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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster I8 - Reproducing Nanoparticle Uptake in Artificial Cells Using Droplet Interface Bilayers

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Mira Patel

Undergraduate Student
The University of Mississippi
Vicksburg, Mississippi, United States

Co-Author(s)

SS

Sarita Shrestha

Graduate Student
University of Georgia, United States

Primary Investigator(s)

Eric C. Freeman, PhD

Primary Investigator
University of Georgia, United States

Introduction: Functionalized nanoparticles are novel tools that are currently being investigated for immunization applications. Unfortunately, the mechanics of nanoparticle interactions with immune cells are not well characterized at this time, given the difficulties of isolating desired interactions in the complex environment of living cells. To address this limitation, researchers have developed various in-vitro platforms to replicate nanoparticle binding on immune cells. While several artificial membrane platforms exist that allow for the replication of cellular phenomena, some of these model membranes are still overly simplified compared to their natural counterparts and not adequate in the replication of specific phenomena.

The droplet interface bilayer (DIB) method, however, is a model platform for understanding cell membrane mechanics that relies on the self assembly of lipids at an oil-water interface. Lipid membranes are formed between lipid-coated aqueous droplets in an oil reservoir. DIBs allow for simultaneous electrical and mechanical characterization of the membrane and may provide new insights to nanoparticle-membrane interactions. Preliminary measurements that are needed for this technique include equilibrium interfacial tension (IFT) values, which are obtained through pendant drop tensiometry. In this study, several concentrations of lipids in varying oil solvents were examined to determine the most effective combination of creating a functional DIB that replicates nanoparticle adsorption to the membrane. This research project examines the addition of biotinylated lipid headgroups in a DIB platform for investigating nanoparticle binding to cell membranes as shown in Figure 1.

Materials and

Methods: Lipid solutions including 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),Cholesterol (Chol), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl)(sodium salt) (Biotinyl PE) were prepared by mixing the lipids in chloroform at desired ratios, evaporating the chloroform, and dispersing the lipids in varying oils. Two lipid compositions were investigated, one without the biotinylated headgroups (4:1 DOPC:Chol molar ratio), and one with the biotinylated headgroups (8:2:1 DOPC:Chol:Biotinyl PE). The selected oils were combinations of Squalene, Hexadecane, and Silicone Oil AR20, and 5 mg/mL lipid-in-oil mixtures were prepared. Two buffer solutions were prepared for the aqueous phase, both including 250 mM Potassium Chloride (KCl) and 10 mM 3-(N-Morpholino) propane sulfonic acid (MOPS). One buffer solution included Avidin (0.1 mg/mL), and one did not.

The various oil and water solutions were combined, and the tension of their interface was tracked over time using pendant drop tensiometry. Changes in the tension of the interface indicate packing of the lipid monolayer, with lipids assembling from the oil phase. The hypothesis was that the simultaneous presence of biotinylated lipids in the oil phase and avidin in the aqueous phase would result in rapid monolayer formation, demonstrating successful incorporation of the biotinylated headgroups.

Once ideal compositions were identified by their rapid and stable monolayer formation, DIBs were formed by suspending aqueous droplets from silver/silver-chloride electrodes connected to a patch clamp amplifier within the lipid-in-oil reservoir. Biotinylated nanoparticles were injected directly into the droplet as shown in Figure 1. and changes in the properties of the membrane were observed using a microscope and the electrical characteristics of the membrane.

Results, Conclusions, and Discussions: The data from the pendant drop tensiometry method was analyzed for all lipid-in-oil solutions, with and without avidin added into the aqueous phase. Our focus primarily lied on the equilibrium interfacial tension (IFT) values for the droplets, which were observed when the droplets were stable in a pendant drop shape. IFT values allow us to understand the extent of packing in the lipid monolayer. The data suggests that the equilibrium IFT for 8:2:1 DOPC:Cholesterol:Biotynl PE in hexadecane with avidin is significantly lower than the equilibrium IFT for the same solution without avidin. This suggests that the addition of avidin in the aqueous/continuous phase allows for the formation of a more tightly packed monolayer, indicating that the biotinylated headgroups are present. Furthermore, the data shows that the equilibrium IFT for 4:1 DOPC:Cholesterol in hexadecane does not vary with avidin, further reinforcing that the measured change in the 8.2.1 mixtures is due to avidin-biotin interactions. However, when squalene is used as the oil, there is no significant difference between avidin and non-avidin trials for all lipid solutions. This suggests that either the monolayer does not include the biotinylated lipid headgroups or that avidin is not necessary for ensuring a well-packed lipid monolayer.

Afterwards, the droplet interface bilayer method was used to create a bilayer between two aqueous droplets in a lipid-in-oil reservoir. The 8:2:1 DOPC:Cholesterol:Biotynl PE lipid solution in either squalene or hexadecane was used as the lipid-in-oil reservoir. After the DIB formed, it was observed that the 8:2:1 DOPC:Cholesterol:Biotinyl PE in squalene formed a larger membrane when compared to the lipid-in-hexadecane solution. Additionally, the success rate for bilayer formation with lipid-in-hexadecane was 50%, whereas the success rate for lipid-in-squalene was 20%. Hexadecane will be used as the oil phase in future work.

Future work will explore various concentrations of lipids-in-oil, further adjust the molar ratio of lipids, and complete measurements of membrane properties with and without the presence of biotinylated lipids, avidin, and the nanoparticles.

Acknowledgements (Optional): 1 . Leptihn, S., Castell, K. O., Cronin, B., Lee, E., Gross, C. M. L., Marshall P. D., Thompson, R. J., Holden, M., Wallace, M. (2013). Constructing droplet interface bilayers from the contact of aqueous droplets in oil. Nature Protocols, 8(6) 1048-1057.
2. Berry, D. J., Neeson, J. M., Dagastine, R. R., Chan, Y.C. D., Tabor, F. R. (2015). Measurement of surface and interfacial tension using pendant drop tensiometry. Journal of Colloid and Interface Science, 454(2015), 226-237.

The authors graciously acknowledge the donation of biotinylated nanoparticles from the Meng Lab at Duquesne University. This material is based upon work supported by a National Science Foundation Research Experiences for Undergraduates (REU) site program under Grant No. 1950581.