Micro-Injection of Amphiphilic Nanoparticles into a Droplet Interface Bilayer Reveals Particle-Induced Reductions in Membrane Capacitance

With their ability to passively enter cells, amphiphilic gold nanoparticles (AmNPs) functionalized with a striped pattern of hydrophilic sulfonate and hydrophobic octanethiol ligands show promise for enhanced drug delivery. However, the specific mechanisms of their passage through cellular membranes remain unclear, in part due to a lack of appropriate experimental tools. Ongoing work in the Sarles group at UTK seeks to reveal these interactions by leveraging the droplet interface bilayer (DIB) technique for model membrane formation and electrophysiological characterization. The advantages of the DIB method for assembling a lipid bilayer include the ability to independently control droplet and lipid leaflet compositions and membrane area. The DIB technique also permits the use of electrophysiology measurements to monitor changes in bilayer structure and transport properties, both of which may aid in the disclosure of NP-membrane interactions.<br/>Herein, we utilize micro-injection to incorporate AmNPs into one droplet of a droplet interface bilayer <i>after</i> bilayer formation occurs and use electrophysiological measurements as well as brightfield imaging to assess NP-induced changes in bilayer properties. Post-bilayer-formation direct micro-injection of NPs allows for maintaining stable membranes needed to study NP-membrane interactions and permits before-and-after comparisons to be made on the same membrane. In this study, DPhPC model membranes are formed between 300 nL droplets in hexadecane. A pulled micropipette connected to a pneumatic injector is used to inject AmNPs without disrupting the membrane. Before injecting AmNPs, the electrical resistance and capacitance of the membrane are electrically tested using wire-type electrodes to establish baseline bilayer characteristics and, thereby, quantify NP-induced changes to the membrane after injection. This procedure reveals that AmNPs with 15 mol% OT ligands decrease the area-normalized capacitance of the bilayer by up to 20%, where the amount of reduction depends on the final AmNP concentration. When combined with other electrical measurements, this trend indicates that AmNPs spontaneously embed into the hydrophobic center of the bilayer, increasing its average thickness and reducing its capacitance per unit area. In contrast, no discernible changes in the bilayer's specific capacitance were observed after adding hydrophilic NPs lacking hydrophobic ligands. This implies that the hydrophobic ligands are responsible for the insertion of AmNPs into the bilayer. Moreover, AmNPs with 30 mol% hydrophobic ligands showed larger decreases in specific capacitance that occurred at a lower AmNP concentration) compared to 15 mol% OT AmNPs. Finally, we observed that AmNPs lowered the membrane resistance, resulting in increases in average ion current through the bilayer under an applied voltage. These changes also coincided with transient, discrete current spikes with durations of 3 and 12 ms. Unlike many works which leveraged qualitative experimental measures, our findings demonstrate how direct microinjection and electrophysiology measurements that specifically incorporate capacitance monitoring can be used to study NP-membrane interactions.