U38 - Sterilization of Electrospun Soy Protein Fibers Loaded with Extracellular Vesicles

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Thea Spellmeyer

Undergraduate Researcher
Carnegie Mellon University, New Jersey, United States

Last Author(s)

NR

Nader Rezazadeh

PhD candidate
Carnegie Mellon University, Pennsylvania, United States

Primary Investigator(s)

PC

Phil Campbell

Research Professor in Department of Biomedical Engineering
Carnegie Mellon University
Pittsburgh, Pennsylvania, United States

Introduction: : Affordable, sustainable, and effective wound dressings are crucial for abrasion healing. In recent years, there has been a push to explore electrospinning polymer solutions of polyethylene oxide (PEO) and soy protein isolate (SPI) into nano-scale fibers to create wound dressing films. Soy is cost-effective, easily scalable, biodegradable, and provides amino acids for cell metabolism. SPI-based films remain preclinical, and none consider advancing the therapeutic potential of the SPI with the addition of extracellular vesicles (EVs) containing growth factors. The objective of this research was to assess three techniques for sterilizing SPI/PEO fibers fabricated using water as a solvent, while preserving the integrity of the incorporated EVs. The three sterilization methods evaluated were autoclaving the polymer solution before electrospinning, adding antibiotics in the solvent, and sterilizing the SPI and PEO with ultraviolet (UV) light prior to mixing with sterilized water. A scanning electron microscope (SEM) showed successful fiber formation only in the antibiotic and UV sterilization techniques. The films from these techniques underwent a contamination test and no statistically significant difference was found between the film groups and the negative control group. A metabolic assay showed the films increased the overall metabolic activity of C2C12 cells after 2 and 4 days. Finally, a fluorescent EV uptake experiment showed EVs remained bioactive after being released from the UV-treated films. The antibiotic-treated film EV uptake is in the works. In conclusion, the antibiotic and UV sterilization techniques were successful in fiber formation and EV preservation.

Materials and

Methods: : Three sterilization methods were evaluated. Each utilized the same polymer composition of 6% SPI and 1.6% PEO. The first sterilization method was autoclaving the polymer solution for 15 minutes at 120 degrees celsius. The second sterilization method was adding penicillin and streptomycin so the solvent was 5% antibiotics. The third method was putting the SPI and PEO powders under UV for an hour before mixing with sterilized water.

The electrospinning process involved a syringe pump, voltage generator, 22 gauge needle, metal plate (collector) wrapped in aluminum foil, and tube for air flow. The following parameters were slightly adjusted to obtain fibers: distance between the needle and collector ~ 20 cm, electric field potential ~ 20 kV, airflow ~ 10 bar, syringe pump speed ~ 0.7 mL/hr.

SEM imaging was used to evaluate fiber morphology.

For the contamination test, the films were placed in transwells with 10% FBS and DMEM media and monitored for visible bacteria and fungal contamination for a week.

The AlamarBlue assay was used to determine the metabolic impact of the films on C2C12 cells. The films were placed in transwell baskets with C2C12 cells and the metabolic activity was assessed with a 4 hour AlamarBlue assay after 2 and 4 days.

Finally, the EV uptake assay was performed to qualitatively show EVs remain bioactive after being released from the films. The EVs were isolated from C2C12 cells, labeled with DiD, added to the solution at the end of the mixing period, and imaged with an EVOS microscope.

Results, Conclusions, and Discussions:: The autoclaved sterilization technique resulted in an heterogeneous solution and could not be effectively electrospun. The SEM image showed no successful fiber formation. Thus, the contamination test, metabolic test, and EV uptake assay were not continued with the autoclaved solution.

The antibiotic-treated solution electrospun well and formed smooth, relatively uniform fibers. The UV-treated solution showed partial fiber formation; however, there were some beads and the diameters were not uniform. Little to no contamination was observed during the contamination tests. A Fisher’s Exact Test indicated no significant difference between the experimental groups and negative control groups. However, the sample size was small which makes it difficult to detect a real difference between the two groups.

This project was part of a collaboration with a company called NeuEsse whose product, OmegaSkin, is in the process of FDA approval. The metabolic impact of the films on C2C12 cells was compared to OmegaSkin and quantified as a percent increase from a negative control group with no films.

As shown in the graph, the OmegaSkin group had the greatest metabolic activity with a 50% increase on both days. The UV-treated film and antibiotic-treated film groups also resulted in increased metabolic activity but less than OmegaSkin. One strange result was the 8% metabolic decrease in the UV-treated film group after 4 days. In the group, one well decreased significantly while the other two wells increased. The well that decreased was not identified as an outlier in a Grubbs Test so could not be removed. This well may have had cells damaged through an extra forceful media change or perhaps contamination.

A fluorescence EV uptake experiment showed the EVs were still bioactive and internalized by C2C12 cells after the UV-treated films dissolved. The EV uptake for antibiotic-treated films is in the works.

In conclusion, using antibiotics in the solvent and sterilizing the powders with UV resulted in successful fiber formation and minimal contamination. The films increased the overall metabolic activity of C2C12 cells over 2 and 4 days.

Future steps would include measuring the metabolic impact of the films with EVs relative to OmegaSkin.

Acknowledgements and/or References (Optional): :