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Surface Coverage-Regulated Cellular Interaction of Ultrasmall Luminescent Gold Nanoparticles Lingshan Gong, Ying Chen, Kui He, and Jinbin Liu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b08103 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on February 4, 2019

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Surface Coverage-Regulated Cellular Interaction of Ultrasmall Luminescent Gold Nanoparticles Lingshan Gong, Ying Chen, Kui He, and Jinbin Liu*

Key Laboratory of Functional Molecular Engineering of Guangdong Province, School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, China

KEYWORDS: surface coverage, imaging, cellular interaction, ultrasmall gold nanoparticles, luminescence

ABSTRACT: Investigations for accurately controlling the interaction between functional nanoparticles (NPs) and living cells set a long-thought benefit in nanomedicine and disease diagnostics. Here, we reveal a surface coverage-dependent cellular interaction by comparing the membrane-binding and uptake of three ultrasmall luminescent gold NPs (AuNPs) with different surface coverages. Lower surface coverage leads to fast

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cellular interaction and strong membrane-binding but low cellular uptake, whereas high surface coverage induces slow cellular interaction and low membrane-binding but major cellular uptake. The slight number increase of cell-penetrating peptide on the surface of AuNPs shows improved cellular interaction dynamics and internalization through direct cellular membrane penetration. Furthermore, the different intrinsic emissions resulted from the surface coverage variation, especially the pH-responsive dual emissions, make the AuNPs as powerful optical probes for subcellular imaging and tracking. The findings advance the fundamental understanding of the cellular interaction mechanisms of ultrasmall AuNPs and provide a feasible strategy for the design of functional NPs with tunable cellular interaction by surface regulation.

The ultrasmall luminescent gold nanoparticles (AuNPs, d < 3.0 nm) with distinct features, such as tunable luminescence, modifiable surface properties, good biocompatibility and metabolic clearance, draw intense interest in optical investigation,13

noninvasive imaging4-7 and biomedical explorations.8-12 Due to the increasing

contributions of surface characteristics with NP size shrinking, the regulation of surface


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ligand densities is an efficient way to vary the NPs’ physicochemical properties and biological behaviors.13-21 For example, the quantum yield of ultrasmall AuNPs could be enhanced largely by increasing the density,22 rigidify23,24 or donating electron capability25 of their surface ligands. We also found that increasing surface coverage of ultrasmall AuNPs induced variation of local binding geometry of thiolate ligands from weak chiral conformation to strong one,26 resulting in blue shift of emission maximum from near-infrared (NIR) to visible wavelength with stable interim dual emissions (NIR/visible). For the larger NPs (from several to hundreds of nanometer), surface coverage has been well demonstrated to govern their cellular interactions.27-31 For instance, the ligand density-mediated ligand conformational fluctuation,28 steric repulsion for protein absorption32 and the adhesion strength for cells33 have been used to regulate the cellular interaction of larger NPs for understanding their biological mechanisms and achieving their biological functions. However, there are few reports about the effect of surface coverage of ultrasmall NPs on their cellular interactions. Whether much larger surface-to-volume ratio of ultrasmall NPs benefits to the surface coverage-regulated cellular interaction is still unknown.


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Herein, we designed and synthesized a series of ultrasmall luminescent AuNPs with different surface coverages ranging from 29% to 47% (Scheme 1) by co-introducing glutathione and CR8 (CR-AuNPs), a cell-penetrating peptide contains a thiol for anchoring on AuNPs and a cell-penetrating region for transporting molecules or small NPs across plasma membrane.34,35 Then, these CR-AuNPs with single 810 nm emission, dual emissions (810/615 nm), and single 615 nm emission, respectively, served as robust fluorescent probes for imaging their interaction with living cells. We discovered that the ultrasmall AuNPs with lower surface coverage showed fast cellular interaction and strong membrane-binding but low cellular uptake, whereas higher surface coverage performed slow cellular interaction and low membrane-binding but major cellular uptake, distinct from the previously reported larger NPs whose stronger membrane interaction leading to a high level of cellular uptake.30,35 In addition, the pHresponsive dual emissions of AuNPs could easily indicate their subcellular location, endocytosis pathways and the states of living cells through ratiometric imaging. These results indeed indicate that surface coverage is a crucial factor in determining the


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cellular interaction mechanisms of ultrasmall luminescent AuNPs besides their optical properties.

RESULTS AND DISCUSSION

Characterization of CR-AuNPs with Different Surface Coverage. We revisited the structure-properties of CR-AuNPs with different surface coverage to reveal their differences in the cellular interactions. Their emission and excitation spectra changed with the increase of surface ligand density: the emission peak displayed a blue shift from 810 nm single emission to 615/810 nm dual emissions to final 615 nm single emission, whereas the excitation maximum showed a red shift from 382 nm of the 07CR-AuNPs to 397 nm (615 nm emission)/408 nm (810 nm emission) of the 09CRAuNPs, and to 417 nm of the 13CR-AuNPs (Figure 1A and S1). The luminescence of the CR-AuNPs was largely depended on the surface coverage and local bonding environment, consistent with our previous report.26 The high surface coverage on the 13CR-AuNPs resulted in strong Au-ligand charge transfer and 615 nm emission, whereas a low surface coverage on the 07CR-AuNPs led to weak charge transfer and


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810 nm emission. However, differentiating from the distinguishing excitation and emission spectra, their absorption spectra and the core sizes (~ 2.0 nm) were almost identical (Figure 1B and S1). As expected, their surface coverages of 07CR-AuNPs, 09CR-AuNPs and 13CR-AuNPs increased from 29% to 47% with the increase of surface ligands after purification (Figure S2 and S3), while a few numbers of CR8 showed a negligible effect on surface coverage after comparing their component of both CR-AuNPs and GS-AuNPs under the similar synthesis conditions (Figure 1C, S4 and S5, Table S1 and S2), respectively. Both zeta potential and gel electrophoresis measurements confirmed that all CR-AuNPs are negatively charged at pH 7.4 (Figure 1D and S7), and their zeta potential gradually increased with the decrease of pH (Figure 1D). The zeta potential measurement results also indicated that the low modification of positively charged CR8 had not distinct effect on the charge of CR-AuNPs, which also slightly affect their migration rate (Figure S6 and S7). In addition, the introduced CR8 did not change the pH-responsive properties of AuNPs: The emissions at both 615 nm and 810 nm of dual-emitting 09CR-AuNPs showed sensitive ratiometric responses (emission intensity ratio values of I615 nm/I810 nm or I810 nm/I615 nm) to pH changes and stable


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reversibility (Figure 1E, 1F and S8), whereas the luminescence of single-emitting CRAuNPs showed lower pH dependency (Figure S9). However, the ratio values of I615 nm/I810 nm

or I810 nm/I615 nm from the mixture of 07CR-AuNPs and 13CR-AuNPs did not

show significant change at different pH values (Figure S10). These results demonstrated that the ratiometric pH response was originated from a single particle of dual-emitting 09CR-AuNP containing two coupled 615 nm and 810 emission centers. As a result, a distinct ratiometric images demonstrated that the synthesized dual-emitting 09CR-AuNPs was capable of efficient identification of the ambient pH condition (Figure S8B).

Acidic Environment Induced Membrane-Binding Enhancement. To explore the pHdependent interaction of various CR-AuNPs with cellular membrane, membrane-binding imaging of living HeLa cells were performed after incubation with CR-AuNPs of 500 nM (Figure S11) for 10 min at an extracellular pH range from 5.8 to 7.4. As shown in Figure 2A, the 810 nm-emitting 07CR-AuNPs had weak interaction with cellular membrane at pH 7.4 in DPBS. As the decrease of the extracellular pH, the decreasing zeta potential


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of the CR-AuNPs led to the lower electrostatic repulsion between CR-AuNPs and cellular membrane. The luminescence intensities of the cells dramatically increased, suggesting the interaction between 07CR-AuNPs and the cellular membrane would significantly enhance in a weakly acidic condition due to significantly decreased surface negative charge of 07CR-AuNPs at the lower pH value from 7.4 to 5.8 (-38.8±0.59 mV to -19.5±0.42 mV, Figure 1D). However, cells after interaction with the 615 nm-emitting 13CR-AuNPs almost had no luminescence with lowered extracellular pH from 7.4 to 6.3. Only when pH dropped to 5.8 could some cells be observed slight luminescence. This weak membrane-binding of 13CR-AuNPs was caused by the strong electrostatic repulsion between highly negative-charged 13CR-AuNPs (-58.5±0.36 mV to -45.3±0.44 mV) and negative-charged cellular membrane. Distinct from the single-emitting CRAuNPs, both the luminescent intensities at 810 nm and 615 nm of the dual-emitting 09CR-AuNPs gradually enhanced with lowering extracellular pH from 7.4 to 5.8 (Figure 2B and 2C). The pH-dependent differences of increasing intensity at 810 nm and 615 nm could also be obviously displayed by ratiometric image of ratio value of I615 nm/I810 nm (R615/810nm, Figure 2B and 2D), implying the pH-responsive dual-emitting CR-AuNPs had


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potential as probes for fluorescent pH imaging of living cells. Similarly, binding of 07GSAuNPs and 09GS-AuNPs with membrane was largely enhanced in a mildly acidic condition (Figure S12) owing to the reduction of global charge repulsion with the decreasing surface potential of NPs.36 But rare accumulation of 13GS-AuNPs was visualized on the surface of cellular membrane even in pH dropped to 5.8, consistent with a previous report for the 605 nm-emitting GS-AuNPs.37 Compared with the results of GS-AuNPs, the much stronger membrane-binding ability of CR-AuNPs especially in neutral pH environment was contributed to the cell penetration ability and positive charge of CR8. These observations not only suggested that the NPs with lower surface coverage exhibited stronger interaction with cellular membrane, which was significantly enhanced in a slightly acidic environment, but also demonstrated that the designed dual-emitting AuNPs could achieve subcellular pH visualization by ratiometric imaging.

Imaging of Cellular Internalization. To further unravel the effect of surface coverage of the CR-AuNPs on the cellular interaction, time-dependent imaging of living cells after incubating with CR-AuNPs with different ligand densities were then investigated. As


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shown in Figure 3, with extension of incubation time, the luminescent intensity of cells significantly enhanced as the increased interaction of CR-AuNPs with cells. The cells showed obvious luminescence after incubating with 07CR-AuNPs and 09CR-AuNPs for 1 h, respectively, whereas there was little luminescence of cells immersed in 13CRAuNPs until prolonged the incubation time to 3 h. Similarly, the interaction of GS-AuNPs with cells tended to be slower as their ligand density increased: 3 h for 07GS-AuNPs, 6 h for 09GS-AuNPs and even rare interaction with cells in 6 h for 13GS-AuNPs (Figure S13). This was because the higher ligand density induced larger repulsion and steric hindrance between AuNPs and cells that led to the lower rate and efficiency in their membrane-binding and endocytosis. These results indicated that the positively charged CR8 could enhanced their interaction rate and efficiency with cells in both membranebinding and endocytosis processes.

Besides the interaction speed, the surface properties of NPs could decide on their binding sites and final distribution. A time lapse of location information for all CR-AuNPs clearly displayed that the CR-AuNPs first accumulated on the surface of cells once


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contacted with cells (Figure 3, Movie 1-3). But their distribution varied over time. As shown in Figure 3D, the z-stack imaging of cells revealed that most 07CR-AuNPs still accumulated on the cellular membrane at 6 h (~82%, Figure 3G) with little internalization, differentiating from larger NPs whose stronger interaction of NPs with membrane always results in a high level of internalization.30,35 While most 09CR-AuNPs and 13CR-AuNPs would then be internalized and homogeneously distributed around the cytoplasm (Figure 3E and F), their amount of membrane binding subsequently reduced to be 40% for 09CR-AuNPs and 36% for 13CR-AuNPs at 6 h, respectively (Figure 3G). The similar surface coverage-dependent cellular interaction processes of the GS-AuNPs were also observed (Figure S13). Both results of the cellular interaction with CR-AuNPs and GS-AuNPs indicated that the AuNPs with lower surface coverage preferred to bind with the cellular membrane but not internalize into the cells. In addition, the much faster and stronger interaction of CR-AuNPs with cells was attributed to the direct binding with cellular membrane or transmembrane functions of CR8, consistent with a non-endocytic mode mediated by cell-penetrating peptide.38


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The fluorescent 3D imaging technique was introduced to explore their uptake mechanism by using dual-emitting 09CR-AuNPs as model, which can clearly display the subcellular locations of the probes. The 3D ratiometric images of cells stained with pHresponsive dual-emitting AuNPs were used to monitor the states and subcellular locations of AuNPs (Figure 4). Due to the increasing R810/615 nm value with the increase of acidity in lysosome formation, these larger R810/615 nm value in ratiometric images could point out the lysosome during endocytosis. Figure 4G showed the statistical values of R810/615 nm larger than 3 from ratiometric images to statistic amount of 09CRAuNPs in lysosome. The lower percentage (R810/615 nm > 3) might attribute to the transmembrane capability of CR8 with a transient nucleation of water-filled pores,34 by which partial 09CR-AuNPs directly cross cytomembrane without undergoing endocytosis. In addition, the colocalization of AuNPs with Lysotracker Green, a specific fluorescent dye for lysosome, was also employed to explore their uptake mechanisms. The high luminescent intensity but lower colocalization percentage of 09CR-AuNPs with Lysotracker Green (~35%, while ~42% for 09GS-AuNPs) reconfirmed 09CR-AuNPs


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was internalized into cells through less endocytosis owing to the transmembrane capability of CR8 (Figure 4).

Endocytosis Pathways. Endocytosis is a general energy-dependent cellular uptake pattern of extracellular materials, which encompasses two broad categories: phagocytosis for large exogenous materials, pinocytosis for NPs, solutes and fluid.39 Pinocytosis can be fall into at least four basic subcategories: macropinocytosis, caveolae-mediated endocytosis, clathrin-mediated endocytosis, and clathrin-/caveolaeindependent endocytosis.39,40 For exploration of endocytosis pathway, the endocytic inhibitors41 including chlorpromazine (ZPC, clathrin inhibitor), methyl-β-cyclodextrin (CD, caveolae inhibitor), amiloride (AML, actin inhibitor), and sodium azide (NaN3, energy distraction) were introduced to evaluate their inhibiting effect on the uptake of CRAuNPs (Figure 5), respectively. Pretreatment of the cells with CPZ did not disable their endocytosis for CR-AuNPs (Figure 5 and S14). In contrast, luminescent intensities of the cells significantly decreased with incubation of 09CR-AuNPs after pretreated by CD (retaining 67.6% for 810 nm emission and 60.7% for 615 nm emission) or AML


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(retaining 45.3% for 810 nm emission and 23.0% for 615 nm emission), suggesting that the cellular internalization of CR-AuNPs involved multiple pathways containing caveolae-mediated endocytosis and micropinocytosis (actin-dependent process). Furthermore, a slight difference (retaining 94.2% for 810 nm emission and 73.8% for 615 nm emission) was observed for the cells with and without NaN3 treatment, thus indicating that the internalization of CR-AuNPs occurs by multiple mechanisms including both energy-dependent and energy-independent processes. Importantly, the difference between the luminescent intensities at 810 nm and 615 nm implied the pH changed after pretreatment of endocytic inhibitors, which further confirmed that the designed dual-emitting CR-AuNPs could be served as indicators for monitoring the states and functions of living cells.

CONCLUSION

In summary, we unraveled a strategy for regulation of cellular interaction with ultrasmall luminescent AuNPs by varying their surface coverage. These AuNPs with low surface coverage showed fast cellular interaction and strong membrane-binding but low


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cellular internalization proportion, whereas AuNPs with high surface coverage displayed a weak cellular interaction and membrane-binding but more cellular uptake. Besides, the surface coverage-regulated dual-emitting AuNPs could serve as self-calibration indicators for monitoring the states and functions of living cells. Furthermore, introduction of some functional ligands (e.g., cell-penetrating peptide) could be an efficient means to tune cellular interaction dynamics and internalized mechanism of NPs. This design not only offers a strategy for generation of multifunctional luminescent AuNPs in bioimaging but also provides a feasible approach to regulate their interaction mechanisms with cells for future diagnostics.

Scheme 1. Schematic illustration of CR-AuNPs with different surface coverage and their interactions with living cell.


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Figure 1. The emission spectra (A), core-size distributions (B), surface coverage (C) and zeta-potentials (D) of 07CR-AuNPs, 09CR-AuNPs and 13CR-AuNPs. The pHdependent fluorescent spectra (E) of dual-emitting 09CR-AuNPs and their linear relationship (F) between ratios of luminescence intensities at 810 nm to 615 nm at different pH values.


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Figure 2. The fluorescent images of living cells after treatment with 07CR-AuNPs (A, left), 13CR-AuNPs (A, right) and 09CR-AuNPs (B) in DPBS with different pH values, and their statistical intensities (C) and ratio values (D), respectively. Scale bar: 10 μm.


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Figure 3. The time-dependent fluorescent images of living cells after treatment with 07CR-AuNPs (A), 09CR-AuNPs (B) and 13CR-AuNPs (C). Their corresponding 3Dimages (D-F) and (G) percentages of statistical intensities around cellular membrane at 6 h. Scale bar: 10 μm.


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Figure 4. The fluorescent and ratiometric 3D-images of living cells after treatment with 09CR-AuNPs (A, B, C) and 09GS-AuNPs (D, E, F). (G) The percentages of statistical ratio values lager than 3 in ratiometric 3D-images. (H) The percentages of the


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colocalization of AuNPs and Lysotracker Green. The red channel for 810 nm, green channel for 615 nm and blue channel for Lysotracker Green.


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Figure 5. The fluorescent images of living cells after treatment with 500 nM dualemitting 09CR-AuNPs as control (A), and additional inhibitors of cellular endocytosis: ZPC (B), CD (C), AML (D), NaN3 (E). Scale bar: 20 μm.


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ASSOCIATED CONTENT

Supporting Information. The following files are available free of charge. Materials and methods, synthesis and characterization of ultrasmall AuNPs, cell imaging and interaction mechanism exploration, and supporting figures (PDF) Movie 1 07CR-AuNP (AVI)

Movie 2 09CR-AuNP (AVI)

Movie 3 13CR-AuNP (AVI)

AUTHOR INFORMATION

Corresponding Author

* E-mail: [email protected]; Tel.: +86-20-22236846.

ORCID

Jinbin Liu: 0000-0002-2046-131X

Lingshan Gong: 0000-0002-4402-1992


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The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant 21573078), Guangdong Natural Science Funds for Distinguished Young Scholars (Grant 2016A030306024) and the Fundamental Research Funds for the Central Universities.

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Table of Contents.


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Surface Coverage-Regulated Cellular Interaction ... - ACS Publications

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