Conducting Polymer-Coated Carbon Cloth Captures and Releases Extracellular Vesicles by a Rapid and Controlled Redox Process

Extracellular Vesicles
/References

Electrochemical techniques offer great opportunities for the capture of chemical and biological entities from complex mixtures and their subsequent release into clean buffers for analysis. Such methods are clean, robust, rapid, and compatible with a wide range of biological fluids. Here, we designed an electrochemically addressable system, based on a conducting terpolymer [P(EDOT-co-EDOTSAc-co-EDOTEG)] coated onto a carbon cloth substrate, to selectively capture and release biological entities using a simple electrochemical redox process. The conducting terpolymer composition was optimized and the terpolymer-coated carbon cloth was extensively characterized using electrochemical analysis, Raman and Fourier transform-infrared spectroscopy, water contact angle analysis, and scanning electron microscopy. The conductive terpolymer possesses a derivative of EDOT with an acetylthiomethyl moiety (EDOTSAc), which is converted into a "free" thiol that then undergoes reversible oxidation/reduction cycles at +1.0 V and -0.8 V (vs Ag/AgCl), respectively. That redox process enables electrochemical capture and on-demand release. We first demonstrated the successful electrochemical capture/release of a fluorescently labeled IgG antibody. The same capture/release procedure was then applied to release extracellular vesicles (EVs), originating from both MCF7 and SKBR3 breast cancer cell line bioreactors. EVs were captured using the substrate-conjugated HER2 antibody which was purified from commercially available trastuzumab. Capture and release of breast cancer EVs using a trastuzumab-derived HER2 antibody has not been reported before (to the best of our knowledge). A rapid (2 min) release at a low potential (-0.8 V) achieved a high release efficiency (>70%) of the captured, HER2+ve, SKBR3 EVs. The developed system and the electrochemical method are efficient and straightforward and have vast potential for the isolation and concentration of various biological targets from large volumes of biological and other (e.g., environmental) samples.

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Background Previous studies found that cigarette smoke (CS) exposure could induce NSCLC malignancy and miRNA dysregulation. Yet, the association of CS-induced miRNA dysregulation and NSCLC malignancy has not been clearly understood. This study aimed to evaluate the effect of CS exposure in smokers on the expression of miR-10b-5p and miR-320b in extracellular vesicles (EVs) from NSCLC patients. Material and methods Bioinformatic analysis was conducted to validate miRNA candidates. Blood and tissue samples were collected from NSCLC patients (n = 21) with smoking and non-smoking history. EVs were isolated from plasma and miRNAs were extracted from the isolated EVs. The miRNAs relative expression was analyzed and compared. Results In silico analysis identified miR-320b and miR-10b-5p as potential biomarkers for diagnosing NSCLC in smokers. Experimental analysis revealed differential expression of EVs-associated miRNAs in NSCLC patients with smoking and non-smoking histories. EVs-associated miR-10b-5p was significantly overexpressed in smoker NSCLC patients (p = 0.000), while miR-320b expression was significantly lower in this group (p = 0.018). Additionally, smoking intensity influenced miRNA expression, with higher smoking intensity correlating with increased miR-10b-5p expression and decreased miR-320b expression. ROC analysis demonstrated that EVs were a superior source of miRNAs compared to plasma for NSCLC diagnostics. miR-10b-5p and miR-320b in EVs showed higher diagnostic performance (AUC 0.878; 0.739) compared to plasma (AUC 0.628; 0.559). Conclusion CS exposure induces different expression of miR-10b-5p and miR-320b in EVs of NSCLC patients with smoking history. EV-related miR-10b-5p and miR-320b showed potential to be utilized as prognostic biomarker for smokers NSCLC patients.

2025

Extracellular vesicles (EVs) have emerged as promising therapeutics with broad clinical applications as diagnostic biomarkers and therapeutic drug delivery systems. Yet, these biopharmaceuticals pose a challenge in terms of manufacturing due to their complexity and heterogeneity. Despite advancements in the field, current purification technologies lack scalability and/or selectivity. Affinity chromatography (AC) − coupling unmatched specificity and scalability − could be used to simplify purification processing and generate clinical-grade EVs with higher titers and purity. In the present work, we report the implementation of an immuno-AC resin to capture and purify EVs directly from clarified cellular feedstocks. Firstly, to guide and support marker selection, vesicle phenotype characterization was conducted using single particle interferometric reflectance image sensing (SP-IRIS) coupled with immunofluorescence. CD81 was the marker which shown to be more present and more likely to have the other markers (CD63 and CD9). Thus, anti-CD81 VHH ligand was generated and evaluated towards recombinant CD81 protein and CD81 bearing EV particles using surface plasmon resonance (SPR). Different chromatographic studies with Anti-CD81 ligand immobilized onto agarose beads resin were conducted to optimize the process parameters (residence time, dynamic binding capacity and impurity clearance). At residence time of 2 min, on average 40 % of pure triple tetraspanin-positive EV fraction was recovered. The enrichment in EV particles herein obtained, based on scale-up calculations, it would be possible to produce 1 × 1013 EVs from a 1L cell culture, while meeting impurity requirements in a single-step purification process (impurity removal over 2 log reduction value). A single-step purification process is possible, enabling the successful isolation of homogeneous EVs population, counting with a final HCP titer of 60 ng/mL and 9 ng/mL of dsDNA impurities. EV’s morphological integrity and internalization ability were also demonstrated, showcasing elution’s efficiency under mild conditions. Overall, this work contributes to the development of a novel, highly specific, AC technology using a camelid-derived affinity ligand which, bridging the scalability requirements demanded of large-scale production, could potentiate the advent of EV-based therapies.

2025

Extracellular vesicles (EVs) have emerged as promising therapeutics with broad clinical applications as diagnostic biomarkers and therapeutic drug delivery systems. Yet, these biopharmaceuticals pose a challenge in terms of manufacturing due to their complexity and heterogeneity. Despite advancements in the field, current purification technologies lack scalability and/or selectivity. Affinity chromatography (AC) − coupling unmatched specificity and scalability − could be used to simplify purification processing and generate clinical-grade EVs with higher titers and purity. In the present work, we report the implementation of an immuno-AC resin to capture and purify EVs directly from clarified cellular feedstocks. Firstly, to guide and support marker selection, vesicle phenotype characterization was conducted using single particle interferometric reflectance image sensing (SP-IRIS) coupled with immunofluorescence. CD81 was the marker which shown to be more present and more likely to have the other markers (CD63 and CD9). Thus, anti-CD81 VHH ligand was generated and evaluated towards recombinant CD81 protein and CD81 bearing EV particles using surface plasmon resonance (SPR). Different chromatographic studies with Anti-CD81 ligand immobilized onto agarose beads resin were conducted to optimize the process parameters (residence time, dynamic binding capacity and impurity clearance). At residence time of 2 min, on average 40 % of pure triple tetraspanin-positive EV fraction was recovered. The enrichment in EV particles herein obtained, based on scale-up calculations, it would be possible to produce 1 × 1013 EVs from a 1L cell culture, while meeting impurity requirements in a single-step purification process (impurity removal over 2 log reduction value). A single-step purification process is possible, enabling the successful isolation of homogeneous EVs population, counting with a final HCP titer of 60 ng/mL and 9 ng/mL of dsDNA impurities. EV’s morphological integrity and internalization ability were also demonstrated, showcasing elution’s efficiency under mild conditions. Overall, this work contributes to the development of a novel, highly specific, AC technology using a camelid-derived affinity ligand which, bridging the scalability requirements demanded of large-scale production, could potentiate the advent of EV-based therapies.

2025
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