The Role of Surface Functionality in Nanoparticle Exocytosis

The exocytosis process is a key factor in determining the biological behavior of engineered nano-carriers in drug delivery, affecting the release rate and half-life of the nano-carriers in cells.[1] Understanding the factors that control exocytosis will contribute to the development of more effective nano-carriers. The exocytosis rate of nano-carriers is affected by multiple factors including size,[2] shape,[3] and surface functionality.[4] Controllable exocytosis rates of nanomaterials leads to tunable retention times in cells, allowing controllable release of drug inside the cells.[5] In our study, nanoparticles featuring identical size and a common structure were used to probe the role of surface properties on particle clearance. We examined the exocytosis of cationic gold nanoparticles (AuNPs) of identical core size (2 nm diameter) and different surface functionalities. Here, we report that surface functionality is an important factor for determining the exocytosis rate of nano-carriers, as quantified by inductively-coupled plasma mass spectrometry (ICP-MS) in both closed and flow systems. To address the specific role of surface functionality in exocytosis, we have designed a series of AuNPs with that allow us to explore the effects of specific chemical functionality on exocytosis. The Brust-Schiffrin two-phase synthesis method[6] and the Murray place-exchange method were used to obtain the functionalized AuNPs 1-5,[7] featuring nanoparticles of identical size, and analogous structure (detailed ligand synthesis, gold nanoparticle synthesis, and characterization are available in the Supporting Information). Breast cancer cells (MCF-7) were incubated with media containing NPs (200 nM) shown in Figure 1a for 3 h before removing and replacing with NP-free media. The concentration (200 nM) of NPs used has been shown to not induce cell damage or membrane leakage.[8] NP exocytosis amounts were then determined over a 6 h period using both a closed system and a flow system in which the media were continuously pumped through the cell culture plate and then collected for analysis using inductively-coupled plasma mass spectrometry (ICP-MS) (Figure 1b). The percentage of exocytosed AuNPs was calculated by Equation 1. Exocytosis(%)=Exo(ng)Cell(ng)+Exo(ng)×100 (Equation 1) Figure 1 (a) Structural illustrations of the NPs used in this study and the calculated hydrophobic values (Log P) of the highlighted headgroups. (b) Percentage of NPs exocytosed compared to endocytosed amounts in a closed and flow system after 6 h. The exocytosis ... In this equation, Exo is the amount of exocytosed AuNPs (ng), and Cell is the amount of AuNPs remaining in the cells (ng). As expected, higher exocytotic efficiency was provided by the dynamic system (Figure 1b).[9] Previous research showed that multiple aspects of NP-biological interaction can be correlated to the hydrophobicity of the NPs using a predicted log P of the head groups as a measure.[10] As shown in Figure 1, there is not an obvious trend in exocytosed gold amount based on ligand hydrophobicity for NP 1, 3, and 4. .In contrast, NP5 with aromatic ligands shows a significant increase in exocytosed gold amount compared to other NPs, suggesting that chemical structure is an important determination of exocytosis. We next explored the kinetics of the exocytosis using a subset of the original particles, We selected NP 1, 4, and 5 (least hydrophobic, most hydrophobic, and aromatic NPs, respectively) to further evaluate the effect of surface functionality (e.g., hydrophobicity or structural functionality) on exocytosis. These NPs were incubated with cells for 3 h and washed using NP-free media three times to remove the NPs attached on the surface of cells. Then, the flow system was employed to measure the extent of exocytosis under dynamic conditions, with the gold amount in the media quantified over time by ICP-MS (Figure 2). The gold concentration in the collected media at different time points was measured (Figure 2a). The amount of excreted AuNPs out of the total uptake amount for each AuNP was determined at different time points with Eq. (1) (Figure 2b). As shown in Figure 2c, most of the AuNPs are exocytosed during the first 3 h with the majority of NP 4 excreted after 1.5 h. NP 5 is exocytosed to the greatest extent, but this NP is also taken up by the cells to a greater extent than NPs 1 or 4. Even so, a higher percentage of NP 5 is excreted than NP 1 or 4 (Figure 1). These results suggest that the aromatic structure of the surface ligand of NP 5 plays an important role in regulating NP cellular exocytosis. Figure 2 Exocytosis amount of NPs monitored by ICP-MS in the flow system. (a) Exocytosis rate measured as gold concentration (ng/mL) in the collected cell culture media at different time intervals. (b) The percentage of the NPs measured in the collected media ... Cellular TEM was used to clarify the fate of AuNPs from the closed and flow systems to better understand the exocytosis process (Figure 3). The cellular TEM images in Figure 3 indicate the existence and aggregation of NPs in endocytotic vesicles after 6 h incubation. Interestingly, the TEM image of NP 4 (the most hydrophobic NP) illustrates that these endocytosed NPs remain in vesicles close to the cellular membrane, while endocytosed NPs 1 and 5 are further from the cellular membrane (Figure 3). It is also worth mentioning that NP 5 is observed by TEM less frequently in the cell than NP 1 and NP 4, which is consistent with the higher exocytosis amount of this NP (Figure 2a and c). Figure 3 Cellular TEM images for NP 1, NP 4, and NP 5. Images were taken 6 h after the incubation of NPs and cells and indicate the intracellular behaviors of these NPs after the 6-h exocytosis period in the flow systems. Scale bar, 100 nm. In summary, we have quantified the exocytosis behavior of NPs with different surface functionalities in both closed and flow systems. These studies indicate that the hydrophobicity does not dramatically affect the exocytosis rate of NPs. We have shown, however, that the surface functionality (e.g., aromatic structure) plays a role in the exocytosis rate of NPs. These findings should lead to better designs for NPs as nano-carriers by enabling more rationale control of the exocytosis process. A deeper understanding of the surface functionality-dependent exocytosis rate of nano-carriers could be employed and tuned for effective payload delivery, enhancing the therapeutic effect of nanomedicines in the future.