Letter pubs.acs.org/NanoLett
Interrogation of Cellular Innate Immunity by Diamond-NanoneedleAssisted Intracellular Molecular Fishing Zixun Wang,† Yang Yang,§ Zhen Xu,† Ying Wang,† Wenjun Zhang,*,‡,¶ and Peng Shi*,†,⊥ †
Department of Mechanical and Biomedical Engineering, ‡Department of Physics and Material Science, and ¶Center of Super-Diamond and Advanced Films (COSDAF), City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR 999077, China § Functional Thin Films Research Center, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China ⊥ Shenzhen Research Institute, City University of Hong Kong, Shenzhen 518057, China S Supporting Information *
ABSTRACT: Understanding intracellular signaling cascades and network is one of the core topics in modern biology. Novel tools based on nanotechnologies have enabled probing and analyzing intracellular signaling with unprecedented sensitivity and specificity. In this study, we developed a minimally invasive method for in situ probing specific signaling components of cellular innate immunity in living cells. The technique was based on diamond-nanoneedle arrays functionalized with aptamer-based molecular sensors, which were inserted into cytoplasmic domain using a centrifugation controlled process to capture molecular targets. Simultaneously, these diamond-nanoneedles also facilitated the delivery of double-strand DNAs (dsDNA90) into cells to activate the pathway involving the stimulator of interferon genes (STING). We showed that the nanoneedle-based biosensors can be successfully utilized to isolate transcriptional factor, NF-κB, from intracellular regions without damaging the cells, upon STING activation. By using a reversible protocol and repeated probing in living cells, we were able to examine the singling dynamics of NF-κB, which was quickly translocated from cytoplasm to nucleus region within ∼40 min of intracellular introduction of dsDNA90 for both A549 and neuron cells. These results demonstrated a novel and versatile tool for targeted in situ dissection of intracellular signaling, providing the potential to resolve new sights into various cellular processes. KEYWORDS: Diamond nanoneedles, intracellular sensing, in situ detection, innate immunity, STING activation
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many of these probes require intracellular delivery to initiate the sensing process and cannot be retrieved afterward, and potentially induce toxicity and repeatability issues. With the convergence of nanotechnology and biomedical research, many nanodevices have been introduced for investigation of intracellular activity.13−17 Nanowire or nanoneedle structures have been exploited to effectively access intracellular domain for various applications.18−26 Particularly, a nanowire-cell sandwich assay was recently developed to probe enzymatic activity in living cells.19 This method is not versatile enough to work with different intracellular signaling components such as transcriptional factors, messenger proteins, or microRNAs, etc. Here, we developed a minimally invasive method for in situ probing of intracellular signaling dynamics in living cells. The biosensing device was based on a diamondnanoneedle array functionalized with aptamer molecular
ost cellular behaviors and functions are associated with intracellular signal transduction. Understanding the signaling cascades and network is one of the core topics in molecular biology. The invention of novel tools for probing signaling activity in living cells has been part of the major thrust advancing our knowledge about different aspects of cell function.1−4 From the traditional in vitro biochemical assays5,6 to the commonly used approaches based on fluorescent measurement,7−11 existing methods for probing intracellular signaling have distinct advantages and drawbacks. The biochemical assays usually involve isolation of signal elements (proteins, enzymes, or nucleotides, etc.) from cell homogenates.6 Though this method is very effective to characterize molecular interactions, even down to single molecular level,12 it does not provide any information in the context of functional cellular environments. Another commonly used strategy for intracellular sensing has been based on fluorescent imaging.8−10 For example, fluorophore−quencher conjugates have been widely employed to visualize intracellular molecular processes;7 fluorescence resonance energy transfer (FRET) has also been used to monitor the kinase activation in living cells.11 However, © XXXX American Chemical Society
Received: August 7, 2015 Revised: September 4, 2015
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DOI: 10.1021/acs.nanolett.5b03126 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 1. Schematic of the “molecular fishing” technique based on functionalized diamond-nanoneedles. The nanoneedles were functionalized with aptamer sequences designed to bind NF-κB, which was trigged to move to nuclear region upon activation of the STING pathway by foreign DNA pathogens (dsDNA90).
Figure 2. Functionalization and characterization of diamond-nanoneedle array. (a) The strategy for cross-linking FITC-labeled aptamer onto diamond-nanoneedles. (b) Scanning electron microscopy image showing the morphology of diamond-nanoneedles. Scale bar, 10 μm. (c) Three dimensional reconstruction of confocal images showing an overview of the functionalized diamond-nanoneedle array. Scale bar, 50 μm. (d) Representative confocal image showing enlarged view of the functionalized diamond-nanoneedles. Scale bar, 10 μm. For panels c and d, green fluorescence was from FITC-labeled aptamer cross-linked to the diamond-nanoneedles.
(dsDNA90).27,28 The delivery of dsDNA90 was simultaneously achieved at the initial nanoneedle insertion, as a result of temporary membrane disruption21 (Figure 1). After certain probing period, the diamond-nanoneedles were retrieved from cytoplasmic domain, and the amount of captured NF-κB protein was analyzed and quantified by immunostaining and confocal microscopy. By repeated probing in living cells at multiple time-points, we were able to reveal the signaling dynamics of NF-κB in both cancer cells (A549) and primary
sensors, which were used to study cellular innate immunity targeting the host-defense response to pathogen molecules such as foreign DNAs. In this proof-of-concept study, diamondnanoneedles were used as “fishing rods” that were inserted into cytoplasmic region of living cells without damaging them; the aptamer sequences on nanoneedles were used as “fishing baits” that were designed to specifically capture NF-κB, which is released upon stimulator of interferon genes (STING) activation by intracellularly delivered double-strand DNAs B
DOI: 10.1021/acs.nanolett.5b03126 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 3. Diamond-nanoneedle-assisted intracellular “molecular fishing”. (a) Schematic of the centrifugation controlled intracellular insertion and probing process. (b) FITC fluorescence image showing diamond-nanoneedles before “molecular fishing”. Scale bar, 20 μm. (c) Cy5 fluorescence image showing diamond-nanoneedles after “molecular fishing” of NF-κB. Immunostainning was performed targeting NF-κB proteins (red) afterward. (d) Combined fluorescence image showing colocalization of NF-κB speckles (red) with diamond-nanoneedles (green). Scale bar in the inset indicates 10 μm.
Figure 4. Interrogation of STING activated innate immune response in living A549 cells. (a) Phase-contrast image of cultured A549 cells. Scale bar, 50 μm. (b) Combined fluorescence images showing colocalization of NF-κB speckles (red) with diamond-nanoneedles (green) at 0, 7, 15, and 40 min after intracellular delivery of dsDNA90. Scale bar, 20 μm. Enlarged views of the boxed region in the top row are shown in the bottom row. Scale bar, 5 μm. (c) Quantitative analysis of NF-κB positive nanoneedles with or without the STING activator (dsDNA90) in A549 cells at different experimental time points. Error bars indicate s.e.m. from three independent experiments. ∗, P < 0.001 by ANOVA analysis.
neurons. Altogether, this study demonstrated a novel and versatile tool for targeted in situ dissection of intracellular signaling, and it can potentially be used to resolve new sights into various cellular processes. Fabrication and Functionalization of Diamond-Nanoneedles. The diamond-nanoneedles were fabricated using bias-assisted reactive ion etching (RIE) by electron cyclotron resonance microwave plasma chemical vapor deposition (ECRMPCVD) and were characterized by scanning electron
microscopy (SEM). Most nanoneedles were fabricated to be a tapered cylindrical morphology with a diameter of ∼300 nm and a height of ∼5 μm (Figure 2). The material, diamond, has been demonstrated with superior biocompatibility due to its inertness and is safe to use with various types of cells including fragile primary neurons.21 To render the diamond-nanoneedles biosensing capability, they were functionalized with the DNA aptamers (5′-GGGGAATCCCC-3′) that were specifically designed to bind NF-κB.29 The chemical modification strategy C
DOI: 10.1021/acs.nanolett.5b03126 Nano Lett. XXXX, XXX, XXX−XXX
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Figure 5. Interrogation of STING activated innate immune response in primary hippocampal neurons. (a) Phase-contrast image of cultured primary hippocampal neurons (9 days in vitro). Scale bar, 50 μm. (b) Combined fluorescence images showing colocalization of NF-κB speckles (red) with diamond-nanoneedles (green) at 0, 7, 15, and 40 min after intracellular delivery of dsDNA90. Scale bar, 20 μm. Enlarged views of the boxed region in the top row are shown in the bottom row. Scale bar, 5 μm. (c) Quantitative analysis of NF-κB positive nanoneedles with or without the STING activator (dsDNA90) in neuron cells at different experimental time points. Error bars indicate s.e.m. from three independent experiments. ∗, P < 0.001 by ANOVA analysis.
implementation of the diamond-nanoneedle-assisted intracellular “molecular fishing”. Interrogation of Cellular Innate Immunity in Living Cells. The aptamer-functionalized nanoneedles were then used to investigate the dynamics of NF-κB signaling in different types of cells, including cancer cells (A549) and primary neurons, to probe their innate immune response after invasion of pathogen dsDNA. A549 cells were cultured in four-well plates to ∼80% confluence before any experiments (Figure 4a). To monitor the NF-κB translocation process, we repeated the “molecular fishing” in living cells at different time points (0, 7, 15, and 40 min) after STING activation by intracellularly delivering dsDNA90 at the first (0 min) probing trial. As shown in Figure 4, panels b and c, upon STING activation, the percentage of NF-κB positive nanoneedles gradually decreased from 52.1 ± 6.7% (n = 3, mean ± s.e.m. (standard error of the mean), same below) to 8.9 ± 0.6% over 40 min, which was likely due to decreased intracellular level of NF-κB as a result of its translocation from cytoplasmic to nuclear regions.28,30 In our control groups without the STING activator (dsDNA90), no such reduction of isolated NF-κB was observed (Figure 4c). The nanoneedle-assisted probing technique was then applied to more fragile cells, primary neurons (9 days in vitro, Figure 5a), to test if neuronal cells have a similar NF-κB signaling dynamics as a result of STING activation. As shown in Figure 5, the amount of NF-κB isolated from neurons also reduced significantly, as indicated by a reduction of NF-κB positive nanoneedles (from 67.2 ± 4.8% to 30.7 ± 5.9%, n = 3, mean ± s.e.m.) within a few minutes after STING activation (Figure 5b,c). These results further proved that our diamondnanoneedle-assisted probing technique is a capable, versatile, and flexible tool for dissecting intracellular signaling. In conclusion, we developed a novel tool for probing signal transduction and for in situ isolation of targeted signaling components from living cells using arrays of diamondnanoneedles functionalized with aptamer-based molecular sensors. Of particular interest, we used the tool to investigate the cell host defense activity, cellular innate immunity, in
was shown in Figure 2, panel a. A diamond-nanoneedle patch was first bathed in heated piranha solution to modify the surface with hydroxyl groups. (3-Aminopropyl)triethoxysilane (APTES) was then cross-linked to the surface, to which NHSbiotin, streptavidin, and biotinylated DNA aptamers were sequentially linked. Because of multiple binding sites on a molecule, streptavidin was used as a bridge for the final functionalization of aptamer DNAs. For visualization purposes, the aptamer sequences were prelabeled with FITC fluorophore on the 3′ end so that functionalized diamond-nanoneedles could be readily examined by laser confocal microscopy as a result of successful cross-linking of FITC-labeled aptamer sequences (Figure 2c,d). Intracellular “Molecular Fishing”. As we designed, the diamond-nanoneedles were used as “fishing rods”, and the cross-linked aptamers were used as “fishing baits”, which can together be used to retrieve intracellular signaling components from living cells in a minimum invasive format. Meanwhile, the nanoneedles also worked as a delivery tool for cytoplasmic introduction of foreign materials (e.g., dsDNA90 in this study) by temporarily disrupting cellular membrane21 (Figure 3a). For proof-of-concept, the functionalized nanoneedles (Figure 3b) were first tested in A549 cells to see if any NF-κB protein could be isolated from intracellular region after STING activation upon invading of foreign dsDNA90. The whole detection and “molecular fishing” processes were performed in a centrifugation induced supergravity environment (Figure 3a, 12.8 g) where the nanoneedles were delicately pressed to poke cells for 90 s to capture NF-κB molecules. The patch of nanoneedle array was then retrieved and immunnostained to examine the amount of successfully isolated NF-κB (Figure 3c). After the “molecular fishing” procedures, NF-κB staining appeared to be concentrated speckles on the nanoneedle patch. The majority of these NF-κB speckles were colocalized with nanoneedles in a highly consistent manner (Figure 3d), whereas no similar NFκB staining was observed in control patches with diamondnanoneedles cross-linked with just Alexa Fluor 488 (no aptamer, Supplementary Figure S1), suggesting successful D
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Nanodiamond films of 7 μm thick were deposited in step one using a commercial ASTeX MPCVD equipped with a 1.5 kW microwave generator. The nanodiamond deposition was performed in the plasma induced in a 10% CH4/H2 mixture at a total pressure of 30 Torr and total gas flow rate of 200 sccm. The microwave power and deposition temperature were maintained at 1200 W and 800 °C, respectively. After the nanodiamond film deposition was finished, the second step of RIE was performed using electron cyclotron resonance MPCVD. The ASTeX microwave source employed a magnetic field of 875 G generated by an external magnetic coil. The RIE conditions were as follows: a mixture of 45% Ar and 55% H2 was used as the reactive gases at a total flow rate of 20 sccm; the substrate bias was −200 V; the reactant pressure was 7 × 10−3 Torr. The etching duration was 3 h and the input microwave power 800 W, respectively. The morphology of diamondnanoneedle patch was characterized by a Philips FEG SEM XL30. The sample was tilted 90° for SEM. Functionalization of Diamond-Nanoneedles. Diamondnanoneedles were first bathed in piranha (3:1, v/v, 98% H2SO4: 27.5% H2O2) solution at 90 °C for 1.5 h and then cleaned by distilled water, methanol, methanol/dichloride methane (DCM) mixture (3:1, v/v), and DCM sequentially. The nanoneedle patch was dried with nitrogen and then immersed in APTES solution (20% in DCM) overnight in nitrogen protected environment. Ethanol, 2-propanol, and distilled water were used to wash the nanoneedle patch step by step, which was followed by nitrogen drying. After cleaning, the nanoneedle patch was sequentially bathed in NHS-Biotin (Sigma-Aldrich) solution (1 μg/mL in PBS) for 1 h, streptavidin (SigmaAldrich) solution (10 μg/mL in PBS) for 2 h, and FITC-labeled biotinylated aptamer (Takara) solution (1 μM in TE-buffer, pH = 8) for 1 h. The patch was rinsed with distilled water between different steps. The functionalized nanoneedles were imaged by confocal microscope equipped with a 40× water-immersion lens (Leica, SP8). Centrifugation Controlled “Molecular Fishing”. Intracellular delivery of dsDNA90 and subsequent signal probing were performed using a centrifugation controlled process as previously described.21 Briefly, the culture medium was first removed, and cells were covered by 50 μL of dsDNA90 solution (25 ng/mL). A nanoneedle patch was placed on top of the liquid with nanoneedles facing toward cells. The whole setup was then placed in a centrifuge with a plate-rotor and spun at 400 rpm (22.8g). After centrifugation, 450 μL of culture medium (containing dsDNA90 at 25 ng/mL) was immediately added to the culture well to lift off the nanoneedle patch, and also completed the first molecular probing at time 0 min. After being incubated for 7, 15, or 40 min, the cells were examined using multiple aptamer-functionalized nanoneedle patches to capture and determine the NF-κB signaling dynamics upon STING activation by dsDNA90. Each “molecular fishing” process was also controlled by centrifugal force and lasted for 90 s in the centrifugation-induced supergravity environment (300 rpm, 12.8g). Immunostaining and Fluorescent Microscopy. After “molecular fishing”, the retrieved diamond-nanoneedle patch was first washed by PBS and blocked with casein solution (1% in PBS) for 30 min at room temperature. The sample was then incubated with anti-p50 primary antibody (Life Technologies) for 30 min and Cy5-labeled secondary antibody (Life Technologies) for 15 min. After thorough rinsing with PBS, confocal microscopy was used to image and quantify the
response to foreign DNA pathogens.30,31 We found that the transcriptional factor, NF-κB, was quickly transferred from cytoplasm to nucleus upon the activation of the STING pathway by a stereotypic pathogen, dsDNA90.27,28 Especially for primary neurons, cells from the central nervous system were thought to be immune-privileged in the past due to the existence of blood−brain barrier; our results directly showed that pathogens, such as foreign DNAs, also trigger significant responses in neurons. By taking advantage of a repeatable and reversible intracellular probing protocol, we directly isolated NF-κB proteins from cell inside without hurting the cells and quantified the STING activation level via a straightforward colocalization assay. Besides the demonstrated “molecular fishing” based on aptamer−protein interaction, the “fishing baits” (aptamer sequence) can be easily replaced with a large repertoire of molecules targeting other intracellular signaling components, such as microRNAs and enzymes, which opens up new possibility for many downstream analyses that could done on freshly isolated and enriched molecular targets. In addition to molecular sensing and fishing, our method also provides an alternative to quickly deliver various materials into cytoplasm, aiming to activate specific intracellular pathways.21 In this case, we showed that dsDNA90 was delivered into A549 and neuron cells within a couple minutes, matching the time scale of STING activation and subsequent NF-κB translocation, which would not be possible if using traditional methods based on cellular endocytosis processes.30 Experimental Section. Cell Culture. All procedures involving animals were approved by the animal ethical committee of City University of Hong Kong. Hippocampal neurons were cultured on 12 mm Germen coverslips (Bellco Glass). Before use, the coverslips were cleaned with concentrated nitric acid (70 wt %/wt) overnight and rinsed with sterile DI water. The coverslips were further coated with polylysine (Sigma) at 100 μg/mL overnight and then laminin at 10 μg/mL (Life Technology) for 4 h before neuron cells were seeded. Hippocampi tissues were dissected from E18 embryonic Sprague−Dawley rats and treated with papain (Sigma) for 30 min at 37 °C. Dissociated neurons were prepared by triturating enzymatic treated tissue with a 1 mL pipet tip in Dulbecco’s modified eagle medium (DMEM) solution containing 10% fetal bovine serum (FBS). Neurons were then seeded onto coated coverslips at a density of 3−5 × 104/cm2 in four-well multidishes. After the initial adhesion of neuron cells (2 h after seeding), the medium was replaced by Neurobasal medium supplemented with B27, L-glutamine, and penicillin/streptomycin. Half of the medium was replaced with fresh medium every 3−4 days. A549 cancer cells were maintained in DMEM (Life Technology) supplemented with 10% FBS (HyClone), Lglutamine, and penicillin/streptomycin. Before any “molecular fishing” experiments, cells were seeded in a four-well multidish (Nunclon, Thermo Scientific) and let grow to ∼80% confluence. Nanoneedle Fabrication and Characterization. The fabrication is based on two processes as previously described.21 Deposition of nanodiamond film and subsequent bias-assisted reactive ion etching (RIE) by electron cyclotron resonance microwave plasma chemical vapor deposition (ECR-MPCVD). N-type (001) silicon wafers of 3 in. in diameter were used as substrate. Before nanodiamond deposition, the substrate was ultrasonically abraded for 60 min in a suspension of nanodiamond powders with a grain size of 5 nm in ethanol. E
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amount of NF-κB protein bonded on the surface of diamondnanoneedles. The colocalization of NF-κB staining speckles and nanoneedle fluorescence were used as indications of successful fishing of molecular targets from cell inside.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b03126. Results from control experiments of “molecular fishing” with diamond-nanoneedles functionalized with Alexafluor 488 instead of aptamers (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions
Z.W. and Y.Y. contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (NSFC81201164, NSFC51402344), Early Career Scheme from RGC Hong Kong (ECS125012), General Research Fund from RGC Hong Kong (GRF11211314, GRF11218015), Innovation and Technology Commission of Hong Kong (ITS/376/13), and grants from City University of Hong Kong (ARG9667120).
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REFERENCES
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