and Characterization of Chiral Graphene Quantum Dots (GQDs)

To enhance the siRNA loading capacity of sEVs, researchers developed chiral graphene quantum dots (GQDs). Using a modified Hummers’ method, GQDs were synthesized and functionalized with D-cysteine to impart chirality. TEM analysis showed that the average size of the GQDs was 6.88 ± 2.16 nanometers. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy further confirmed the chiral functionalization of the GQDs. These chiral GQDs enabled efficient siRNA loading into sEVs through nanoscale chiral interactions while maintaining the structural integrity of the sEVs.

3. Functionalization with pH-Responsive Peptide (GALA)

To enhance the lysosomal escape capability of sEVs, researchers designed a pH-responsive peptide (GALA) and anchored it to the lipid bilayer of sEVs using cholesterol. The GALA peptide undergoes a conformational change from a random coil to an α-helix under acidic conditions, promoting the fusion of sEVs with lysosomal membranes and facilitating the release of siRNA into the cytoplasm. Fluorescence labeling experiments confirmed the successful insertion of the GALA peptide into the sEV membrane and demonstrated significant charge changes under acidic conditions.

4. siRNA Loading and Delivery

Researchers utilized chiral GQDs to load siRNA into sEVs and achieved efficient lysosomal escape through GALA peptide functionalization. Experimental results showed that the siRNA permeation efficiency of the chiral GQD loading method was 63.46 ± 4.07%, 8.82 times higher than that of the traditional sonication method. Additionally, siRNA-loaded sEVs exhibited excellent stability in serum, maintaining siRNA integrity for up to 6 hours.

5. Cellular Uptake and Lysosomal Escape

Using confocal laser scanning microscopy (CLSM), researchers observed that GALA-functionalized sEVs demonstrated significant cytoplasmic delivery in breast cancer cells. Compared to non-functionalized sEVs, GALA-sEVs exhibited a 1.74-fold increase in cytoplasmic delivery efficiency. Furthermore, GALA-sEVs effectively escaped lysosomes under acidic conditions, releasing siRNA into the cytoplasm and achieving efficient gene silencing.

6. TGF-β Gene Silencing and Chemosensitization

Researchers successfully silenced the TGF-β gene in breast cancer cells using siRNA-loaded sEVs, significantly reducing the stiffness of the extracellular matrix (ECM) and thereby enhancing the sensitivity of breast cancer cells to the chemotherapeutic drug doxorubicin (Dox). Experimental results showed that TGF-β gene silencing significantly impaired the migration ability of breast cancer cells and demonstrated a synergistic effect when combined with Dox treatment.

Conclusion and Significance

This study developed an efficient sEV-based gene delivery platform by integrating chiral GQDs and pH-responsive peptides, significantly improving siRNA loading efficiency and targeted delivery capability. This platform not only preserves the natural biological advantages of sEVs but also achieves efficient gene silencing through enhanced lysosomal escape. The results demonstrate the great potential of this system in overcoming chemotherapy resistance in breast cancer, providing new insights for future cancer treatments.

Research Highlights

1. Efficient siRNA Loading: Achieved highly efficient siRNA loading into sEVs using chiral GQDs, with a loading efficiency 8.82 times higher than traditional methods.
2. pH-Responsive Lysosomal Escape: Functionalization with the GALA peptide significantly enhanced the lysosomal escape capability of sEVs, improving cytoplasmic delivery efficiency of siRNA.
3. Synergistic Chemosensitization: TGF-β gene silencing significantly reduced ECM stiffness in breast cancer cells, enhancing the chemotherapeutic efficacy of Dox.

This study not only provides new technical approaches for the application of sEVs in gene therapy but also offers a potential solution to overcome chemotherapy resistance in breast cancer, holding significant scientific and practical value.