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Semi-permeable functional DNA-encapsulated nanocapsules as protective bioreactors for biosensing in living cells Cheng-Yi Hong, Shu-Xian Wu, Shi-Hua Li, Hong Liang, Shan Chen, Juan Li, Huang-Hao Yang, and Weihong Tan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b00081 • Publication Date (Web): 11 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017
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Analytical Chemistry
Semi-permeable functional DNA-encapsulated nanocapsules as protective bioreactors for biosensing in living cells Cheng-Yi Hong,†,§ Shu-Xian Wu,† Shi-Hua Li,† Hong Liang,† Shan Chen,† Juan Li,*,†,‡ Huang-Hao Yang*,† and Weihong Tan*,‡,§ †
The Key Lab of Analysis and Detection Technology for Food Safety of the MOE, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350002, China ‡ Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering and College of Biology, Collaborative Innovation Center for Molecular Engineering and Theranostics, Hunan University, Changsha 410082, China § Department of Chemistry and Department of Physiology and Functional Genomics, Center for Research at the Bio/Nano Interface, UF Health Cancer Center, University of Florida, Gainesville, FL 32611-7200, USA Fax: (+1) 352-846-2410 ABSTRACT: The development of functional DNA-based nanosensors in living cells has experienced some design challenges, including, for example, poor cellular uptake, rapid nuclease degradation and high false positives. Herein, we designed selectively permeable poly (methacrylic acid) (PMA) nanocapsules to encapsulate functional DNAs for metal ions and small-molecules sensing in living cells. Since functional DNAs are concentrated in the nanocapsules, an increasing reaction rate is obtained in vitro. During endocytosis, polymeric capsules simultaneously improve cellular uptake of functional DNAs and preserve their structural integrity inside the confined capsule space. More importantly, selective shell permeability allows for the free diffusion of small molecular targets through capsule shells, but limits the diffusion of large biomolecules, such as nuclease and nonspecific protein. Compared to the free Zn-DNAzyme, PMA nanocapsules could reduce false positives and enhance detection accuracy. Furthermore, PMA nanocapsules are biocompatible and biodegradable. Through the controllability of wall thickness, permeability and size distribution, these nanocapsules could be expanded easily to other targets, such as microRNAs, small peptides and metabolites. These nanocapsules will pave the way for in situ monitoring of various biological processes in living cells and in vivo.
Functional nucleic acids (functional DNAs), such as aptamers and DNAzymes, which can be designed and selected in vitro, provide molecular recognition and catalytic activities, enabling them to play important roles in biological analysis and biomedical applications.1-4 However, these functional DNAs also encounter some challenges, especially with respect to bioanalytical applications in living cells. Two common challenges have been noted in this area. First, functional DNAs are negatively charged molecules and have low binding affinity with the cell membrane, which complicates entry into living cells.5 Second, degradation by nucleases is a frequent occurrence.6 To address these challenges, many efforts have been made, including the use of gold nanoparticles (AuNPs), graphene oxide (GO) and carbon nanotubes (CNTs), as both carriers and fluorescence quenching materials in sensor design.7-9 While these results are encouraging, some unaddressed issues remain, such as the generation of false positive signals in the case of nonspecific binding by DNA/RNA-binding proteins.10 Moreover, it may cause distinct reduction in the activity of functional DNAs, especially DNAzyme due to steric hindrance of nanoparticle surface attached to the DNAzyme.11 In addition, some of the above materials are not biodegradable, causing safety issues at high concentration. Therefore, the design and construction of intracellular biosensors must take into account
biocompatibility, as well as protection against degradation and a reduction in false positives. Owing to their unique features, polymeric capsules have attracted considerable research attention in recent years with promising applications in the fields of biosensors, biocatalysts, gene silencing, drug delivery and cancer therapy.12-14 In particular, micro- or nanosized polymeric capsules have attracted great interest as versatile carriers in the area of materials science, chemistry and pharmaceuticals.15-17 These capsules are made by stepwise layer-by-layer (LBL) adsorption of polymers onto a temporary template core, which can be removed to produce hollow capsules. Based on the controllability of their wall thickness, permeability and size distribution, many biomolecules such as proteins, enzymes, DNA, and drugs, have been encapsulated inside the hollow interior.18-20 Stimulated by external factors, like temperature, pH, and light, these biomolecules are released by gradual biodegradation.21,22 Moreover, semi-permeable polymeric capsules can simultaneously protect biomolecules against nuclease degradation and nonspecific protein binding, while preserving their structural integrity inside the confined hollow capsule space.23 However, despite increased research activities, the use of polymeric capsules in intracellular sensing has been treated in only a few studies.24,25
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Therefore, inspired by the selective permeability and biodegradability of nanocapsules, we designed poly (methacrylic acid) (PMA) nanocapsules to encapsulate functional DNAs (DNAzyme in this case) for biosensing in living cells. As illustrated in Scheme 1a, the DNAzyme was first adsorbed onto positively charged silica templates via electrostatic interactions. After that, poly (N-vinyl pyrrolidone) (PVP) and thiolated PMA multilayers were built up according to previous publications.26,27 After dissolving silica cores and releasing PVP multilayers, the singlecomponent, crosslinked PMA nanocapsules were formed. Semipermeable PMA capsules allowed small solute molecules, such as metal ions, to diffuse through the capsule membrane, while limiting the diffusion of large biomolecules, such as nuclease. As expected, functional DNAs encapsulated in PMA nanocapsules maintain its functional integrity, which shown high and specific response to their target (Scheme 1b). Furthermore, PMA nanocapsules serve as protective bioreactors to protect functional DNAs from nuclease degradation and improve cellular uptake, making functional DNAs suitable for intracellular sensing.
Scheme 1. Schematic illustration of (a) encapsulation of functional DNAs into PMA nanocapsules, (b) the PMA nanocapsules for intracellular imaging of metal ions in living cells.
EXPERIMENTAL SECTION Materials and Apparatus. Both 400 nm and 700 nm SiO2 particles were obtained from Aladdin as a suspension (2.5% w/v) and used as received. Poly(methacrylic acid, sodium salt) (PMA), Mw = 15 kDa, was purchased from Polysciences (USA), and poly(vinylpyrrolidone) (PVP), Mw = 55 kDa, cysteamine hydrochloride, N-(3-dimethyl-amino-propyl)-N'ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), 3aminopropyltrimethoxysilane (APTS), dithiothreitol (DTT), chloramine T trihydrate, magnesium (II) chloride, zinc (II)
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acetate dihydrate, lead (II) acetate trihydrate, calcium (II) chloride, barium (II) chloride, copper (II) chloride, manganese (II) chloride, ferrous (II) sulfate heptahydrate and mercury (II) acetate were purchased from Sigma-Aldrich. Ultrapure water obtained from a Millipore water purification system (18.2 MΩ resistivity) was used in all runs. Transmission electron microscopy (TEM) images were taken on the FEI Tecnai G20 transmission electron microscope with an accelerating voltage of 200 kV. Dynamic light scattering (DLS) data were measured using Zetasizer (Nano ZS, Malvern, Worcestershire, England). The fluorescence measurements were performed with a Hitachi F-4600 fluorimeter (Hitachi Co. Ltd., Japan) equipped with a xenon lamp under room temperature. The fluorescence images of cells were taken on a Confocal Laser Scanning Microscope (CLSM) (Nikon, Japan). Preparation of Amine-Functionalized SiO2 Particles. The suspension of 700 nm SiO2 particles in 5 mL of ethanol was charged with 250 µL of 30% ammonia solution and 1 mL of APTS and incubated for 2 h. Afterwards, the particles were recovered via centrifugation and washed with sodium acetate buffer (pH 4, 10 mM). Thiolated poly (methacrylic acid) (PMA) Synthesis. The sample of thiolated poly (methacrylic acid) was synthesized as described previously.26 In brief, a PMA solution (250 mg of 30 wt % solution) was diluted into 5 mL of potassium phosphate buffer (0.1 M, pH 7.2). The resulting solution was charged with EDC (70 mg) and NHS (45 mg), and the mixture was stirred for 15 min. Following this, 7.5 mg of cysteamine hydrochloride preoxidized in air for three days was added to the mixture. The reaction was allowed to proceed overnight. The resulting mixture was dialyzed extensively against distilled water, and the polymer was isolated via freeze-drying. Before the assembly of PMA/PVP bilayers, thiolated poly (methacrylic acid) was incubated in potassium phosphate buffer (1 M, pH 8) containing DTT for 12 h, and the thiolated poly (methacrylic acid) solution was diluted with sodium acetate buffer (10 mM, pH 4) to the desired concentration. Assembly of PMA/PVP bilayers. A suspension of the amine-functionalized SiO2 particles was incubated with 1 µM Zn2+-DNAzyme-Cy3-BHQ2 for 12 h followed by three wash steps. The DNA-coated SiO2 particles (1 mg/mL) were redispersed with a solution of thiolated poly (methacrylic acid) (1 mg/mL) in sodium acetate buffer (10 mM, pH 4), and adsorption of thiolated poly (methacrylic acid) was allowed to proceed for 15 min with constant shaking at 4 °C. After that, the particles were washed with sodium acetate buffer three times and redispersed with a solution of PVP (1 mg/mL) in sodium acetate buffer. PVP adsorption was allowed to proceed for another 15 min at 4 °C, after which the particles were washed with sodium acetate buffer. This procedure described the assembly of a single bilayer, and the process was repeated until forming eight bilayers. Preparation of Zn-DNAzyme-PMA nanocapsules. After forming eight PMA/PVP bilayers, the SiO2 particles were treated with 2 mM chloramine T (CaT) in 2morpholinoethanesulfonic acid buffer (50 mM, pH 6) for 1 min. The SiO2 particles were then dissolved by treatment with HF/NH4F solution. After centrifugation, the Zn-DNAzymePMA nanocapsules were resuspended in 50 mM HEPES buffer (containing 100 mM NaCl, pH 7.4). The washing cycles were repeated until the pH of the Zn-DNAzyme-PMA nanocapsules suspension became identical to the pH of
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Analytical Chemistry
HEPES buffer. Pb-DNAzyme-PMA, Zn-Pb-DNAzyme-PMA and ATP-PMA were prepared using different DNAzymes. Other procedures were the same as above. Fluorescence Measurements. A certain concentration of Zn2+ stock solution was added into Zn-DNAzyme-PMA nanocapsules and incubated for 30 min. The dye (Cy3) was excited at 540 nm, and the fluorescence emission spectra were recorded from 558 to 700 nm. The time-dependent fluorescence changes of Cy3 were followed by exciting the dye at 540 nm and monitoring the fluorescence at 563 nm. Selectivity Experiment. The Zn2+ stock solutions were added into Zn-DNAzyme-PMA nanocapsules with a final concentration of 1 µM. For the other metal ions, the concentrations were the following: 5 µM for Mg2+ and 25 µM for Pb2+, Ca2+, Ba2+, Cu2+, Mn2+, Fe2+ and Hg2+. Measurements were performed as described above. Imaging Intracellular Zn2+ and Pb2+. First, MCF-7 cells were cultured in RPMI-1640 medium for 24 h. Next, the cells were incubated with Zn2+ and Pb2+ (25 µM) at 37 °C in 5% CO2 for 60 min, followed by washing with PBS. Finally, ZnPb-DNAzyme-PMA nanocapsules were delivered into MCF-7 cells for 3 h, and the cells were washed three times with PBS. Fluorescence images were acquired on a confocal laser scanning microscope (CLSM). Zn2+ was recorded by FAM in the green channel with 488 nm excitation. Pb2+ was recorded by Cy5 in the red channel with 637 nm excitation. MCF-7 cells were incubated with 25 µM Zn2+ and 25 µM Pb2+, respectively, or without metal ions, as control. ATP imaging. For cell imaging experiments, MCF-7 cells were cultured in RPMI-1640 medium for 24 h. Prior to the addition of ATP-PMA nanocapsules, cells were incubated with or without 10 µg/mL oligomycin or 5 mM Ca2+ for 30 min. Then ATP-PMA nanocapsules were delivered into MCF7 cells. After 3 h, the cells were washed three times with PBS and imaged with CLSM under excitation at 480 nm. RESULTS AND DISSCUSION Characterization of PMA nanocapsules. To demonstrate the utility of our design, we selected Zn2+ as the first model. Negatively charged SiO2 particles (-26.2±2.00 mV, Figure S1) with a diameter of approximately 700 nm (Figure 1a, b) were chosen in this assay. The successful modification of amino to SiO2 particles was confirmed by z-potential (10.9±1.02 mV, Figure S1) and FT-IR spectra (Figure S2). Then the aminofunctionalized SiO2 particles were exposed to solutions of Cy3-labeled Zn2+-specific DNAzyme (Table S1). The change of DNAzyme fluorescence indicated successful adsorption of DNAzyme on the SiO2 particles via electrostatic interaction (Figure S3). Next, the PMA/PVP multilayers were built up by the sequential deposition of PMA and PVP polymer onto SiO2 particles according to the previously published procedure.26,27 Transmission electron microscopy (TEM) revealed that eight PMA/PVP bilayers approximately 22±4 nm in thickness were deposited on the surface of SiO2 particles (Figure 1 c, d), indicating a thickness of one PMA/PVP bilayer to be approximately 2.75±0.5 nm. This result was confirmed by depositing the PMA and PVP polymer onto SiO2 particles of different sizes (Figure S4). After silica core dissolution and the release of PVP multilayers, PMA nanocapsules were finally formed (Figure 1 e, f). Next, we investigated the stability of the PMA nanocapsules in a range of physiological solutions. PMA nanocapsules
exhibited good dispersion in HEPES buffer and cell medium, even after 8 hours (Figure S5a, b). Moreover, these capsules were stable in reduced glutathione solution for 3 hours, while swelling of nanocapsules was observed, indicating that the disulfide linkages were gradually destroyed in a reducing environment (Figure S6).26-28 These results revealed that the PMA nanocapsules will be degradable in the intracellular environment, showing good biocompatibility.
Figure 1. TEM images of amine-functionalized SiO2 particles (a,b) nondeposited and (c,d) deposited with eight PMA/PVP bilayers. (e,f) TEM image of PMA nanocapsules.
Figure 2. (a) Fluorescence emission spectra of Zn-DNAzymePMA nanocapsules treated with various concentrations of Zn2+ (0, 0.1, 0.25, 0.5, 1, 2.5, and 5 µM). Inset: the plot of F/F0 ratio as a function of Zn2+ concentrations. (b) Selectivity studies of Zn-DNAzyme-PMA nanocapsules over other ions (N=5). The in vitro response of Zn2+-specific DNAzymeencapsulated PMA (Zn-DNAzyme-PMA) nanocapsules was then investigated. As illustrated in Figure 2a, fluorescence intensity increased with increasing Zn2+ concentrations from 0 to 5 µM. These results revealed that Zn-DNAzyme-PMA nanocapsules could effectively signal the presence of Zn2+. Furthermore, Zn-DNAzyme-PMA nanocapsules responded to targets more rapidly than free Zn2+-specific DNAzyme probes (Zn-DNAzyme) (Figure S7), indicating the increased reaction rate compared to the traditional DNAzyme-based detection method. Next, the selectivity of Zn2+ detection was tested by comparing Zn2+ with some divalent metal ions, such as Mg2+, Pb2+, Ca2+, Ba2+, Cu2+, Mn2+, Fe2+ and Hg2+. The result shown in Figure S8 suggested that Zn-DNAzyme-PMA nanocapsules treated with Zn2+ had a higher level of fluorescence intensity
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than other divalent metal ions. The difference of fluorescence enhancement between the Zn2+ and other divalent metal ions was statistically significant (p
