Cytosolic Transport of Nanoparticles through Pressurized Plasma

Nov 21, 2016 - Transporting nanoparticles into live cells is important for drug delivery and other related applications. We found that cells exposed t...
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Cytosolic Transport of Nanoparticles through Pressurized Plasma Membranes for Molecular Delivery and Amplification of Intracellular Fluorescence Yoshihisa Kaizuka,*,† Tomoto Ura,† Shaowei Lyu,†,‡ Ling Chao,‡ Joel Henzie,† and Hidenobu Nakao† †

National Institute for Materials Science, 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047, Japan, and Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan

‡

S Supporting Information *

ABSTRACT: Transporting nanoparticles into live cells is important for drug delivery and other related applications. We found that cells exposed to hypoosmotic pressures can internalize substantial quantities of gold nanoparticles. Importantly, these nanoparticles can circumvent normal intracellular traffic and be transported directly into the cytosol, without the need for surface functionalization. In contrast, nanoparticles endocytosed at physiological osmolality are segregated inside endocytic organelles and are not able to reach the cytosol. Cytosolic internalization was observed for nanoparticles of various sizes and materials, with minimal short- or longterm damage induced by the internalized particles. Thus, our strategy can be used as a delivery platform for a range of applications from therapeutics to medical imaging. As examples, we demonstrated rapid delivery of membrane-impermeable molecules to the cytosol by using nanoparticles as carriers and the use of nanoparticles assembled within the cytosol as plasmonic nanoantenna to enhance intracellular fluorescence. We propose a model for the mechanisms behind nanoparticle internalization through pressurized plasma membranes via the release of lateral pressures. Such characterizations may constitute a foundation for developing new technologies, including nanoparticle-based drug delivery.

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INTRODUCTION

While these methods can deliver molecules to the cytosol, they do so with varying and sometimes very low efficiencies. The endocytic pathway captures most macroscopic carriers including nanoparticles and viruses, which are then trapped inside endosomes and lysosomes. In this scenario, some form of “endosomal escape” must occur for the cargo molecules to reach the cytosol.1−3,7 However, the mechanisms for endosomal escape are not well understood and the efficiency of escape is not well controlled. Furthermore, molecules may be degraded during their stay inside the endosomes or lysosomes prior to their escape. Among the other methods, peptide tags are convenient but are not yet applicable to all biomolecules, and physical methods tend to damage cells as holes are created in plasma membranes.6 Herein, we used gold nanoparticles (GNPs) and other metal and inorganic nanoparticles as a model cargo and investigated how to deliver macroscopic particles to the cytosol. By virtue of their stability and nontoxicity, GNPs have been previously used as delivery vehicles by attaching molecules to their surfaces.1 GNPs are also useful as agents for imaging and spectroscopy, because they support collective excitations of free electrons called local surface plasmon polaritons (LSPs).8,9 LSPs can focus light to nanometer length scales, creating strong

Efficient and controlled delivery of materials to the cytosol of live cells is one of the central challenges of drug and gene delivery and is also useful for many biochemical or imaging applications.1−3 The plasma membrane is the first barrier for entry into the cell by most molecules, with the endocytic machinery acting as a second layer of defense. A healthy cell uses endocytosis to segregate external materials into endosomes and lysosomes, which are then disassembled with digestive enzymes and low pH or exocytosed. Signaling proteins act as the third layer of defense, where pattern recognition receptors (PRRs) and DNA/RNA sensors recognize pathogens and initiate cellular reactions.4 All of these systems limit the access of external materials to the cytosol, where most of the important biochemical reactions in the cytoplasm take place. Many transport technologies have been developed to overcome these cellular defenses. Liposomes and viral particles are frequently used as cargo carriers for delivering genetic materials.3 Synthetic nanoparticles are another major carrier of biomolecules inside the cell, and numerous types of polymeric, inorganic, and metal nanoparticles have been synthesized with complicated surface modification technologies.1,5,6 Additionally, cell penetrating peptide tags containing multiple positive amino acids can deliver proteins to the cytosol,6 while physical methods, such as microinjection, electroporation, or gene gun technology, have also been used for this purpose. © XXXX American Chemical Society

Received: September 18, 2016 Revised: November 18, 2016 Published: November 21, 2016 A

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Figure 1. Spatial localization of GNPs internalized in cells under different conditions. Representative fluorescence microscopy images of mCherryconjugated 5 (A) and 100 nm (B) diameter GNPs that were internalized in HeLa cells expressing Lamp1-GFP. Cells were under 300 mOsm/L (near physiological osmolality). Whole-cell merged images (left) and blow up images. (C, D) 5 or 20 μg/mL bare 100 nm diameter GNPs internalized in HeLa-Lamp1GFP cells under 100 mOsm/L solution and imaged by simultaneous epi-fluorescence and reflection microscopy after washing out excess GNPs. Images of multiple cells at each condition in panels A−D are shown in Figure S1. (E) Density of internalized 100 nm bare GNPs in the section images of cells under different incubation conditions (5 or 20 μg/mL; 100, 200, or 300 mOsm/L). To calculate density, the number of GNPs and areas of cell sections in the images were yielded by semi-automatic image analysis. More than 20 cells were analyzed under each condition. (F) Co-localizations of Lamp1-GFP on the GNPs detected in the image analysis as in panel E counted and compared.

electromagnetic (EM) fields that amplify the emission properties of fluorescent molecules located near the metal surface. This effect has been used in various types of chromatography-based diagnostics, nanoscale molecular detection, and in vivo photoacoustic imaging.10−12 Thus, a wide range of characterization and detection methods is available for studying GNPs. We found that live cells under hypoosmotic pressures can rapidly internalize large amounts of GNPs into the cytosol, the space outside of endocytic organelles, with minimal damage to the cells. Inhibitors of endocytosis did not perturb this cytosolic internalization, and plasma membranes under osmotic pressure exhibited enhanced permeability toward hydrophobic dyes. These observations suggest that GNPs directly penetrate to the cytosol via the pressurized

plasma membranes and can bypass endosomes and lysosomes. Using this system, we successfully introduced different types of nanoparticles to the cytosol of live cells, including mesoporous silica nanoparticles that enabled the rapid and efficient delivery of membrane-impermeable molecules to the cytosol. We also show that this system enabled the assembly of protein−GNP conjugations in the cytosol. These assembled structures behaved as plasmonic nanoantenna and exhibited enhanced intracellular fluorescence, which will be useful for imaging and analytical applications. Finally, we describe a physical model of how nanoparticles can penetrate cell membranes under hypoosmotic pressure, which may aid our understanding of the phenomena and the development of novel technologies, for applications including in vivo drug delivery. B

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Figure 2. Representative fluorescence/reflection images of HeLa cells that expressed wild type and engineered mCherry proteins and internalized GNPs under different conditions: (A) 20 μg/mL of 100 nm GNPs internalized at 100 mOsm/L in HeLa expressing wild type mCherry; (B) 20 μg/ mL of 100 nm GNPs internalized at 300 mOsm/L in HeLa expressing engineered C−mCherry−C protein; (C) 20 μg/mL of 100 nm GNPs internalized at 100 mOsm/L in HeLa expressing C−mCherry−C. (D) Schematic of spatial localization of GNP and mCherry proteins at differential osmotic pressures. Images of multiple cells at each condition (A-C) are shown in Figure S1.

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RESULTS Imaging the Internalization and Localization of Gold Nanoparticles in Live Cells. We first tested how GNPs were internalized in cells under normal conditions by fluorescence microscopy. To create a fluorescence label for GNPs, we expressed recombinant mCherry fluorescent proteins in Escherichia coli (E. coli) and purified them accordingly. Wild type mCherry proteins do not have internal cysteine residues, so we genetically introduced a cysteine at the N-terminus of the peptide to help bind the protein to the gold nanoparticle via a sulfur−gold bond. To image the fluorescent nanoparticles internalized in the cells, HeLa cells were incubated with 5 μg/ mL of the mCherrry-conjugated GNPs in culture medium for 2 h. After rinsing, individual fluorescent GNPs could be imaged inside the cells, including both 5 and 100 nm diameter particles (Figure 1A,B and Supporting Information Figure S1A,B). This result confirms that the labeling was efficient, and fluorescence quenching of mCherry by the Au surface was not a significant issue for detection. By dual-color imaging, we observed that most of the GNPs were co-localized with Lamp-1 GFP (lysosome marker, Figure 1A,B), but not with Rab5-GFP (early endosome marker, Figure S2A,B). Thus, GNPs with a wide range of sizes were internalized in cells and were likely transported to the lysosomes through endocytosis, as expected.13 As an imaging control, we examined the localization of nanoparticles inside cells treated with detergent Triton X-100. The detergent creates pores in the plasma and intracellular membranes, making cells not viable, and endocytosis does not function. As expected, mCherry GNP entered the cytoplasm of the Triton X-100-treated HeLa cells after a brief incubation (∼10 min), but they were located outside of the lysosome

(Figure S2C). These GNPs likely entered via diffusion through the holes in the plasma membrane. In addition, we added Triton X-100 to cells that already contained GNPs in their lysosomes and found some of those trapped nanoparticles appeared to leak into the cytosolic compartment (Figure S2D). These results again confirm that co-localization of GNP and Lamp1 was due to the trapping of GNPs inside lysosomes. Enhanced Internalization of GNPs into the Cytosol of Live Cells via Pressurized Plasma Membranes. To introduce nanoparticles into the cytosol of live cells, we used two different approaches. First, the concentration of nanoparticles used during incubation was increased. Second, osmotic pressure was applied to the cells during the incubation. Through these treatments, we expected to saturate the capacity of the lysosomal pathway, so that excess particles leaked into the cytosol. Additionally, osmotic forces applied to the plasma membrane might allow some particles to directly traverse the membrane. In these experiments we used unmodified 100 nm GNPs to avoid potential artifacts that can be caused by surface modifications. These unmodified GNPs could be imaged using a reflection filter because they scatter light much more strongly than background reflections (details are described in Materials and Methods). Strikingly, we observed that the 100 nm diameter GNPs could only be transported into the cytosol, space outside of the lysosomes, when cells were exposed to hypoosmotic pressure. Such cytosolic transport of GNPs was observed at various GNP concentrations in the extracellular media (Figure 1C,D and Figure S1C,D). To quantify these observations, the number of internalized GNPs in each image was counted. The total number of intracellular GNPs could be enhanced either by increasing the extracellular GNP concentration or by changing osmolality from 300 mOsm/L (near physiological osmolality) C

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Figure 3. (A) Proliferations of HeLa cell at three conditions (cells under medium and cells that experienced 2 h of incubation with 20 μg/mL GNPs at 300 mOsm/L (medium) or 100 mOsm/L (1:2 mix of medium and water)), measured by the absorbance of WST-8 formazan at 460 nm at the different time points after incubation with GNPs. Medium was replaced with fresh DMEM in all cases after the incubation period with GNPs. (B) Image of cells incubated with 20 μg/mL GNPs at 100 mOsm/L, at the time after the 2 h incubation with GNPs and at 2 days after the incubation. (C−E) Time-lapse imaging of calcein fluorescence loaded in HeLa cells. Cells were subjected to different osmotic pressures by replacing media. (C) Images of cells at 100 mOsm/L at two time points. (D) Plot of normalized sections area of cells that were measured from time-lapse images of calcein in cells. (E) Plot of normalized integral intensities of calcein fluorescence in whole images.

plasmid vector lacking any signal sequence. Thus, these proteins were expressed in the cytosol and not in endosomal compartments or in organelles, as long as the protein expression levels in cells were kept low enough that overexpressed proteins did not form aggregates or accumulate within endosomes and other organelles. We found that GNPs internalized in the cytosol (under 100 mOsm/L) co-localized with C−mCherry−C (Figure 2C and Figure S1E), strongly indicating direct binding. In contrast, GNPs within endosomes (internalized under 300 mOsm/L) and cytosolic C−mCherry−C did not co-localize, suggesting spatial segregation of the two (Figure 2B and Figure S1F). As a control, we also observed that GNPs in the cytosol (internalized under 100 mOsm/L) and wild type mCherry (lacking cysteine) in the cytosol did not exhibit clear colocalization (Figure 2A and Figure S1G), confirming GNP− protein binding via the gold−sulfur bond shown in Figure 2C. We concluded that GNPs enter the cytosol under osmotic pressure (Figure 2D). Next, we examined the short- and long-term effects of osmotic stresses on cells. We first prefilled HeLa cells with calcein to monitor the effect of hypoosmotic pressure by fluorescence microscopy (Figure 3C−E). These pressurized cells exhibited instantaneous swellings as expected (Figure

to 100 mOsm/L (hypoosmotic). At 300 mOsm/L, most GNPs were trapped in lysosomes even at the higher extracellular GNP concentration, as shown for mCherry-labeled GNPs (Figure 1A,B, showing that the labeling of GNPs with mCherry did not alter the internalization pathway). This observation demonstrates that the capacity of the lysosomal pathway was not filled in these conditions. In other words, although increasing the number of nanoparticles during incubation did promote more endocytosis, only cells under hypoosmotic stress increased the number of GNPs delivered into the cytosol, in addition to those GNPs that were endocytosed in lysosomes. We sought to prove definitively whether these nanoparticles were indeed located within the cytosol rather than contained within other cellular organelles. Co-localization studies can indicate where GNPs are located because binding of cytosolic proteins cannot occur when GNPs are segregated into separate intracellular compartments. We used two proteins: wild type mCherry, which intrinsically lacks cysteine, and a genetically modified version of mCherry that had triple cysteine residues introduced at both the N- and C-termini (i.e., C−mCherry−C). The cysteine-rich version of mCherry was expected to bind to gold surfaces via gold−sulfur bonding, whereas the wild type protein lacks such specific binding capacity. Genes of both proteins were cloned in a regular mammalian expression D

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Figure 4. Measurements of the GNP internalization in cells by flow cytometry. (A) Dot plots of cells under each condition. One dot represents a signal from a single cell. Fluorescence (red) and back scattering signals of single HeLa cells incubated with 5 μg/mL of 100 nm GNP (bare (dark) or mCherry-labeled) at 300 mOsm/L (medium) for 2 h and control cells (no GNP). Mean and standard deviation of signals are plotted. (B) Histograms of cell fractions for HeLa cells measured in panel A. (C) Cell fraction histograms for red fluorescence and BSC signals from HEK293 cells (GNPs were incorporated under the same condition as in panel A for HeLa cells). (D) BSC signals of HEK293 cells incubated with 0 (control), 2, 5, and 20 μg/mL of 100 nm GNPs for 2 h at 100 and 300 mOsm/L. (E, F) HEK293 cells incubated with 5 μg/mL of 100 nm GNPs for 2 h at 300 (E) and 100 (F) mOsm/L, with or without 5 μM brefeldin A (BFA) or 10 μM colchicine. Both inhibitors were added to the control cells without GNPs. Cells treated with inhibitors were preincubated with the inhibitors for 1 h at 37 °C before adding GNPs. (G−I) Cell fraction histogram in fluorescence (green) signals of HEK293 cells incubated with or without 5 μM of GFP (G), calcein (H), or DiBAC4(3) (I) at 100 or 300 mOsm/L. Signal gains in the detector differed in each measurement, and thus the background signals (null) varied in these histograms. More than 2000 cells were measured in all conditions, and the cell fraction was calculated for normalization (A−I).

cytometry-based measurements to statistically analyze the incorporation of GNPs into the cytosol of individual cells. Previous work showed that backscattering from cells can be used to measure the internalization of metal nanoparticles within cells.14 To examine whether GNPs inside the cells could be detected by flow cytometry, we first compared the fluorescence and backscattering (BSC) signals from cells that internalized either mCherry-labeled or unmodified GNPs. HeLa cells incubated with GNPs at 5 μg/mL for 2 h were measured with a flow cytometer. We found that the cell populations incubated with both unmodified and mCherrylabeled GNPs exhibited enhanced backscattering signals compared with control cells without GNPs, while only the

3C,D). However, the total amount of calcein trapped in the cells barely changed (Figure 3E), demonstrating that leakage of calcein from the cells was minimal. We concluded that cells under hypoosmotic pressure do not rupture or form macroscopic pores in their plasma membranes. In addition, we monitored the long-term effect by cell proliferation assays (Figure 3A,B). A 2 h treatment of hypoosmotic stresses used for GNP uptake did not have a critical impact on cell proliferation over a period of 48 h. Overall, we did not detect any significant damage to cells induced by the hypoosmotic stresses and internalized GNPs in our experiments. Mechanisms of Transport of Gold Nanoparticles in the Cytosol under Osmotic Pressure. We used flowE

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proteins (GFPs) incubated at 5 μM barely changed at different osmotic pressures (Figure 4G), so the plasma membranes were essentially impermeable to highly hydrophilic molecules even under high osmotic pressures. However, in the case of membrane permeable dyes, we observed significant enhancements of the entry of both calcein and DiBAC4(3) (Figure 4H,I). Calcein was added to cells in the form of a nonfluorescent hydrophobic ester (Figure 4H). Esterase digested this form of calcein in the cytoplasm to generate fluorescent hydrophilic calcein that was no longer permeable to the plasma membranes (Figure 3C−E). DiBAC4(3) is a bisoxonol type anionic dye frequently used as a fluorescent indicator for membrane potential. Increased uptake of DiBAC4(3) is indicative of depolarization of the plasma membrane. However, the DiBAC4(3) internalization was not caused by the instantaneous depolarizations of the plasma membranes that might be caused by sudden fluctuations in the ionic concentration in the extracellular media, because these dyes were added after cells were continuously exposed to hypoosmotic stress for 2 h. As for long-term depolarization of cell membrane potentials, previous measurements showed that hypoosmotic pressure (177−200 mOsm/L) induced only minor changes (5−10 mV depolarization or hyperpolarization) in membrane potentials,17,18 which is smaller than the change we observed.19 Thus, another mechanism could have enhanced the DiBAC4(3) internalization, such as a change in membrane structure, which may be common to the enhanced uptake of various hydrophobic molecules as well as nanoparticles. We also observed rapid cell swelling (Figure 3B), which would enhance the lateral tension and might even stretch the plasma membranes. Under such stretching, the basic structure and dynamics of the cell membranes could change and the hydrophobic layers may become less fluid and thinner. We speculate that such changes may effectively enhance the permeability of plasma membranes and induce direct penetration of heavy GNPs through the membranes under the influence of gravity. Enhanced permeability also promotes penetration of hydrophobic dyes (Figure 4H,I), but it is not enough to enhance the uptake of highly hydrophilic proteins (Figure 4G). In the Discussion, we describe a model of how nanoparticles but not proteins could penetrate through pressurized plasma membranes. Rapid Delivery of Molecules into the Cytosol Using Nanoparticles. Our understanding of the pressure-induced internalization mechanism tells us that this process is not material-specific and other materials may also be internalized in the cytosol by osmotic pressure. Indeed, we observed enhanced internalization of silver (30 nm diameter), titanium oxide (40 nm diameter), and mesoporous silica nanoparticles (200 nm diameter, 4 nm pore size) (Figure 5A). Nanoparticles were incubated with cells at constant concentration, 33 pM, which is equivalent to the concentration of 100 nm GNPs at 20 μg/mL. Thus, the osmotic pressure-induced nanoparticle internalization occurred independently of material properties and particle sizes. These results indicate that surface interactions between nanoparticles and plasma membranes may not be very important for internalization under hypoosmotic conditions. Mesoporous nanoparticles with various materials have been developed as cargo carriers for the delivery of molecules into cells.20−22 In most of the previous studies, these particles were internalized by endocytosis and slow endosomal escape was required to deliver molecules to the cytosol. Such a slow process may be beneficial when observing long-term effects

cells incubated with mCherry−GNPs showed higher red fluorescence as expected (Figure 4A). In addition, we observed higher backscattering signals coming from individual cells that had enhanced red fluorescence signals, further indicating that these contained mCherry−GNPs (Figure 4A). These results demonstrate that we could statistically measure the incorporation of GNPs in an unbiased manner by measuring the increase in average BSC signals of whole-cell populations. Uptake of gold nanoparticles in both HeLa and HEK293 cells were similarly detectable (Figure 4B,C), but the BSC signals of cells containing mCherry−GNPs were slightly lower than cells containing unmodified GNPs in both cell types. It is possible that modification with mCherry might induce aggregation of a portion of GNPs, creating unwieldy aggregates that are too large to enter cells (Figure 4B,C). The flow-cytometry-based assay was used to measure GNP uptake induced by osmotic pressure. The BCS signals from cells incorporated with GNPs under hypoosmotic pressures were enhanced to a much greater degree than cells exposed to 300 mOsm/L in both HEK293 and HeLa cells (Figure 4D and other data not shown). Hypoosmotic pressures enhanced the BSC signals from cells at all GNP concentrations, and more than 2000 cells were measured under each condition. The mean BSC signals for cells internalizing GNPs at either 300 or 100 mOsm/L were 5.9 × 105 or 7.9 × 105 (2 μg/mL GNP), 7.6 × 105 or 14.7 × 105 (5 μg/mL GNP), and 12.6 × 105 or 24.0 × 105 (20 μg/mL), respectively, while the mean background BSC signals from control cells without GNPs was 4.9 × 105 (Figure 4D). Thus, these results statistically validated our observation by microscopy imaging that hypoosmotic pressures increase the uptake of GNPs (Figure 1E). To investigate how GNPs can enter the cytosol, we examined whether the inhibitors of endocytosis might affect internalization. Although many inhibitors exist, we chose brefeldin A (BFA) and colchicine because they do not affect the plasma membrane structures but only modulate the cytoplasmic components. BFA is an inhibitor for GBF-1, which is a guanine exchange factor involved in ER-Golgi retrograde transport,15 and colchicine is a macropinocytosis inhibitor that alters tubulin GTPase activity.16 As expected, both BFA and colchicine significantly attenuated the BSC signals of the cells when most GNPs were endocytosed and trapped in lysosomes (300 mOsm/L and incubation with 5 μg/mL GNPs, as in Figure 1A,B). Average BSC signals were 4.2 × 105 (control, cells with both inhibitors only), 7.7 × 105 (cells with GNPs), 5.8 × 105 (GNP and BFA), and 5.5 × 105 (GNP and colchicine) (Figure 4E). These results demonstrate that both BFA and colchicine inhibited the endocytosis of GNPs. In contrast, at 100 mOsm/ L, we repeatedly observed that these inhibitors did not alter the level of BSC. Average BSC signals were 6.6 × 105 for the control cells with both inhibitors only, 10.0 × 105 for cells with GNPs, 9.8 × 105 for cells with GNP and BFA, and 10.4 × 105 for cells with GNP and colchicine (Figure 4F). This result demonstrates that the inhibition of endocytosis did not block the internalization of GNPs into the cells under hypoosmotic pressure. Because inhibition of endocytosis did not block the internalization of GNPs into cells, we hypothesized that GNPs could diffuse directly through plasma membranes when cells were exposed to hypoosmotic stress. To further examine this hypothesis, we tested whether molecules could be internalized through pressurization of the plasma membrane. We found that the internalization of soluble green fluorescent F

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the Cytosol. We sought to use the cytosolic GNPs to modulate the fluorescence signals of cells, which could be useful for therapeutics and imaging applications. We attempted to develop these applications by examining the interactions between GNPs and fluorescent proteins (Figure 2). In addition to mCherry proteins (wild type and C−mCherry−C), we also expressed green and near-infrared (near-IR) fluorescent proteins, AcGFP and iRFP, and examined how GNPs could modulate the fluorescence of these proteins inside the cells. As we did previously with C−mCherry−C, multiple cysteine residues were added to AcGFP and iRFP (i.e., C−GFP−C and C−iRFP−C, hereafter), although wild type AcGFP and iRFP proteins both contain internal cysteine residues. We found that the fluorescence signals of the cells expressing engineered proteins (C−GFP−C, C−mCherry−C, and C−iRFP−C) were enhanced by the internalized cytosolic GNPs (Figure 6A). In contrast, no such enhancement was observed for cells expressing wild type mCherry (Figure 6A, mCherry(WT)). Earlier we showed that wild type mCherry did not bind to GNPs, contrary to C−mCherry−C (Figure 2A). Thus, these results demonstrate that the interaction between GNPs and fluorescence proteins functioned to enhance fluorescence. Noble metal nanoparticles behave as optical antennae that can amplify the absorption and emission properties of adjacent fluorescent molecules via plasmon-enhanced fluorescence.23,24 Thus, the overall fluorescence enhancement of cells can be interpreted as the ensemble plasmonic antenna effect caused by all fluorescent proteins that are coupled to GNPs. Plasmonenhanced fluorescence of single molecules is strongest when the excitation wavelength overlaps with the local surface plasmon resonance (LSPR), and the emission wavelength of the fluorophore is red-shifted from the LSPR of the particle.23,25 We indeed found that the degree of amplification varied among fluorophores that have distinct excitation wavelengths and that the amplification of emission of each fluorophore was slightly stronger at longer emission wavelengths (Table 1). The peak excitation wavelengths of the proteins were 475 nm (AcGFP), 587 nm (mCherry), and 690 nm (iRFP), respectively. Moreover, previous studies using purified fluorescent proteins (GFP and mCherry) showed that the fluorescence intensities of single fluorescent proteins were enhanced by metal surfaces in vitro.26,27 Collectively, these results showed that the conjugation of fluorescent proteins and GNPs can be used to amplify fluorescence signals of cells and that the enhancement is most likely caused by the plasmonic antenna effect. In addition, there could be fluorophore-specific contributions, because different lasers (488, 561, and 638 nm) were used to excite each protein and the structure of each protein may be differentially affected by the GNP surface. Gold Nanoparticles Clustering in the Cytosol. The absorption spectrum of our 100 nm GNPs in aqueous solution had a maximum at 550570 nm, regardless of whether or not they were partially bound by serum proteins (Figure S3). Because LSPR underlies the absorption of GNPs, the intensity of the electromagnetic (EM) field created by LSPR of single GNPs is the strongest around these wavelengths.8 The plasmonic-enhanced fluorescence is also expected to be strongest when the excitation wavelength of the fluorophore is near these wavelengths. However, we observed stronger plasmonic enhancement for iRFP (excited at 690 nm) than for mCherry (excited at 587 nm) (Table 1). This discrepancy could be explained by the red shift of LSPR of GNPs in the cytosol, and the modified LSPR might be better overlapped

Figure 5. (A) HEK293 cells incubated with 33 pM nanoparticles of Ag (30 nm diameter), mesoporous silica (200 nm diameter), and itanium oxide (40 nm diameter) for 2 h at 300 and 100 mOsm/L and the backscattering signals of cells measured by flow cytometry and compared with control cells under 100 mOsm/L without nanoparticles (null). More than 2000 cells were measured under all conditions, and the cell fraction was calculated for normalization. (B− D) Mesoporous silica nanoparticles (200 nm diameter with 4 nm size holes) were loaded with Alexa 555-labeled phalloidin. Then, 33 pM of the nanoparticles were incubated with HeLa cells at 300 mOsm/L (B) and 100 mOsm/L (C) at 37 °C. Cells were imaged after 1 h. Asterisks and arrows indicate individual cells and phalloidin-stained F-actins. The schematic shown in panel D depicts these results.

such as cellular changes over several days caused by delivered genetic materials but are not ideal for systems requiring quick delivery of molecular imports.1,3,20 In contrast, our experimental system enables the direct delivery of nanoparticles to the cytosol within a few hours. To create a model molecular delivery system, we loaded fluorescent phalloidin in mesoporous silica nanoparticles (200 nm diameter, 4 nm pore size) and used osmotic pressure to internalize them into HeLa cells. Within 1 h, filamentous staining appeared in the cells, showing that phalloidin was transported into the cytosol and was bound to filamentous actins (Figure 5C). In contrast, we only observed spots of fluorescence wihtin cells under 300 mOsm/L (near physiological osmolality), suggesting that the phalloidin-loaded nanoparticles were trapped in lysosomes and the diffusion of phalloidin was restricted within lysosomes (Figure 5B). These results demonstrate that we could deliver molecules rapidly and efficiently to the cytosol by exposing cells to nanoparticles under hypoosmotic pressures (Figure 5D). Note that the cells in the images were still alive and thus may look slightly different from the fixed and permeabilized cells that are stained with phalloidin. Creating Plasmonic Nanoantenna Inside Cells by Assembling Protein−Gold Nanoparticle Conjugates in G

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Figure 6. (A) Flow cytometry measurements for fluorescence signals of cells expressed with each fluorescence protein (mCherry, C−GFP−C, C− mCherry−C, or C−iRFP−C) with or without GNPs internalized at 100 mOsm/L for 2 h. (D, E) Dark-field microscopy images of HeLa cells recorded by a color-CCD camera. GNPs were internalized at 300 mOsm/L (B) or 100 mOsm/L (C). Magnified images of GNPs in individual cells are displayed on the right. (D) Representative spectra of one of brightest GNP spots in the cytosol and typical single GNPs in the lysosome and outside the cell (on a glass plate). The bar in the inset image is 1 μm. (E) Relative scattering intensities of single GNPs (monomer, d = 100 nm), dimers, and trimers modeled with the finite difference time domain (FDTD) method. A refractive index of n = 1.35 was chosen so that the model closely matches the environment of the cytosol. A full description of the simulated particles is included in the Supporting Information. (F) Histogram of average integral RGB signals of individual GNP spots in the images of panels B and C. (G) Histogram of the ratios between red and blue signals in individual GNP spots in the images of panels B and C. (H) Schematic of GNP clustering in the cytosol and the localized antenna effect on the color of fluorescent proteins.

Table 1. Fluorescence Enhancement of Cells by Internalized GNP in Detectors for Distinct Emission Wavelength Ranges fluorescence enhancement in detectors for distinct wavelength ranges fluorophore in cells

Ex peak (nm)

500−550 nm

570−630 nm

650−680 nm

690−750 nm

755−815 nm

C−GFP−C C−mCherry−C C−iRFP−C

475 587 690

1.223

1.227 1.801 2.125

1.285 1.815 2.398

1.331 1.824 2.456

1.857 2.457

binding or GNP clustering.8,9 Protein binding to the surface of GNPs modifies the refractive index, resulting in a red shift of

with the iRFP excitation spectrum. Such a red shift of LSPR can be induced by surface modifications of GNPs, including protein H

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Figure 7. Physical model of how nanoparticles can penetrate through the pressurized plasma membranes. Gravitational pressure could induce a local break between lipid bonds to trigger insertion. Once inserted, GNPs could release the lateral tension, although the newly formed GNP−lipid interface could be an energy cost.

the LSPR wavelength by ∼10 nm or less,8,9,28 as was also shown as a slight shift in the absorption spectra of GNPs in serum-containing media (Figure S3). In contrast, it is wellknown that GNP clusters support stronger EM fields than single particles and that the LSPR of clusters tends to red shift and broaden at larger scales.8,9,28 Because the excitation wavelength for iRFP exceeds the LSPR wavelength of single GNPs by ∼100 nm, clustering of GNPs might better explain our observations. To characterize the modulations of LSPR, we measured the scattering of GNPs in the cytosol by dark-field microscopy. In this setup, only scattered light from the GNPs is detected, and the color of the light can be recorded as signals split into the red, green, and blue (RGB) pixels of a color-CCD camera. Because scattered light is also substantially enhanced near the peak LSPR wavelength, the color of scattered light should appear more red if the LSPR of particles are red-shifted.8,9 To determine the colorimetric difference between GNPs in the cytosol versus GNPs in the lysosomes, we measured GNPs internalized in HeLa cells under either 100 or 300 mOsm/L, respectively (Figure 1F). These measurements were conducted in the absence of exogenous fluorescent proteins. In the image constructed from all RGB channels, we indeed observed the red color shift of GNPs in the cytosol, compared with the largely yellowish GNPs trapped in the lysosomes (Figure 6B,C). We also observed a similar red shift of GNPs that had diffused into the cytosol of necrotic cells, where cell membranes were ruptured (Figure S4). The spectra of scattered light from single GNP spots were measured (Figure 6D). The spectra of the brightest GNPs in the cytosol were red-shifted by roughly 100 nm, compared with the spectra of yellow GNPs within lysosomes. These yellow GNPs within lysosomes were considered to be single GNPs, because their spectra were very similar to the spectra of single GNPs outside the cells (Figure 6D). In addition, the intensity of spectra was much stronger in the red-shifted GNPs within the cytosol, suggesting that the GNP spots in the cytosol probably contained more than one GNP (Figure 6D). However, the physical sizes of GNP spots that appeared in

the images were similar, both in the cytosol and in the lysosomes, suggesting that even highly red-shifted GNP spots in the cytosol did not contain many 100 nm GNPs. To examine whether a cluster containing a few GNPs can induce a red shift, we simulated the scattering spectra of monomers, dimers, and trimmers of GNPs by the finite difference time domain (FDTD) method28 (Figure 6E and Figure S5). These simulations showed that even dimers can yield a ∼100 nm red shift in the scattering spectrum and several-fold amplification of scattering intensity, compared with single GNPs (Figure 6E). In addition, we measured the intensity in RGB pixels for all GNP spots in the images shown in Figure 6B,C. We found that both the mean RGB intensities and the ratio of red to blue intensities of all GNPs were enhanced for cytosolic GNPs (Figure 6F,G). These analyses statistically confirmed that the GNP spots in the cytosol contained more GNPs and their scattering spectra were red-shifted, compared with the GNP spots within lysosomes. Collectively, these results suggest that GNPs in the cytosol tend to form clusters and induce a red shift in the LSPR. Because the cytosol is highly crowded with proteins, both the surface neutralization of GNP by protein absorption as well as the depletion attractive force can induce GNP clustering in the cytosol.29,30 In contrast, in lysosomes, the protein density may be lower due to degradation caused by digestive enzymes, and furthermore, the number of GNPs per single lysosome is lower, thus less clustering may occur. Observations of such differential clustering phenomena as well as of the interaction of GNPs and fluorescent proteins in the cytosol suggested that the surfaces of GNPs were not completely covered with serum proteins or totally passivated before the entry to the cells. EM fields are localized in nanometer-scale gaps in the clusters, and when a fluorophore is trapped inside these gaps, it can generate a fluorescence enhancement that is many times stronger than that of a single particle.23 Our FDTD simulation also showed that the enhanced EM field created by clustered GNPs is spatially larger at longer wavelengths (Figure S5). Therefore, the GNP clustering may underlie the fluorophore-dependent amplification of cellular fluorescence (Figure 6H). I

DOI: 10.1021/acs.langmuir.6b03412 Langmuir XXXX, XXX, XXX−XXX

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Our studies used a combination of microscopy and flow cytometry techniques, including simultaneous fluorescence− reflection imaging for nanoparticle localization in live cells, statistical flow-cytometry-based measurements of internalized nanoparticles, and colorimetric imaging of nanoparticle clustering to understand the conditions under which nanoparticles can directly traverse the cell membrane. We applied these multiple methods to a single experimental system; thus our studies may also be useful for assay development and fundamental characterizations of nanoparticles within cells. Other techniques could be introduced for further analysis. For example, our analysis of GNP−protein interactions in cells was insufficient to provide the molecular-scale views of how GNPs form clusters and how GNPs interact with proteins. In vitro biochemical experiments using GNPs and purified proteins would be a useful future direction of study. Our strategy enables efficient and rapid delivery of nanoparticles to the cytosol, and thus it could be useful for various applications, particularly in vitro, including molecular transport (Figure 5) and nanoantenna assembly (Figure 6). Formation of nanoantenna could provide insights, in addition to its usefulness in bio-optical assays, because such intracellular structures may regulate cellular biochemical systems. Cytosolic nanoparticles conjugated with specific biomolecules could selectively regulate reactions in a highly localized manner within the cytosol. Thus, such a system may be useful as a novel tool for basic cell biology. In contrast to these in vitro applications, applying our strategy in vivo is not straightforward. Nonetheless, further characterizations of the internalization process could lead to the development of new strategies to alter the plasma membrane structures in vivo and thus enable the cytosolic internalizations of nanoparticles for drug delivery.

DISCUSSION We demonstrated that the internalization of nanoparticles within the cytosol was induced by hypoosmotic pressure. Our analysis suggests that a large portion of those internalized nanoparticles directly penetrate through pressurized plasma membranes. How such penetration under osmotic stress would occur has been a fundamental question in physical chemistry,31 although the penetration of larger objects, such as GNPs, has rarely been studied. We propose that penetration of nanoparticles occurred through the insertion of GNPs via gravitational pressure and by the release of lateral tension within stretched plasma membranes (Figure 7). The membranes of swelled cells under hypoosmotic pressure were stretched without creating large holes (Figure 3C−E), resulting in strong lateral tension. Such macroscopic tension derived from the expansion of the average distance between neighboring lipids and the weakened interactions between hydrophobic lipid tails, in the microscopic view. In such a stretched state, the pressure applied by objects with higher density such as GNPs could disrupt the local integrity of lipid−lipid connections and induce the insertion. Note that, in fluid lipid bilayers, local fluctuation in the density of lipids would aid the insertion of GNPs and such a localized disruption of molecular bonds alone does not necessarily lead to the rupture of the whole membrane structure. In contrast, light objects such as soluble GFP proteins would not easily trigger such an insertion. Furthermore, the insertion of nanoparticles within lipid bilayers could be energetically favorable, because the inserted objects could release the lateral tension by compensating for the enhanced membrane surface area of swelled cells and effectively shortening the spacing between lipids, although the newly formed GNP−lipid interface should be a free energy cost. Inserted nanoparticles could pass through the membrane by gravitational force, and then another GNP would insert to contentiously release the tension. In such a manner, GNPs could penetrate the membranes without the creation of permanent holes (Figure 3E). Other factors could also contribute. Membrane thinning could occur upon stretching. However, such a change would have a higher impact on smaller sized objects, because the typical width of lipid bilayers is 4−5 nm, and thus it is not consistent with our observations: internalization of soluble GFP (