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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session B

Poster I4 - Enhancing the Potency of anti-Biofilm Extracellular Vesicles

Thursday, October 24, 2024
2:45 PM – 3:45 PM EST
Location: Exhibit Hall E, F & G

Introduction:  The focus of my project has been to develop a way to have extracellular vesicles (EVs) prevent Pseudomonas aeruginosa (PA) biofilm formation. Many biofilms are caused by PA which produces surface polysaccharides that enhance adhesion and thus infection. The WHO has recognized PA as a critical first priority pathogen. Based on this, the focus has been on preventing a PA infection from forming. Also, due to the increasing rate of antimicrobial resistance, looking at other options besides antibiotic drugs to fight infections is essential. Biofilms are composed of an extracellular matrix that enhances microbial adherence to surfaces and they have an increased resistance to antimicrobials. In order to see the effect that EVs have on PA14 biofilms, we were able to isolate the EVs and perform biofilm assays. A second focus of the project has been differentiating THP1 monocytes into macrophages and then differentiate the macrophages into inflammatory and regenerative phenotypes. We hypothesized that the different stages of the inflammatory response could present higher levels of preventing the PA biofilms. The overall goal is to enhance the potency of anti-biofilm extracellular vesicles and to explore different options for therapeutic developments.

Materials and

Methods:  The isolated EVs come from the THP1 monocyte cell line. Each day, we count the cells and change media in order to keep the culture around 600,000 cells/mL. Once the EVs have been isolated by tangential flow filtration (TFF), they are concentrated using a centrifuge. Once isolated, we perform assays to see their potential in preventing the formation of the PA14 biofilm. The main biofilm assay that I have been performing this semester is a crystal violet quantification. The biofilms consist of different treatments of the isolated EVs and the PA14 strain. We allow it to sit overnight, then stain the plate with crystal violet, and the third day, add acetic acid to quantify on the plate reader.
In order to differentiate cells, we add PMA into the flasks containing THP1 cells. This culture then sits overnight and differentiates the respective flasks into macrophages. The concentrations that were used to differentiate into M1 were 20 ng/mL of IFN-gamma and 10pg/mL of LPS. For M2, we incubated with 20 ng/mL of interleukin 4 and 20 ng/mL of interleukin 13. After the cells have grown in the media overnight, we aspirate the old media and add EV depleted media into the flasks. The cultures incubate for another night and we isolate their EVs using the TFF the following day. The differentiated cells derived from EVs are then used in the crystal violet biofilm assays as three new treatments against PA.

Results, Conclusions, and Discussions: I have been able to perform an initial study that includes treatment of EVs isolated from macrophages and THP1 cells to prevent biofilm formations. All of the assays control consists of isolated EVs from the media. The expected result from the media is that it will not provide any prevention against the PA biofilms. This enables us to compare the EV results to the amount of biofilms that grew when not being prevented due to lack of EVs present.

This experiment was able to display results between the differentiated cells. Although there was not a significant difference, this data can still be used in future studies. We hope to begin troubleshooting in the fall and looking at different conditions or time to grow the biofilms in. This may change the effect that the EVs have on the biofilm formation. Another experiment we plan to perform to confirm the degree of macrophages polarization in the cell culture to check that the lack of differences is not due to our polarization protocol. If necessary, we will work on our differentiation technique in order to increase phenotypic differences in the cells.
In conclusion, antimicrobial resistance has been steadily increasing and this research will directly have a positive impact on this issue by potentially providing an alternative to antibiotics, reducing their use in clinical settings. These studies can provide another way to administer protection against biofilms without the use of more antibiotics.

Acknowledgements (Optional):