Effect of nanoparticle surface charge at the plasma membrane and beyond

Abstract

Herein, we demonstrate that the surface charge of gold nanoparticles (AuNPs) plays a critical role in modulating membrane potential of different malignant and nonmalignant cell types and subsequent downstream intracellular events. The findings presented here describe a novel mechanism for cell-nanoparticle interactions and AuNP uptake: modulation of membrane potential and its effect on intracellular events. These studies will help understand the biology of cell-nanoparticle interactions and facilitate the engineering of nanoparticles for specific intracellular targets.

Figure 1

Summary of gold nanoparticles (AuNPs) effects on cellular membrane potential. AuNPs of different surface charges were generated by chemical modification of the terminal portion of the ligand bonded to the nanoparticle core. Four types of AuNPs were examined (A): neutral (⁰AuNP), positive (⁺AuNP), negative (⁻AuNP) and zwitterionic (^±AuNP). The diameter (*) and surface charge (**) were measured by dynamic light scattering and zeta potential, respectively. Using the cell-permeant fluorescent membrane potential indicator RH414 and real-time fluorescence microscopy, membrane potential changes following exposure to AuNPs of different surface charges were measured for two ovarian cancer cell lines (CP70, A2780), human bronchial epithelial cells (BEC) and human airway smooth muscle (ASM) cells (also see supplemental Figure S1). ⁺AuNPs (1.2 μM) produced rapid and significant membrane depolarization (panel B; bars; comparable to that induced by 40 mM KCl, diamonds). The extent of membrane potential change was dependent on ⁺AuNPs concentration (C), with minimal changes at 10 nM. In comparison to ⁺AuNPs, those with other charges had negligible effects on membrane potential in any cell type (B). Values are means ± SE. * indicates significant AuNP effect (p<0.05).

Figure 2

AuNP uptake and membrane potential. (A) In CP70 cells, only ⁺AuNPs (0.4 μM) showed significant uptake, with substantial intracellular levels present at 5 min, followed by a higher level beyond 2h. (B) Prior exposure to KCl (inducing membrane depolarization) significantly (p<0.05) reduced the extent of uptake of ⁺AuNPs (1.2 μM for 30 minutes treatment) in four different types of cells. (C) In all the cells investigated, prior exposure to KCl significantly blunted the extent of membrane depolarization subsequently induced by ⁺AuNPs. Values are means ± SE. * indicates significant AuNP effect (p<0.05).

Figure 3

Summary of ⁺AuNPs effects on intracellular Ca²⁺ ([Ca²⁺]_i) levels. (A, B) In ovarian cancer cells (CP70, A2780) and airway cells (BEC, ASM) loaded with the ratiometric fluorescent Ca²⁺ indicator fura-2, ⁺AuNPs (1.2 μM) produced substantial increases in [Ca²⁺]_i levels that reached a maximum value in ~5 minutes and in some cell types decayed to a lower level above baseline (see supplemental Figure S2). In comparison, AuNPs with other charges had negligible effects on [Ca²⁺]_i levels. The effect on [Ca²⁺]_i levels was dependent on the concentration of ⁺AuNPs (C), with even 10 nM AuNPs producing a substantial increase in [Ca²⁺]_I (compared to small effects on membrane potential). In ovarian cancer CP70 cells, the role of specific [Ca²⁺]_i regulatory mechanisms were examined by first inhibiting specific Ca²⁺ regulatory mechanisms, and then exposing cells to ⁺AuNPs (1.2 μM) (D). Inhibition of plasma membrane Ca²⁺ influx via L-type Ca²⁺ channels resulted in significant reduction in positively charged AuNP effects on [Ca²⁺]_i levels. AuNP effects were even more reduced in the absence of extracellular Ca²⁺ (0 Ca HBSS) suggesting that Ca²⁺ influx (partly via L-type channels) contributes to the observed change in [Ca²⁺]_i levels with AuNPs. The remainder of the [Ca²⁺]_i response appears to be derived from endoplasmic reticulum Ca²⁺ release (since the response persists in the absence of extracellular Ca²⁺) especially via IP3 receptor channels (evidenced by lack of AuNP effects when the channels are inhibited by Xestospongin C). Lack of effect of mitochondrial Ca²⁺ pathways (CGP 37,157 for mitochondrial Na+/Ca²⁺ exchange and Ru360 for mitochondrial Ca²⁺ uniporter) suggests that AuNPs may not be affecting mitochondria. Values are means ± SE. * indicates significant AuNP effect, and # indicates significant effect of 0 Ca HBSS, and % indicates significant effect of inhibitor (p<0.05).

Figure 4

Effect of AuNPs on cellular proliferation vs. apoptosis. (A) Even brief (30 min) exposure of cells to ⁺AuNPs substantially blunted the proliferation of human BECs, but did not affect the proliferation of ovarian cancer CP70 or A2780 cells. (B). Apoptosis of BEC by ⁺AuNPs was concentration-dependent, and substantial. In contrast, some degree of apoptosis (< 10 %) of CP70 cells occurred only at a higher ⁺AuNP concentration. (C) The viability of both BECs and ASM cells was substantially reduced by ⁺AuNPs, with a concentration-dependence for ASM. * indicates significant effect (p<0.001)

Figure 5

(A) Transmission electron microscopy of ⁺AuNP (concentration = 0.4 μM) interactions with plasma membrane. Top panels demonstrate localization of ⁺AuNPs within the plasma membrane (CP70 cells). Bottom panels demonstrate lack of plasma membrane disruption following ⁺AuNP uptake (BEC cells). (B) Schematic of AuNP effects on cellular function. Based on our findings using ⁺AuNPs and different types of cells, we propose that AuNPs are taken up intracellularly, based on membrane potential. Upon uptake, ⁺AuNPs produce membrane depolarization, and increase [Ca²⁺]_i by enhancing Ca²⁺ influx and inducing release of intracellular Ca²⁺ stores (e.g. via IP₃ receptor channels of the endoplasmic reticulum; ER). These changes can result in increased apoptosis and decreased cellular proliferation, depending on cell type. Further modulation of apoptosis and proliferation may involve direct nanoparticle effects on intracellular signaling mechanisms.